Revision as of 18:26, 21 October 2019

Sortase A7M (Ca²⁺-independent variant)

Profile

Name	Sortase A7M
Base pairs	450
Molecular weight	17.85 kDa
Origin	Staphylococcus aureus, synthetic
Properties	Ca²⁺-independent, transpeptidase, linking sorting motif LPXTG to poly-glycine Tag

Structure

Figure 1 : Modeled 3D-Structure of Sortase A7M. To find out more, visit our modeling page.

Usage and Biology

Transpeptidase: Sortase

Sortases belong to the class of transpeptidases and are mostly found in gram-positive bacteria. The high rate of resistance to several antibiotics targeting gram-positive bacteria is also based on the property of this enzyme class. Sortases can non-specifically attach virulence and adhesion‐associated proteins to the peptidoglycans of the cell-surface.
In general, sortases are divided into six groups (A-F) that have slightly different properties and perform three tasks in cells. Group A and B attach proteins to the cell-surface while Group C and D help building pilin-like structures. Group E and F are not properly investigated yet which is why their exact function is not known. For our project we are especially interested in the sortases of the group A since they covalently attach various proteins or peptides on the cell membrane as long as their targeting motif is at the C-terminus of the corresponding protein. In comparison to other transpeptidases Sortase A has the advantage that it is rather stable regarding variations in pH Sortase A catalyzes the formation and cleavage of a peptide bond between the C-terminal LPXTG amino acid motif and an N-terminal poly-glycine motif. The enzyme originates from Staphylococcus aureus and is able to connect any two proteins as long as they possess those matching target sequences. In the pentapeptide motif LPXTG, X can be any amino acid except cysteine. Sortase A is rather promiscuous with regard to the amino acid sequence directly upstream of this motif, a fact that makes it optimal for labeling applications. Even better, amino acids C-terminal of the poly-glycine motif are not constrained to a certain sequence.

Reaction

To better understand how the enzymatic reaction works it is necessary to look at the crystal structure of Sortase A. The enyzme consists of an eight-stranded β‐barrel fold structure. The active site is hydrophobic and contains the catalytic cysteine residue Cys184 as well as a key histidine residue H120 that can form a thiolate-imidazolium with the neighboring cysteine. An additional structural property that also other sortases show is the calcium binding site formed by the β3/β4 loop. The binding of a calcium ion slows the motion of the active site by coordinating to a residue in the β6/β7 loop. This helps binding the substrate and increasing the enzymatic activity nearly eightfold. When a substrate gets into the active site, the cysteine attacks the amide bond between the threonine and the glycine in the LPXTG motif. After this the protonated imidazolium serves as an acid for the departing glycine with unbound NH₂ of the former amide bond while the rest of the motif is bound to the cysteine residue. Another glycine nucleophile is then necessary in its deprotonated form to attack the thioester and re-establish an amide bond at the LPET-motif. This reaction is dead-ended if the used nucleophile is water. Due to the fact that the mechanism is based on protonated forms of the catalytic residues the reaction is quite pH-dependent. Although the Sortase A in general is relatively stable between pH 3 and 11 the reaction works best around pH 8.

Sortase variants

Due to the fact that the wildtype Sortase A shows rather slow kinetics, a pentamutant has been developed (Sortase A5M). This version of the enzyme carries mutations in P94R/D160N/D165A/K190E/K196T which lead to a 140- fold increase in activity. Thereby, reaction rates are improved even at low temperature, however, Sortase A5M is still Ca²⁺-dependent. This dependence interferes with potential in vivo usage, as the concentrations of calcium in living cells can vary considerably. Hence a sortase mutant that acts across high differences in calcium concentrations or even works completely Ca²⁺-independently would be required for in vivo applications of sortase. To attain a high yield enzyme which is also calcium-independent Ca²⁺-independent mutations were combined with the Sortase A5M resulting in Sortase A7 variants such as the Sortase A7M. The newly achieved calcium-independence of these variants enable sortase applications not only in vitro but in vivo as well.

Sortase A7M

For our project we chose to work with this optimized Sortase A7M. Its size is about 17.85 kDa and it has been shown to be stable for several weeks in the fridge at 4 °C. It also possesses the same properties of pH stability like other sortases but comes with the advantage of being calcium independent. "Sortagging" applications have included the cyclization of proteins and peptides , modification and labeling of antibodies and the synthesis of protein conjugates with drugs, peptides, peptide nucleic acids and sugars.Moreover it poses a lot of advantages for the binding of two proteins in vivo since it has relatively small tags which avoids putting too much metabolic burden on the cells when expressing the proteins of interest. This also avoids disturbing the folding of the proteins of interest and the later biological functions since the Sortase A7M is able to work under physiological conditions. Other methods like the intein- based labeling of surfaces require large fusion-proteins with the intein domain which puts stress on the living cells and might cause folding and solubility issues. Another application for sortase-mediated systems is the anchoring of proteins on the cell wall of gram-positive bacteria which can be used for display of heterologous proteins. It is also possible to attach non-biological molecules to the respective tag. The accessibility and flexibility determine the ability of a sortase enzyme to recognize the sorting motif and catalyzing the transacylation.

Methods

Cloning

The methods used for cloning of the different mutants of the sortase were restriction and ligation via NdeI and SalI and Gibson assembly. The vector posesses a kanamycin resistance and the srta7m is controlled through a T7 promoter, which can be induced with IPTG. Sortase A7M is controlled by the same T7 promoter. The product was checked via sequencing.

Expression and purification

After successfully transforming our sortase genes in BL21 cells, we inoculated 100 mL overnight cultures, with the respective antibiotic. The next day 1 L cultures were inoculated with the overnight culture to reach OD₆₀₀ = 0.1. Subsequently the cultures were incubated under constant shaking at 37 °C until they reached OD₆₀₀ = 0.6. At OD₆₀₀ = 0.6 the cultures were induced with 0.5 mL of 1 M Isopropyl-β-D-thiogalactopyranosid (IPTG). The gene expression was performed at 30 °C under constant shaking overnight. After expression of Sortase A7M in BL21 cultures the cells were crushed via EmulsiFlex (Avestin) and proteins were purified through affinity chromatography via Fast Protein Liquid Chromatography (FPLC) with the ÄKTA pure (GE Healthcare, Illinois, USA). His-Tag was used for purification of Sortase A7M and Sortase A (Stockholm) and Strep-Tag II was used for purification of Sortase A5M.

SDS-Page

To verify the successful production of of Sortase A7M and others SDS-PAGEs were performed. The resulting bands were compared to the molecular weight of the different sortase variants. Also, SDS-PAGEs were completed to verify enzymatic activity in assays prior to measuring sortase properties via Fluorescence Resonance Energy Transfer (FRET).

Flourescence Resonance Energy Transfer (FRET)

To determine the kinetics of our transpeptidase variants, FRET assays were performed in 384 well-plates (dark) using a Tecan plate reader. A FRET relies on the phenomenon that an excited fluorophore (donor) transfers energy to another fluorophore (acceptor), thereby exciting it. This process only works if both fluorescent molecules are in close proximity and depends on the FRET-Pair. By transferring the energy from donor to acceptor, the donor's emission is reduced and the intensity of the acceptors emission is increased . The efficiency depends on the distance between the fluorophore, the orientation and the spectral characteristics . You can see the principle of FRET in Fig. 2.

Figure 2 : Jablonski diagram showing the energy transfer between a FRET-pair (inspired by Alex M. Mooney).

Mass Spectrometry

To estimate the product yield of catalyzed reactions by Sortase A7M we performed mass spectrometry. The tested molecules can be distinguished between products and educts due to desorption and ionization. Therefore, we used the electrospray ionization (ESI) technique for the mass spectrometry. This technique has a low resolution but is a very soft ionization method, which makes it an optimal method for biological molecules.

Enzyme-linked Immunosorbent Assay (ELISA)

The enzyme-linked immunosorbent assay (ELISA) is an analytical assay frequently utilized for immobilization and verification of different macro molecules. Immobilizing the recognition tag of the sortase on a surface allows us to verify the coupling efficiency of Sortase A7M under certain conditions. Firstly, we functionalized paper to presenting a poly-G peptide sequence on the surface. Using the Sortase A7M, a ZZ-domain carrying a LPETG amino acid sequence is coupled to the short peptide sequence of GGGßA. The ZZ-domain itself shows high affinity to the human IgG antibody FC-domain and therefore allows the following immobilization of IgG. The secondary antibody is an anti-human IgG, Fab-specific antibody carrying the horseradish peroxidase enzyme (HRP). HRP is capable of converting 4-chloro-1-naphthol to benzo-4-chlorocyclohexandienone using hydrogen peroxide. This color-reaction allows us to draw a conclusion about the previous ZZ-domain’s sortase-mediated coupling efficiency since the turnover of the HRP is directly connected to the ligated ZZ-domains.

Results

Characterization of Sortase A7M (and comparison to BBa_K3187016)

How do we measure if our purified sortases are active?

After purification of the sortases, we first performed SDS-PAGEs to verify that they are pure and monomeric. You can see in Fig. 3 that the purifications were successful. Next, we tested if the purified sortases connect two proteins that carry the important Sortase-recognition tags, N-terminal polyG and C-terminal LPETGG. Therefore, we added the sortases to a mix of GGGG-mCherry and mCherry-LPETGG. The reactions were performed in different buffers, at different enzyme-to-substrate ratios and for different time spans. We performed an SDS-PAGE, and prior to Coomassie staining, we recorded fluorescent images of the gel. Thereby, we could identify mCherry bands in the gel.

Figure 3 : SDS-PAGE of Sortase A7M and Sortase A5M where the bands show up at approximately 15 kDa. Our estimated size for Sortase A7M was 17.85 kDa, and for Sortase A5M 18.07 kDa. This confirms the result shown on the gel, since the band of Sortase A5M is a little higher than the one of Sortase A7M.

Figure 4 : a) Fluorescence gel of the sortase-reaction of GGGG-mCherry and mCherry-LPETGG mediated by Sortase A7M incubated for 2 h and 4 h each. Reaction solutions were mixed with different ratios from enzyme to substrate concentration(1:3;1:10) and each incubated in two different buffers(Tris-HCl and Ammoniumdicarbonat). Product bands at a height of about 57 kDa can be seen in lane 4, 5, 6, 8, 9 (from left to right). The bands below the product at about 38 kDa could be semi-denatured mCherry dimers.
b) Fluorescence gel on top of the coomassie-stained gel of the sortase-reaction of GGGG-mCherry and mCherry-LPETGG mediated by Sortase A7M incubated for 2 h and 4 h each. Reaction solutions were mixed with different ratios from enzyme to substrate concentration(1:3;1:10) and each incubated in two different buffers (Tris-HCl and Ammoniumdicarbonat). Product bands at a height of about 57 kDa can be seen in lane 4, 5, 6, 8, 9 (from left to right). The bands below the product at about 38 kDa could be semi-denatured mCherry dimers. Additionally, Sortase A7M can be seen at 17 kDa.The unprocessed mCherry monomers can be seen at 28 kDa.

As shown in Fig. 4, under certain conditions, a product band appeared at the expected size of 57.3 kDa (28.5+28.8 kDa). From this first activity test, we draw three conclusions:

Our purified Sortase A7M is active
The enzyme-substrate ratio affects the product yield
The duration of the reaction affects the product yield

Additionally, TRIS buffer seems to alter the coomassie staining efficiency of Sortase A7M. This endpoint measurement gave us a first impression that our Sortase A7M works nicely. Of course, we wanted to further characterize the parameters of the reaction. When we understand the Sortase better, modification of our VLPs will become more straightforward.

How do we measure sortase reaction kinetics

In the above described assays, we noticed the impact of enzyme-substrate ratio and reaction duration on the overall product yield. We thought about how to further measure the kinetics of the sortase reaction. In the literature, sortase reaction kinetics are often measured by FRET-assays. Therefore, we designed a suitable FRET-assay.

Development of a new FRET pair

For characterization of the reaction kinetics of Sortase A7M, Sortase A5M and Sortase A, we decided to develop a suitable FRET pair. In order to find an optimal FRET pair, we first recorded an emission and absorption spectrum of 5-Carboxytetramethylrhodamin-LPETG (TAMRA) and GGGG-mCherry to verify the suitability for the FRET effect, checking for a possible overlap between the donor's emission and the acceptor's excitation.

Figure 5 : Design of a FRET-pair of 5-TAMRA-LPETG (TAMRA) and GGGG-mCherry (mCherry). In this configuration TAMRA acts as donor and mCherry as acceptor. When the two fluorophores are not linked via the substrates of the sortase only TAMRA is being excited. After sortase mediated ligation of the two substrates mCherry is the fluorophore being excited via the FRET and the emission of mCherry intensifies. Meanwhile, the emission of TAMRA decreases.

TAMRA is a chemical fluorophore that has an absorbance maximum at 543 nm and an emission maximum at 570 nm. The terminal carboxy group of the dye was linked via a lysine linker to the LPETG sequence (see Fig. 5). mCherry has an N-terminal poly-glycine sequence and can therefore be linked to the LPETG motif of TAMRA via the Sortase A. For a sufficient FRET-effect, it is also necessary that the distance between donor and acceptor is lower than the Förster radius.
First, we wanted to identify which concentrations are needed for our experiment, then set up the reaction and measured fluorescence intensities. Over time, a decline in the emission of TAMRA can be observed as Sortase A7M/A5M is converting more educts to products.

Figure 6 : The graph shows the excitation and emission spectra of TAMRA and mCherry. Due to the large overlap of TAMRA emission and mCherry excitation it is possible to perform a FRET with this pair of fluorophores. The graph show the relative fluorescence unit (RFU[%]) in relation to the excited/emitted wavelength [nm]. The peaks are normalized to 100 %.

The emission and excitation spectra of TAMRA and mCherry exhibit an overlap of emission of TAMRA and excitation of mCherry. Based on this output, a FRET-assay for the kinetics of Sortase A7M was performed to confirm whether the FRET-pair is working. As TAMRA is excited with light of a lower wavelength than mCherry, the former serves as FRET donor and the latter as acceptor. We chose the excitation wavelength at 485 nm to prevent unnecessary “leak” excitation of mCherry. Nevertheless, an excitation of mCherry could not be excluded and may have negative effects on the visibility of the FRET.

Figure 7 : Spectrum of the negative control of TAMRA and mCherry, without Sortase A7M, over the course of 20 min in 5 min intervals. Depicted are the emission wavelengths against the RFU.

Figure 8 : Spectrum of TAMRA and mCherry, with Sortase A7M, over the course of 20 min in 5 min intervals. Depicted are the emission wavelengths against the RFU. The sortase-mediated ligation results in a decline of both emission peaks.

The analysis of the data shown in Fig. 7 confirmed the aforementioned suspicion that mCherry is also excited at 485 nm, which makes differentiation of the fluorescence more difficult. Furthermore, Fig. 8 shows that the difference in the decline of TAMRA is not significant. Accordingly, a decline in the emission maximum of TAMRA over time is also visible in the negative control. One reason might be bleaching of TAMRA through the excitation by the laser. Nevertheless, conversion by the Sortase A7M can be observed by comparing the results with the negative control.

Figure 9 : Sortase reaction in TAMRA-mCherry-FRET after subtracting the negative control. Depicted is the difference in RFU over time [min]. Within the first 20 min of the substrate conversion is the quickest. At 30 min a plateau is reached. After 60 min starts catalyzing the reverse reaction. The mean ΔRFU value was normalized to zero for better visualization. The ΔRFU refers to the difference between the negative control without the respective sortase at 570 nm.

To confirm the functionality of the Sortase A7M, another more sufficient FRET-pair was developed. The measured absorbance and emission spectra indicated that TAMRA and superfolder green fluorescence protein (sfGFP) are a possible FRET-pair.

Figure 10 : The graph shows the excitation and emission spectra of TAMRA and mCherry. Due to the large overlap of sfGFP emission and TAMRA excitation it is possible to perform a FRET with this pair of fluorophores. The graph show the relative fluorescence unit (RFU[%]) in relation to the excited/emitted wavelength [nm]. The peaks are normalized to 100 %.

Figure 11 : Design of a FRET-pair of 5-TAMRA-LPETG (TAMRA) and GGGG-sfGFP (sfGFP). In this configuration sfGFP acts as donor and TAMRA as acceptor. When the two fluorophores are not linked only sfGFP is being excited. After sortase-mediated ligation of the two substrates, TAMRA is the fluorophore being excited via FRET and the emission of TAMRA intensifies. Meanwhile, the emission of sfGFP decreases.

The transfer of energy from sfGFP to TAMRA can be seen by the decrease in emission of sfGFP and increase in emission from TAMRA. Compared to TAMRA as an acceptor, the sfGFP bleaches significantly less and is consequently more suitable as a donor for FRET. Furthermore, the afore mentioned problem of simultaneous donor and acceptor excitation seems to be solved. It seems that we have found a FRET-pair with superior properties.

Figure 12 : Spectrum of the negative control of TAMRA and sfGFP, without Sortase A7M, over the course of 25 min in 5 min intervals. Depicted are the emission wavelengths against the RFU.

Figure 13 : Spectrum of TAMRA and sfGFP, with Sortase A7M, over the course of 25 min in 5 min intervals. Depicted are the emission wavelengths against the RFU. The sortase-mediated ligation results in a decline of both emission peaks.

Due to the collected data of both FRET-pairs we decided to use the TAMRA-LPETG and GGGG-sfGFP FRET-pair for further characterization of our Sortase A variants. Two reasons justify this decision:

TAMRA bleaches stronger than sfGFP when excited with a laser.
The spectral overlap between TAMRA and mCherry disturbs “clean” energy transfer, thus the FRET-effect would be less visible and could not be used for analysis of the sortase-mediated reaction.

For recording of sortase reaction parameters we recommend using the FRET-pair sfGFP-TAMRA. As this pair of fluorophores proved to have near perfectly aligned spectra and since the bleaching effect is visibly lower on sfGFP than on TAMRA, we chose to use this FRET-pair in most of our following assay. Nevertheless, we do not rule out the use of TAMRA-mCherry as a FRET-pair since we used it in several FRET-assays as well.

Figure 14 : Animation of Sortase A7M enzyme kinetics over the course of 3 h. The reaction speed increases radically in the beginning moving from RFU 8000 to RFU 6000 at λ = 550 nm where a plateau is reached (blue). The negative control (orange) is also reduced in its RFU due to bleaching. Nevertheless, a peak at λ = 580 nm arises already after short reaction time. This peak indicates the successful Fluorescence Resonance Energy Transfer.

Why are enzyme-substrate ratio and duration important parameters of the sortase reaction?

In one of our first FRET experiments, we addressed the simple theory: More sortase in the reaction mix improves the initial product formation. For this, we used the TAMRA-LPETG : GGGG-mCherry FRET pair. We measured the FRET change over time in a multiwell platereader (Fig. 15).

Figure 15 : Reaction kinetics of Sortase A7M in different concentrations at same level of substrate concentration (6 µM TAMRA-LPETG and (M)GGGG-mCherry). The light blue graph shows the reaction when the sortase concentration is higher (5 µM) and lower concentration (1 µM), in the dark blue graph. Wen the enzyme concentration is lower the maximum in substrate conversion is reached later. The light blue graph also shows a slight decline in product concentration from 60 min onwards.The ΔRFU refers to the respective negative control without each sortase at 570 nm. The mean ΔRFU value was normalized to zero for better visualization.

However, in this assay we observed a striking feature of the sortase reaction. In the reaction with more Sortase A7M present, the FRET change started to decrease after a certain maximum was reached! We suspected some kind of dead-end product formation, as the sortase does also catalyze the reverse reaction of product to educts. Therefore, the overall reaction duration is a very important parameter. We gathered more details about the role of the reverse reaction during our comparison of Sortase A7M and Sortase A5M. Just keep reading if you want to know more!

Who wins - Sortase A7M or Sortase A5M

In our introduction we described that Sortase A7M and Sortase A5M are both fascinating enzymes, although each of them has a unique „selling point“. Sortase A5M is faster, whereas Sortase A7M is Ca²⁺-independent. We confirmed both of these points in extensive FRET-assays. According to the literature, Sortase A5M works best with a Ca²⁺-concentration of 2 mM. In contrast, Sortase A7M is a calcium-independent mutant of the enzyme. Moreover, Ca²⁺ even seems to inhibit this enzyme variant slightly .

Firstly, we confirmed that in contrast to Sortase A5M, Sortase A7M is Ca²⁺-independent. The results are shown in Fig. 16 Sortase A7M also works in presence of Ca²⁺, but these FRET experiments made us suspect that Ca²⁺ may even inhibit Sortase A7M.

Figure 16 : Sortase A7M FRET-assay of connecting TAMRA-LPETG with GGGG-sfGFP with and without Ca²⁺. The Sortase A7M reaction was measured with 6 mM Ca²⁺ every minute. Sortase A7M reaction without Ca²⁺ was measured every three minutes. It is shown that this enzyme variant works with calcium and without calcium as well, although it seems like Sortase A7M is slightly inhibited due to the presence of calcium which explains why the left graph is lower than the right one. The ΔRFU refers to the respective negative control without each sortase at 514 nm. The mean ΔRFU value of the duplicates was normalized to zero for better visualization.

According to the results of this assay, Sortase A7M is definitely Ca²⁺-independent, since it shows linking activity without calcium in the vicinity. The enzyme mutant also works in presence of Ca²⁺, but these FRET experiments made us suspect that Ca²⁺ may even inhibit Sortase A7M, since it shows less activity with calcium around than without calcium.

To better address this question, an ELISA was performed. Therefore, a piece of paper functionalized with GGGβA was connected to a protein domain, which binds antibodies to the LPTEG-tag. The results are shown in Fig. 18.

Figure 18 : Absorbance at 450 nm at a temperature of 23.8˚ C
In well 1 additional 10 mM Ca²⁺ were added which was not the case in well 2. Well 3 serves as a negative control since the enzyme is missing in this reaction

As shown in Fig. 18, the highest absorption was measured in well 2. Thus, Sortase A7M works more efficiently when no Ca²⁺ is around. The absorption is also relatively high for the negative control, which can be explained by poor washing before the substrate for Horeseradish peroxidase (HPR) was added. This assay shows the functionality of Sortase A7M even in context of surfaces since we confirmed that Sortase A7M is able to connect tags attached to paper. This shows that the surface structure is not a relevant factor for the enzyme.

Figure 19 : Comparison of the reaction speed of Sortase A5M with Ca²⁺ and Sortase A7M without Ca²⁺, therefore each working under optimal conditions. The kinetics were measured via a FRET connecting TAMRA-LPETG and GGGG-sfGFP. The ΔRFU refers to the respective negative control without each sortase at 514 nm. The mean ΔRFU value of the triplicates was normalized to zero for better visualization.

When we compare the reaction speed of Sortase A5M and Sortase A7M, Sortase A5M is the clear winner (see Fig. : 19). However, this means of course that the reverse reaction is also faster in the case of Sortase A5M. Consequently, Sortase A7M is the best variant for in vivo modification of our VLPs as it is Ca²⁺-independent. On the other hand, Sortase A5M is a suitable enzyme variant for in vitro modification due to its high efficiency.

What about other substrates?

Primary Amines

The literature describes Sortase A7M as somewhat „promiscuous“ towards other substrates than GGGG(polyG) as long as the substrate possesses a primary amine. The Sortase A7M used for this assay was stored in the fridge at 4 °C for two weeks. The substrates were TAMRA with a KLPETG bound to TAMRA via the lysine side chain and 3-azidopropanamine as the example for a primary amine. The reaction was performed for two hours at 37 °C.
It was then analyzed by electron spray ionization mass spectrometry (ESI-MS) (Fig. 20).

Figure 20 : Mass spectrum before the reaction of TAMRA-LPETG with 3-azidopropanamine showing the educt at 1054 g/mol.

Fig. 20 shows the educt-peak in the mass spectrum. TAMRA with the LPETG-tag weighs 1054 g/mol. Shown above in green is the single charged molecule at 1054.27 g/mol and the double charged molecule at 528.75 g/mol.

Figure 21 : Mass spectrum after the reaction of TAMRA-LPETG with 3-azidopropanamine showing the product at 1079.37g/mol.

Fig. 21 shows the product-peak in the mass spectrum. The primary amine that was taken as an example has a molecular weight of 100 g/mol. After the reaction the glycine of the LPETG-tag has been removed and therefore the product only consists of TAMRA-KLPET-3-azidopropanamine. When adding the two molecular weights and subtracting the weight of the glycine it adds up to a total weight of 1078 g/mol which can be seen in the single loaded 1079.37 g/mol peak (Fig. 21), since the ESI-MS we used has a small error margin. The peak in black again is the double loaded peak at 541.55 g/mol. This clearly shows that the sortase reaction took place.
Furthermore, we can conclude that the Sortase A7M accepts any primary amine as a substrate. However, mass spectrum does not show the ratio of educt and product, which is why we cannot estimate whether the turnover is as high as when using a polyG-tag as substrate. Additionally this assay confirms our suspicion that the Sortase A7M is stable at 4 °C and still functional if stored at said temperature for at least two weeks.

Yield

For the characterization of Sortase A7M an assay was designed to show the coupling efficiency between the TAMRA-LEPTG and the tetrapeptide GGG-Beta-Alanin (GGGβA) catalyzed by the Sortase. The Sortase reaction was performed for 1h at 30˚C and was stopped by enzyme separation through centrifugal filtration. For analysis mass spectrometry (ESI-MS) was used. The mass spectrometry enables differentiation between products and educts. It allowed us to make an estimate of the product yield. The calculated theoretical molecular masses are 1054 g/mol for TAMRA and 1240 g/mol for TAMRA-LPETGGGβA. Therefore, peaks are expected at mass/n, with n ∈ N. By comparison of the number of corresponding peaks, estimation of the product yield is possible as both molecules possess the same amount of ionizable groups and thus the difference in the ionizability of both molecules is negligible.

Figure 22 : Mass spectrum of the sortase-mediated ligation of TAMRA-LPETG and GGGβA showing the difference in height of the educt-peak and the product-peak which can be used to estimate the yield of our Sortase A7M.

In Fig. 22 the 621.56 peak can be assigned to the TAMRA-LEPTGGGβA and the 528.85 to the TAMRA-LPETG. The count ratios of the two molecules mentioned show an excess of the product.

Is Sortase A7M able to attach cargo to P22 coat protein?

We performed the linking reaction with CP-LPETGG and GGGG-mCherry as substrates and applied them to an SDS-PAGE. We saw products at the expected size (28 kDa + 49 kDa = 77 kDa) thus the requirement is fulfilled. However, a lot of additional bands appeared that we did not expect. These bands also appeared when only Sortase A7M and CP were mixed.

Figure 23:

a) Sortase A7M band is at expected height (17.85 kDa). The two negative controls containing only GGGG-mCherry (28 kDa) and CP-LPETGG (49 kDa) at the expected respective heights. b) Shown are sfGFP-SP and CP-LPETGG each incubated with both Sortase A7M and Sortase A5M. Both gels display multimers when coat and a sortase variant are in a sample together.

To investigate this issue, we had a look at the literature and found a matching description in the publication of Patterson et al.. They performed a similar experiment with P22 capsid proteins and observed the same multimers in their SDS-PAGEs . Comparing both SDS-PAGEs, we came to the following assumption:

Because of the promiscuity of Sortase A7M to accept primary amines as substrates, as we discussed previously, the formation of CP multimers occurs, unspecifically catalyzed by Sortase A7M.

Parallel to these experiments, we successfully modified the exterior of pre-assembled VLPs in vitro (VLP assembly). These modified VLPs were homogenous and overall correctly assembled. Therefore, we conclude that the described multimer problem only occurs when Sortase A7M encounters free CP.

Does methionine affect Sortase linking?

Sortase A7M preferably attaches N-terminal poly-G to C-terminal LPETGG. However, the first amino acid of a protein is methionine (to be specific, formylmethionine in bacteria). For our constructs that possess N-terminal polyG-tags, we have to ask ourselves the question: If the initial methionines are not cleaved off after the proteins have been produced, will this interfere with the Sortase reaction?

To investigate this, we cloned and purified two other proteins: TVMVsite-GGGG-mCherry and TEVsite-GGGG-sfGFP. Then we treated these proteins with the respective proteases, resulting in *GGGG-mCherry and *GGGG-sfGFP. Following this *GGGG-mCherry was then compared to (M)GGGG-mCherry which we used in all previous assays. Assays were also conducted on Fig. 24 the processed *GGGG-sfGFP substrate. Fig. 24 confirmed our assumptions that the unprocessed substrate cannot be linked to the sorting motif via Sortase A7M. Subsequently, *GGGG-sfGFP (after protease digest) demonstrate successful linkage via sortase-mediated ligation.

Due to these findings we modified our VLPs with *GGGG-sfGFP.

Figure 24 : Sortase-mediated ligation of TAMRA-LPETG and GGGG-sfGFP (with TEV cleavage site) one cut with TEV protease and one not. The sample with the unprocessed substrate shows no increase in RFU. In contrast the processed substrate shows a clear increase in ΔRFU. After 90 min the reverse reaction begins.The ΔRFU refers to the respective negative control without each sortase at 514 nm. The mean ΔRFU value of the duplicates was normalized to zero for better visualization.

We performed FRET-assays with TAMRA-LPETG and either of the following reaction partners:

(M)GGGG-mCherry, a protein sample that might still carry an N-terminal methionine
*GGGG-mCherry that does not carry any additional N-terminal residue

Before the FRET-assay was started, we adjusted the mCherry-concentrations of both fluorescent protein solutions to the same level. To do so, we diluted them until both showed the same fluorescence at 610 nm.

Figure 25: FRET of the sortase reaction connecting TAMRA-LPETG and GGGG-sfGFP mediated by Sortase A7M. The concentration of the Sortase A7M and TAMRA-LPETG was kept at the same level why the concentration of sfGFP was either 10,7 µM or 1,4 µM. The graphs show that the reverse reaction happens earlier if the GGGG-substrate concentration is lower. The ΔRFU refers to the respective negative control without each sortase at 514 nm. The mean ΔRFU value of the duplicates was normalized to zero for better visualization.

Strikingly, only the (M)GGGG-mCherry construct showed a clear decrease in delta RFU after the maximum delta RFU was reached (at about 160 min).

Figure 26 : Sortase-mediated ligation of TAMRA-LPETG and GGGG-mCherry one cut with TVMV protease and one with a methionine infront of the GGGG-tag. As visible the reverse reaction happens earlier if the methionine is not cleaved of the GGGG-tag. The ΔRFU is referring to the negative controls without Sortase A7M at 570 nm. The mean ΔRFU value of the tripicates was normalized to zero for better visualization.

We assume the following: Although we adjusted the overall mCherry concentration by fluorescence, we cannot determine the absolute amount of MGGGG-mCherry in the (M)GGGG-mCherry sample. However, if this amount was relatively high, the effective substrate concentration that could enter the sortase reaction would be low. That is because MGGGG is a worse sortase substrate than GGGG – if any at all. If we furthermore consider that a low substrate concentration correlates with a faster reverse reaction, we can explain the observed decrease in delta RFU for the (M)GGGG-mCherry sample that contrasts the delta RFU trend of the *GGGG-mCherry sample.

On this basis we can assume that a certain, yet unknown portion of the (M)GGGG-mCherry sample still carries an N-terminal methionine.

These FRET-assays let us assume that methionine disturbs or at least interferes with the sortase reaction mechanism. Indeed, our modeling suggests that methionine affects the interaction of polyG and the flexible loop near the active site of Sortase A7M. Click here if you want to know more about our modeling results!

This strengthens our hypothesis: If there is any amino acid in front of the poly-glycine sequence, substrate binding to Sortase A7M is negatively influenced.

Modeling

Introduction

In synthetic biology, theoretical models are often used to gain insights, predict and improve experiments. In our project we are modifying Virus-like particles (VLPs) by attaching proteins to the surface of the P22 capsid through a linker. The linking is catalyzed using the enzyme Sortase A7M, which is a calcium-independent mutant of the wild type Sortase A from Staphylococcus aureus. We performed modeling to predict the unknown structure of the Sortase A7M, to improve the linker between proteins and therefore optimizing the modification efficiency.
Here Rosetta Comparative Modeling was performed to predict the structure of Sortase A7M. The generated structure was characterized in terms of the molecular mechanisms and binding affinities of ligand peptides using molecular dynamics and docking simulations.

Structure determination

In silico modeling and simulation of proteins requires a 3D structure, which can be obtained from the RCSB Protein Data Bank. However, if no 3D structures are annotated, as it is the case with sortase A7M, the structure has to be determined by other means. The structure prediction of sortase A7M was done using two different approaches.

RosettaCM

Results

The simulation of our Sortase A7M yielded 15,000 structures which have been compared using the Rosetta scoring functions (talaris2013). From the 15,000 structures generated, we inspected the ten best scoring structures.

As can be seen in Fig. 27, the most prominent differences can be found in the regions close to the N- and C-terminus. As fluctuations in those regions are not untypical, we decided to use the best scoring structure, candidate S_14771 (Fig. 28), as the input for the simulations to follow.

Figure 27 : The structural alignment of the ten best scoring sortase structures displaying minor differences with the exception of the C- and N-terminal regions. N- and C-terminal regions tend to show strong fluctuations, thus it is unsurprising to find the terminal regions to be unaligned

Figure 28 : Sortase A7M candidate S_14771 created through RosettaCM.

In order to evaluate the secondary structure of the Sortase A7M candidate S_14771 a Ramachandran plot has been created and compared to the five sortases used as input for the comparitive modeling.

Figure 29: The Ramachandran plot of randomly sampled proteins and the input structures of the comparative modeling show similar secondary structures. Secondary structure analysis of both sortase candidates reveals absence of secondary structures for the ML candidate. This is not the case with candidate S_14771 as the Ramachandran plot shows all relevant structures.

The Ramachandran plot (Fig. 29) showing α-helices and β-sheets is a strong indicator of a successful structure determination, as those secondary structures are crucial for the functionality of sortases.

Conclusion

We used monte-carlo simulations to determine the structure of the mutated transpeptidase Sortase A7M. As a first investigation of the secondary structure a Ramachandran plot of candidate S_14771 was made. The plot showing secondary structures was a first indicator of successful structure prediction. The approach, using Rosetta Comparative Modeling, yielded 15,000 structures scored with the talaris2013 scoring function. The ten best structures were aligned and exhibited almost identical secondary structures (Fig. 27). The greatest structural differences are present in the N- and C-terminal regions. Since terminal regions tend to fluctuate more strongly than non-terminal segments of the protein, we deemed those fluctuations non-relevant for the proteins functionality.
Being the best scoring candidate, structure S_14771 was analyzed structurally using a Ramachandran plot (Fig. 29). The plot shows all the relevant and typical structures sortases exhibit and serves as an indicator for a successful structure prediction.
In the steps to follow, a molecular dynamics (MD) simulation was performed on both structures.

Molecular dynamics

Results

The first possible indicators of a stable protein structure are converging root-mean-square deviation (RMSD), small root-mean-square fluctuation (RMSF) values as well as converging radii of gyration. Using the Python software package and the module Biotite we calculated these quantities and plotted the result for candidate S_14771.

Figure 30 : The RMSD is one of three main indicators of a stable protein structure of the MD simulation of S_14771 over the period of 200,000 ps. As time progressed the RMSD increased with a smaller slope. The value stabilizes at a time of 110,000 ps and fluctuated around the value of 6 Å.

Figure 31 : The prominent fluctuations of the residues from ranges 105 to 115 might indicate a binding site or another form of functional structure. The radius of gyration, just as the RMSD Fig. 30, stabilizes around a simulation time of of 110,000 ps and converges towards a value of 16.7 Å.

Figure 32 : The fluctuations (RMSF) of most residues appear insignificant compared to the first, the last residues and the residues close to residue 110 . Typically the N- and C-terminus tend to fluctuate more intensively due to the lack of stabilizing structures. The prominent fluctuations in the range of residue 105 to 115 can indicate a binding site or another form of functional structure.

Typical RMSDs and radii of gyration converge towards a value dependent on the size of the protein. Convergence of those quantities can be interpreted as a stable state of the protein structure. As it can be seen in Fig. 30 and Fig. 31 both the RMSD and the radius of gyration stabilize at the same time as the simulation reaches 110,000 ps (110 ns), suggesting a now stabilized structure of candidate S_14771 solvated in water. Another indicator of a functional protein is the RMSF. Instead of being averaged over all atoms, the RMSF is averaged over time with respect to each amino acid. It provides insights in both protein stability and functionality. Fig Fig. 32 reveals the RMSF of residues 105 to 115 to be significantly higher than that of other residues. This hints at the presence of a functional unit along these residues. As commented on in the section describing our structure prediction approaches, the N- and C-terminal regions tend to fluctuate more strongly as a result of the absence of stabilizing structures.

To further analyse the potential binding site, we performed a Principle Component Analysis.

Principle component analysis

To analyze our system further Principle Component Analysis (PCA) was performed using GROMACS. By applying PCA to a protein it is possible to gain insights into the relevant vibrational motions and thereby the physical mechanism of the protein.

Animation 33: A Principle Component Analysis of a fast (blue) and a slow (red) mode showing the most prominent movements of the Cα-chain of candidate S_14771. Both modes show movement of the β6/β7 loop consisting of residues 105 to 115 towards the active site . Thus we can assume that the closing β6/β7 loop is involved in the reaction mechanism.

The results from the Principle Component Analysis of candidate S_14771 (Amination 33) show a movement of the residues 105 to 115 towards the active site, supporting our theory that residues 105 to 115 are important for the reaction mechanism. Since the most relevant eigenvector (red), which shows the most relevant movement of the sortase, moves further towards the active site, it is possible that the β6/β7 loop either closes the binding site of the ligand peptides or even transports one peptide towards the other.

Conclusion

Candidate S_14771 that was generated using RosettaCM appears to be a fitting candidate not only due to successful analyses, but also due to the residues of the active site being in close proximity. Not the RMSF but also Principle Component Analysis indicate that residues 105 to 115 forming the β6/β7 loop are crucial for the molecular mechanism of our model.

Docking

Now that the binding site of the Sortase had been found, the peptide ligand needed to be inserted into the binding site to create a peptide-protein complex. The procedure of choice for the introduction of a ligand into the binding site of a protein is called docking. In the following sections, we will present the protocol and methods we used as well as the results they yielded.

Results

For sequences MGGGGPPPPPP(M-polyG), GGGGPPPPPP(polyG) and PPPPPPLPETGG(LPETGG) 50,000 structures have been created and clustered. After the clustering the sample consisted of 100 structures of docked complexes.

Figure 34 : The three best scoring structures (total score, interface score, reweighted score) of the LPETGG-tag are shown. Only two results are visible as the best reweighted score candidate is identical to the best interface score candidate. The reacting section of the LPETGG-tag namely glycine is colored yellow as is the active site. The glycin of both ligand peptides is facing the active site.

Analysis of the scores has shown a similar score for all the three dockings. The best scoring results of the LPETGG docking show a tendency of the glycines to face the active site while also being in close proximity to the active site.

Figure 35: The three best scoring structures (total score, interface score, reweighted score) of the polyG peptide are shown. Only two results are visible as the best reweighted score candidate is identical to the best interface score candidate. Instead of facing the active site (yellow) the reacting glycines (yellow) appear to interact with the β6/β7 loop of the sortase.

Figure 36: The three best scoring structures (total score, interface score, reweighted score) of the poly-g peptide are shown. Only two results are visible as the best reweighted score candidate is identical to the best interface score candidate. Concerning the M-poly-G peptide no uniform directional orientation can be observed. The structure with the best interface score (light blue) is oriendted towards the loop while the structure with the best total/reweighted (dark blue) is oriented towards the β-sheets.

Fig. 34 shows the docking result of the LPETGG peptide to the sortase. The results shown are the best scoring structures of the clustering with respect to the total score, interface score and reweighted score. As the best scoring structure is the same for the total score and the reweighted score only two peptides are shown. This also applies to Fig. 35 and Fig. 36. For both results the reacting glycin residues (yellow) are facing the active site. Additionally, the same residues are in close proximity to the active site.

The figures Fig. 35 and Fig. 36 show the docking of the both polyG and M-polyG. While polyG results align well and seem to be interacting with the β6/β7 loop rather than with the active site, this does not seem to be the case for M-polyG. Instead of both structures interacting with the β6/β7 loop or active site one (best interaction score; dark blue) interacts with the β6/β7 loop and the other (best reweighted/total score; light blue-gray) appears to interact with the active site.

Figure 37: The close up of the M-polyG peptide (best total/reweighted score) indicates an interaction of methionine with arginine₁₃₉ and cysteine₁₂₆.

Figure 38: Methionine of the result with the best interface score interacted with the β6/β7 loop rather than the active site. Still the reactive glycine residues appear to be bound to the β6/β7 loop.

As can be seen in Fig. 37 visualizing the result of the the docking simulation (total/reweighted score) suggests an interaction of methionine and two of the active sites namely arginine₁₃₉ and cysteine₁₂₆. Fig. 38 shows the interaction of M-polyG with the β6/β7 loop. The glycines still interact with the β6/β7 loop. Instead of binding above the β6/β7 loop, which is the case for polyG as illustrated in Fig. 36, the interaction seems to be influenced by methionine. By interacting with the residues in the β-helix methionine could potentially hinder binding of glycine to the β6/β7 loop by partial immobilization of the peptide. Overall peptide binding and orientation is less uniform compared polyG without the leading methionine, which could be an indicator of lesser binding affinity of M-PolyG towards the β6/β7 loop.

Conclusion

To computationally investigate binding affinities of the polyG and M-polyG as well as the LPETGG tags we performed docking simulations using the Rosetta FlexPepDock application. We used a modified version of the recommended protocol as the modified version was easier to automate and served our purpose better than the standard protocol. From the calculated scores only, we could not see a difference in binding affinities. Thus, we inspected the best scoring structures regarding the total score, the interface score and the reweighted score using PyMOL. Since the best structures with respect to total score and reweighted score were the same for all simulations, only two structures have been inspected per run. A polyproline tag was appended to all the peptides to simulate the modification of the VLPs with a small peptide.

As expected, the results showed that for LPETGG, the glycines of both peptides oriented towards the active site. This is unsurprising as peptides with the sequence LPXTGG are known to be substrate of the Sortase. It was more surprising to see the polyG tag oriented away from the active site since polyG also is a known substrate of the sortase. Both polyG peptides were facing the β6/β7 loop (residues 105 to 115) uniformly and appeared to be interacting with it. The M-polyG peptides did not show a uniform orientation or interaction scheme. On one hand the visualization of the best result concerning the total and reweighted score has shown interaction of methionine with the cysteine₁₂₆ and arginine₁₃₉, two residues of the active site. On the other hand, the visualization of the best result with respect to the interface score shows the M-polyG facing the mobile β6/β7 loop. In contrast to the polyG peptide the lacking the methionine, the M-polyG peptide is pulled down below the β6/β7 loop by the methionine interacting with one of the β-sheets leading to the active site. This is not the case with the polgG results, which lie aligned in one plane with the β6/β7 loop.

Modeling Conclusion

For our project it was key to understand and characterize Sortase A7M. As there is no annotated 3D structure for this specific Sortase, an in silico structure determination was performed. Comparative modeling with Rosetta produced valid structures. We used the best structure, candidate S_14771, for extensive characterization. We evaluated the model with regard to its secondary structure using Ramachandran plots. The Ramachandran plot suggested plausible secondary structures.

Molecular Dynamics simulations were used to investigate stability and dynamic properties of the candidate. The RMSD and radius of gyration stabilized over the course of the simulation, a first indicator of an equilibrated structure. Interestingly, RMSF analysis showed strond fluctuations of residues 105 to 115. We further investigated this by performing Principle Component Analysis. Doing so, we extracted the principle movements of the model. We could observe movement of the β6/β7 loop towards the active site, suggesing the presence of a binding site. Consequently, we performed docking simulations.

FlexPepDock was used to conduct the docking simulations with target peptides. Each run yielded 50,000 structures. In multiple steps we reduced the amount of complexes to 100 clusters with respect to total, reweighted and interface score. We extracted the best scoring complexes and investigated interactions.

For LPETGG we observed a uniform binding to the active site, fullfilling our expectation. Strikingly, polyG appeared to bind to the β6/β7 loop in a uniform manner. As it is know from literature polyG is a functioning ligand of sortase. Supported by literature and our data, we postulate the following mechanism: the β6/β7 loop transports bound polyG towards the active site of Sortase A7M, thereby lowering the activation energy of the linking reaction.

As the theory is neither backed up by nor contradicts experimental data, further research is required.

References

For our references, please have a look at our wiki.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal XhoI site found at 445
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

@@ Line 40: / Line 40: @@
                          Figure 1 :
                      </b>
-D-Structure of Sortase A7M. To find out more, visit our<a
+                     Modeled 3D-Structure of Sortase A7M. To find out more, visit our<a
                          href="https://2019.igem.org/Team:TU_Darmstadt/Model" target="_blank"> modeling page.
                      </a>
@@ Line 159: / Line 159: @@
                          <p>The methods used for cloning of the different mutants of the sortase were restriction and
-                             ligation via <i>NdeI</i> and <i>SalI</i> and Gibson assembly. The Sortase&nbsp;A5M was
+                             ligation via <i>NdeI</i> and <i>SalI</i> and Gibson assembly. The vector posesses a
-                            cloned
+                             kanamycin resistance and the <i>srta7m</i> is controlled
-                            into pET24(+) vector via restriction and ligation <i>NdeI</i> and <i>SalI</i> as restriction
-                             enzymes. The vector posesses a kanamycin resistance and the <i>srta7m</i> is controlled
                              through a T7 promoter,
                              which can be induced with IPTG. Sortase&nbsp;A7M is controlled by the same T7&nbsp;promoter.
-                             Sortas&nbsp;A, introduced by iGEM Stockholm 2016, was cloned via Gibson assembly into
+                             The product was checked via sequencing.
-                            PSB1C3.
-                            This has a chloramphenicol resistance and is also controlled under a T7&nbsp;promoter.
-                            Cloning
-                            of all products was checked via sequencing.
                          </p>
@@ Line 184: / Line 178: @@
                              was
                              performed at 30&nbsp;°C under constant shaking overnight. After expression of
-                             Sortase&nbsp;A7M,
+                             Sortase&nbsp;A7M in BL21 cultures the
-                            Sortase&nbsp;A5M, and Sortase&nbsp;A from Stockholm (BBa_K2144008) in BL21 cultures the
                              cells
                              were crushed via EmulsiFlex (Avestin) and proteins were purified through affinity
@@ Line 197: / Line 190: @@
                          <h3>SDS-Page</h3>
-                         <p>To verify the successful production of of Sortase&nbsp;A7M, Sortase&nbsp;A5M, and
+                         <p>To verify the successful production of of Sortase&nbsp;A7M and others
-                            Sortase&nbsp;A
                              SDS-PAGEs were performed. The resulting bands were compared to the molecular weight of the
                              different sortase variants.
@@ Line 231: / Line 223: @@
                                      Figure 2 :
                                  </b>
-                                 Jablonski diagram showing the energy transfer between a FRET-pair (design based on
+                                 Jablonski diagram showing the energy transfer between a FRET-pair (inspired by
-                                 Wikipedia).
+                                 <a href="https://en.wikipedia.org/wiki/F%C3%B6rster_resonance_energy_transfer#/media/File:FRET_Jablonski_diagram.svg"
+                                    target="_blank">Alex M. Mooney</a>).
                              </p>
                          </div>
@@ Line 331: / Line 324: @@
                                  Product bands at a height of about 57&nbsp;kDa can be seen in lane 4, 5, 6, 8, 9 (from
                                  left to right). The bands below the product at about 38&nbsp;kDa could be semi-denatured
-                                 mCherry dimers. Additionally, Sortase&nbsp;A7M can be seen at 17&nbsp;kDaA7M.The
+                                 mCherry dimers. Additionally, Sortase&nbsp;A7M can be seen at 17&nbsp;kDa.The
                                  unprocessed mCherry monomers can be seen at 28&nbsp;kDa.
                              </p>
@@ Line 378: / Line 371: @@
                              designed
                              a suitable
-                             FRET-assay. In the
+                             FRET-assay.
-                            end, we came
-                            up with a new FRET pair not described in the literature to date: 5-TAMRA-LPETG and
-                            GGGG-sfGFP.
                          </p>
                          <h4>Development of a new FRET pair</h4>
@@ Line 394: / Line 384: @@
                              FRET&nbsp;effect, checking for a possible overlap between the donor's emission and the
                              acceptor's
-                             extinction.
+                             excitation.
                          </p>
                          <img class="img-fluid center"
@@ Line 427: / Line 417: @@
                              Sortase&nbsp;A. For a sufficient FRET-effect, it is also necessary that the distance between
                              donor and
-                             acceptor is lower than the Förster radius. The Förster radius describes the distance between
+                             acceptor is lower than the Förster radius.
-                            two
-                            fluorophores at which 50&nbsp;% of the energy is transferred.
                              <br>
                              First, we wanted to identify which concentrations are needed for our experiment, then set up
@@ Line 447: / Line 435: @@
                                      Figure 6 :
                                  </b>
-                                 The graph shows the extinction and emission spectra of TAMRA and mCherry. Due
+                                 The graph shows the excitation and emission spectra of TAMRA and mCherry. Due
-                                 to the large overlap of TAMRA emission and mCherry extinction it is possible to perform
+                                 to the large overlap of TAMRA emission and mCherry excitation it is possible to perform
                                  a FRET with this pair of fluorophores. The graph show the relative fluorescence unit
-                                 (RFU[%]) in relation to the extincted/emitted wavelength [nm]. The peaks are normalized
+                                 (RFU[%]) in relation to the excited/emitted wavelength [nm]. The peaks are normalized
                                  to 100&nbsp;%.
                              </p>
                          </div>
                          <p>
-                             The emission and extinction spectra of TAMRA and mCherry exhibit an overlap of emission of
+                             The emission and excitation spectra of TAMRA and mCherry exhibit an overlap of emission of
                              TAMRA
                              and
-                             extinction of mCherry. Based on this output, a FRET-assay for the kinetics of
+                             excitation of mCherry. Based on this output, a FRET-assay for the kinetics of
                              Sortase&nbsp;A7M
                              was performed
@@ Line 467: / Line 455: @@
                              “leak”
                              excitation of mCherry.
-                             Nevertheless, an extinction of mCherry could not be excluded and may have negative effects
+                             Nevertheless, an excitation of mCherry could not be excluded and may have negative effects
                              on
                              the visibility
@@ Line 520: / Line 508: @@
                                      Figure 9 :
                                  </b>
-                                 ortase reaction in TAMRA-mCherry-FRET after subtracting the negative control. Depicted
+                                 Sortase reaction in TAMRA-mCherry-FRET after subtracting the negative control. Depicted
                                  is the difference in RFU over time [min]. Within the first 20&nbsp;min of the substrate
                                  conversion is the quickest. At 30&nbsp;min a plateau is reached. After 60&nbsp;min
@@ Line 531: / Line 519: @@
                              To confirm the functionality of the Sortase&nbsp;A7M, another more sufficient FRET-pair was
                              developed. The measured absorbance and emission spectra indicated that TAMRA and superfolder
-                             green fluorescence protein (sfGFP) are a possible FRET-pair. The sfGFP has an N-terminal
+                             green fluorescence protein (sfGFP) are a possible FRET-pair.
-                            polyglycine sequence and can therefore be linked to TAMRA with the sorting motif, in the
-                            same
-                            way as mCherry was connected. However, the small overlap between the extinction spectra of
-                            sfGFP
-                            and TAMRA could solve the previous “simultaneous excitation” problem we observed for the
-                            mCherry-TAMRA FRET-pair. Because of the lower excitation maximum of sfGFP compared to TAMRA,
-                            sfGFP was chosen as donor and TAMRA as acceptor. sfGFP was excited at 465&nbsp;nm to
-                            minimize
-                            the unnecessary leak excitation of sfGFP.
                          </p>
                          <img class="img-fluid center"
@@ Line 551: / Line 530: @@
                                      Figure 10 :
                                  </b>
-                                 The graph shows the extinction and emission spectra of
+                                 The graph shows the excitation and emission spectra of
                                  TAMRA and mCherry. Due to the large overlap of sfGFP emission and TAMRA
-                                 extinction it is possible to perform a FRET with this pair of fluorophores.
+                                 excitation it is possible to perform a FRET with this pair of fluorophores.
                                  The graph show the relative fluorescence unit (RFU[%]) in relation to the
-                                 extincted/emitted
+                                 excited/emitted
                                  wavelength [nm]. The peaks are normalized to 100 %.
                              </p>
@@ Line 749: / Line 728: @@
                                  </p>
                              </div>
-                            <p> Secondly, we confirmed that <b>Sortase&nbsp;A5M is inactive if Ca<sup>2+</sup> is
-                                    absent</b>, which can be seen in <b>Fig. 17</b>
-                                As expected, Sortase&nbsp;A5M shows increasing enzymatic activity with increasing
-                                Ca<sup>2+</sup> levels. The reaction runs fastest with 2&nbsp;mM Ca<sup>2+</sup>, and
-                                the
-                                maximal FRET change
-                                (in terms of ΔRFU) is reached after 37.5&nbsp;min. Strikingly, the FRET change decreases
-                                afterwards. We observed this phenomenon before and assume this to be due to dead-end
-                                product
-                                formation caused by the reverse reaction.
-                            </p>
-                            <img class="img-fluid center"
-                                src="https://2019.igem.org/wiki/images/3/38/T--TU_Darmstadt--CalciumFRET.png"
-                                style="max-width:65%" />
-                            </a>
-                            <img class="img-fluid center"
+        </div>
-                                src="https://2019.igem.org/wiki/images/4/4d/T--TU_Darmstadt--FRET_SrtA7M_ohneCa2%2B.png"
-                                style="max-width:65%" />
-                            </a>
-                            <div class="caption">
-                                <p>
-                                    <b>
-                                        Figure 17 :
-                                    </b>
-                                    Both figures show Sortase&nbsp;A5M FRET assay connecting TAMRA-LPETG and GGGG-sfGFP
-                                    with different
-                                    Ca<sup>2+</sup>-concentrations. The right graph is
-                                    showing that Sortase&nbsp;A5M does not work without calcium. The negative ΔRFU
-                                    measurements are probably caused by measuring errors by the
-                                    Tecan platereader. The ΔRFU refers to the respective negative control without each
-                                    sortase at 514 nm.
-                                    The mean ΔRFU value of the duplicates was normalized to zero for better
-                                    visualization.
-                                </p>
-                            </div>
-                            <p>
+        <p>
-                                According to the results of this assay, Sortase&nbsp;A7M is definitely
+            According to the results of this assay, Sortase&nbsp;A7M is definitely
-                                Ca<sup>2+</sup>-independent, since it shows
+            Ca<sup>2+</sup>-independent, since it shows
-                                linking activity without calcium in the vicinity. The enzyme mutant also works in
+            linking activity without calcium in the vicinity. The enzyme mutant also works in
-                                presence
+            presence
-                                of Ca<sup>2+</sup> (<b>Fig.&nbsp;17</b>),
+            of Ca<sup>2+</sup>,
-                                but these FRET experiments made us suspect that Ca<sup>2+</sup> may even inhibit
+            but these FRET experiments made us suspect that Ca<sup>2+</sup> may even inhibit
-                                Sortase&nbsp;A7M, since it shows less activity with calcium around than without calcium.
+            Sortase&nbsp;A7M, since it shows less activity with calcium around than without calcium.
-                            </p>
+        </p>
-                            <p>
+        <p>
-                                To better address this question, an ELISA was
+            To better address this question, an ELISA was
-                                performed. Therefore, a piece of paper functionalized with GGGβA was connected to a
+            performed. Therefore, a piece of paper functionalized with GGGβA was connected to a
-                                protein
+            protein
-                                domain, which binds
+            domain, which binds
-                                antibodies to the LPTEG-tag. The results are shown in <b>Fig.&nbsp;18</b>.
+            antibodies to the LPTEG-tag. The results are shown in <b>Fig.&nbsp;18</b>.
-                            </p>
+        </p>
-                            <img class="img-fluid center"
+        <img class="img-fluid center"
-                                src="https://2019.igem.org/wiki/images/7/77/T--TU_Darmstadt--Absorbance_450_Sortase_yield.png"
+            src="https://2019.igem.org/wiki/images/7/77/T--TU_Darmstadt--Absorbance_450_Sortase_yield.png"
-                                style="max-width:30%" />
+            style="max-width:30%" />
-                            </a>
+        </a>
-                            <div class="caption">
+        <div class="caption">
-                                <p>
+            <p>
-                                    <b>
+                <b>
-                                        Figure 18 :
+                    Figure 18 :
-                                    </b>
+                </b>
-                                    Absorbance at 450&nbsp;nm at a temperature of 23.8˚&nbsp;C <br>
+                Absorbance at 450&nbsp;nm at a temperature of 23.8˚&nbsp;C <br>
-                                    In well 1 additional 10&nbsp;mM Ca<sup>2+</sup> were
+                In well 1 additional 10&nbsp;mM Ca<sup>2+</sup> were
-                                    added which was not the case in well 2. Well 3 serves as a negative control since
+                added which was not the case in well 2. Well 3 serves as a negative control since
-                                    the enzyme is missing in this reaction
+                the enzyme is missing in this reaction
-                                </p>
+            </p>
-                            </div>
+        </div>
-                            <p>
+        <p>
-                                As shown in <b>Fig. 18</b>, the highest absorption was measured in well 2. Thus,
+            As shown in <b>Fig. 18</b>, the highest absorption was measured in well 2. Thus,
-                                Sortase&nbsp;A7M works more efficiently
+            Sortase&nbsp;A7M works more efficiently
-                                when no Ca<sup>2+</sup> is around. The absorption is also relatively high for the
+            when no Ca<sup>2+</sup> is around. The absorption is also relatively high for the
-                                negative
+            negative
-                                control,
+            control,
-                                which can be explained by poor washing before the substrate for Horeseradish peroxidase
+            which can be explained by poor washing before the substrate for Horeseradish peroxidase
-                                (HPR) was added. This assay shows
+            (HPR) was added. This assay shows
-                                the functionality of Sortase&nbsp;A7M even in context of surfaces since we confirmed
+            the functionality of Sortase&nbsp;A7M even in context of surfaces since we confirmed
-                                that
+            that
-                                Sortase&nbsp;A7M
+            Sortase&nbsp;A7M
-                                is able to connect tags attached to paper. This shows that the surface structure is not
+            is able to connect tags attached to paper. This shows that the surface structure is not
-                                a
+            a
-                                relevant factor for the enzyme.
+            relevant factor for the enzyme.
-                            </p>
+        </p>
-                            <img class="img-fluid center"
+        <img class="img-fluid center"
-                                src="https://2019.igem.org/wiki/images/c/c7/T--TU_Darmstadt--Vergleich_A7M_A5M_bei_opt_Bedingung.png"
+            src="https://2019.igem.org/wiki/images/c/c7/T--TU_Darmstadt--Vergleich_A7M_A5M_bei_opt_Bedingung.png"
-                                style="max-width:60%" />
+            style="max-width:60%" />
-                            </a>
+        </a>
-                            <div class="caption">
+        <div class="caption">
-                                <p>
+            <p>
-                                    <b>
+                <b>
-                                        Figure 19 :
+                    Figure 19 :
-                                    </b>
+                </b>
-                                    Comparison of the reaction speed of Sortase&nbsp;A5M with Ca<sup>2+</sup> and
+                Comparison of the reaction speed of Sortase&nbsp;A5M with Ca<sup>2+</sup> and
-                                    Sortase&nbsp;A7M without Ca<sup>2+</sup>, therefore each working under optimal conditions.
+                Sortase&nbsp;A7M without Ca<sup>2+</sup>, therefore each working under optimal conditions.
-                                    The kinetics were measured via a FRET connecting TAMRA-LPETG and GGGG-sfGFP. The ΔRFU refers
+                The kinetics were measured via a FRET connecting TAMRA-LPETG and GGGG-sfGFP. The ΔRFU refers
-                                    to the respective negative control without each sortase at 514 nm. The mean ΔRFU value of
+                to the respective negative control without each sortase at 514 nm. The mean ΔRFU value of
-                                    the triplicates was normalized to zero for better visualization.
+                the triplicates was normalized to zero for better visualization.
-                                </p>
+            </p>
-                            </div>
+        </div>
-                            <p>
+        <p>
-                                When we compare the reaction speed of Sortase&nbsp;A5M and Sortase&nbsp;A7M,
+            When we compare the reaction speed of Sortase&nbsp;A5M and Sortase&nbsp;A7M,
-                                Sortase&nbsp;A5M is the
+            Sortase&nbsp;A5M is the
-                                clear winner (see <b>Fig. : 19</b>). However, this means of course that the reverse
+            clear winner (see <b>Fig. : 19</b>). However, this means of course that the reverse
-                                reaction is also faster in the
+            reaction is also faster in the
-                                case of Sortase&nbsp;A5M.
+            case of Sortase&nbsp;A5M.
-                                Consequently, Sortase&nbsp;A7M is the best variant for in vivo modification of our VLPs
+            Consequently, Sortase&nbsp;A7M is the best variant for in vivo modification of our VLPs
-                                as
+            as
-                                it is Ca<sup>2+</sup>-independent.
+            it is Ca<sup>2+</sup>-independent.
-                                On the other hand, Sortase&nbsp;A5M is a suitable
+            On the other hand, Sortase&nbsp;A5M is a suitable
-                                enzyme variant for in vitro modification due to its high efficiency.
+            enzyme variant for in vitro modification due to its high efficiency.
-                            </p>
+        </p>
-                            <h3>What about other substrates?</h3>
+        <h3>What about other substrates?</h3>
-                            <h4>Primary Amines</h4>
+        <h4>Primary Amines</h4>
-                            <p>The literature
+        <p>The literature
-                                describes Sortase&nbsp;A7M as somewhat „promiscuous“ towards other substrates than
+            describes Sortase&nbsp;A7M as somewhat „promiscuous“ towards other substrates than
-                                GGGG(polyG) as long as the substrate possesses a primary amine. To confirm this, we
+            GGGG(polyG) as long as the substrate possesses a primary amine. The
-                                performed additional assays with other substrates in the lab of Prof. Kolmar. The
+            Sortase&nbsp;A7M used for this assay was stored in the fridge at 4&nbsp;°C for two
-                                Sortase&nbsp;A7M used for this assay was stored in the fridge at 4&nbsp;°C for two
+            weeks.
-                                weeks.
+            The substrates were TAMRA with a KLPETG bound to TAMRA via the lysine side chain and
-                                The substrates were TAMRA with a KLPETG bound to TAMRA via the lysine side chain and
+-azidopropanamine as the example for a primary amine. The reaction was performed for
--azidopropanamine as the example for a primary amine. The reaction was performed for
+            two
-                                two
+            hours at 37&nbsp;°C. <br>
-                                hours at 37&nbsp;°C. It was then analyzed by electron spray ionization mass spectrometry
+             It was then analyzed by electron spray ionization mass spectrometry
-                                (ESI-MS) <b>(Fig.&nbsp;20)</b>. </p>
+            (ESI-MS) <b>(Fig.&nbsp;20)</b>. </p>
-                            <img class="img-fluid center"
+        <img class="img-fluid center"
-                                src="https://2019.igem.org/wiki/images/9/96/T--TU_Darmstadt--SortaseFigure1TAMRA_KE.png"
+            src="https://2019.igem.org/wiki/images/9/96/T--TU_Darmstadt--SortaseFigure1TAMRA_KE.png"
-                                style="max-width:100%" />
+            style="max-width:100%" />
-                            </a>
+        </a>
-                            <div class="caption">
+        <div class="caption">
-                                <p>
+            <p>
-                                    <b>
+                <b>
-                                        Figure 20 :
+                    Figure 20 :
-                                    </b>
+                </b>
-                                    Mass spectrum before the reaction of TAMRA-LPETG with 3-azidopropanamine
+                Mass spectrum before the reaction of TAMRA-LPETG with 3-azidopropanamine
-                                    showing the educt at 1054 g/mol.
+                showing the educt at 1054 g/mol.
-                                </p>
+            </p>
-                            </div>
+        </div>
-                            <p><b>Fig.&nbsp;20</b> shows the educt-peak in the mass spectrum. TAMRA with the LPETG-tag
+        <p><b>Fig.&nbsp;20</b> shows the educt-peak in the mass spectrum. TAMRA with the LPETG-tag
-                                weighs 1054 g/mol. Shown above in green is the single charged molecule at 1054.27 g/mol
+            weighs 1054 g/mol. Shown above in green is the single charged molecule at 1054.27 g/mol
-                                and
+            and
-                                the double charged molecule at 528.75 g/mol.</p>
+            the double charged molecule at 528.75 g/mol.</p>
-                            <img class="img-fluid center"
+        <img class="img-fluid center"
-                                src="https://2019.igem.org/wiki/images/7/7a/T--TU_Darmstadt--SortaseFigure2TAMRA_KE.png"
+            src="https://2019.igem.org/wiki/images/7/7a/T--TU_Darmstadt--SortaseFigure2TAMRA_KE.png"
-                                style="max-width:90%" />
+            style="max-width:90%" />
-                            </a>
+        </a>
-                            <div class="caption">
+        <div class="caption">
-                                <p>
+            <p>
-                                    <b>
+                <b>
-                                        Figure 21 :
+                    Figure 21 :
-                                    </b>
+                </b>
-                                    Mass spectrum after the reaction of TAMRA-LPETG with 3-azidopropanamine
+                Mass spectrum after the reaction of TAMRA-LPETG with 3-azidopropanamine
-                                    showing the product at 1079.37g/mol.
+                showing the product at 1079.37g/mol.
-                                </p>
+            </p>
-                            </div>
+        </div>
-                            <b>Fig. 21</b> shows the product-peak in the mass spectrum. The primary amine that was taken
+        <b>Fig. 21</b> shows the product-peak in the mass spectrum. The primary amine that was taken
-                            as an example has a molecular weight of 100 g/mol. After the reaction the glycine of the
+        as an example has a molecular weight of 100 g/mol. After the reaction the glycine of the
-                            LPETG-tag has been removed and therefore the product only consists of
+        LPETG-tag has been removed and therefore the product only consists of
-                            TAMRA-KLPET-3-azidopropanamine. When adding the two molecular weights and subtracting the
+        TAMRA-KLPET-3-azidopropanamine. When adding the two molecular weights and subtracting the
-                            weight
+        weight
-                            of the glycine it adds up to a total weight of 1078 g/mol which can be seen in the single
+        of the glycine it adds up to a total weight of 1078 g/mol which can be seen in the single
-                            loaded
+        loaded
 .37 g/mol peak <b>(Fig. 21)</b>, since the ESI-MS we used has a small error margin. The
-                            peak
+        peak
-                            in black again is the double loaded peak at 541.55 g/mol. This clearly shows that the
+        in black again is the double loaded peak at 541.55 g/mol. This clearly shows that the
-                            sortase
+        sortase
-                            reaction took place. Furthermore, we can conclude that the Sortase A7M accepts any primary
+        reaction took place. <br>Furthermore, we can conclude that the Sortase A7M accepts any primary
-                            amine
+        amine
-                            as a substrate. However, mass spectrum does not show the ratio of educt and product, which
+        as a substrate. However, mass spectrum does not show the ratio of educt and product, which
-                            is
+        is
-                            why we cannot estimate whether the turnover is as high as when using a polyG-tag as
+        why we cannot estimate whether the turnover is as high as when using a polyG-tag as
-                            substrate.
+        substrate.
-                            Additionally this assay confirms our suspicion that the Sortase A7M is stable at 4&nbsp;°C
+        Additionally this assay confirms our suspicion that the Sortase A7M is stable at 4&nbsp;°C
-                            and
+        and
-                            still functional if stored at said temperature for at least two weeks. <p></p>
+        still functional if stored at said temperature for at least two weeks. <p></p>
-                            <h5>Yield</h5>
+        <h5>Yield</h5>
-                            <p>For the characterization of Sortase A7M an assay was designed to show the coupling
+        <p>For the characterization of Sortase A7M an assay was designed to show the coupling
-                                efficiency
+            efficiency
-                                between the TAMRA-LEPTG and the tetrapeptide GGG-Beta-Alanin (GGGβA) catalyzed by the
+            between the TAMRA-LEPTG and the tetrapeptide GGG-Beta-Alanin (GGGβA) catalyzed by the
-                                Sortase. The Sortase reaction was performed for 1h at
+            Sortase. The Sortase reaction was performed for 1h at
 ˚C and was stopped by enzyme separation through centrifugal filtration. For analysis
-                                mass
+            mass
-                                spectrometry (ESI-MS) was used. The mass spectrometry enables differentiation between
+            spectrometry (ESI-MS) was used. The mass spectrometry enables differentiation between
-                                products and educts. It allowed us to make an estimate of the product yield.
+            products and educts. It allowed us to make an estimate of the product yield.
-                                The calculated theoretical molecular masses are 1054 g/mol for TAMRA and 1240 g/mol for
+            The calculated theoretical molecular masses are 1054 g/mol for TAMRA and 1240 g/mol for
-                                TAMRA-LPETGGGβA. Therefore, peaks are expected at mass/n, with n ∈ N. By comparison of
+            TAMRA-LPETGGGβA. Therefore, peaks are expected at mass/n, with n ∈ N. By comparison of
-                                the
+            the
-                                number of corresponding peaks, estimation of the product yield is possible as both
+            number of corresponding peaks, estimation of the product yield is possible as both
-                                molecules
+            molecules
-                                possess the same amount of ionizable groups and thus the difference in the ionizability
+            possess the same amount of ionizable groups and thus the difference in the ionizability
-                                of
+            of
-                                both molecules is negligible. </p>
+            both molecules is negligible. </p>
-                            <img class="img-fluid center"
+        <img class="img-fluid center"
-                                src="https://2019.igem.org/wiki/images/3/36/T--TU_Darmstadt--results_BC_mass_spectrum.png"
+            src="https://2019.igem.org/wiki/images/3/36/T--TU_Darmstadt--results_BC_mass_spectrum.png"
-                                style="max-width:90%" />
+            style="max-width:90%" />
-                            </a>
+        </a>
-                            <div class="caption">
+        <div class="caption">
-                                <p>
+            <p>
-                                    <b>
+                <b>
-                                        Figure 22 :
+                    Figure 22 :
-                                    </b>
+                </b>
-                                    Mass spectrum of the sortase-mediated ligation of TAMRA-LPETG and GGGβA
+                Mass spectrum of the sortase-mediated ligation of TAMRA-LPETG and GGGβA
-                                    showing the difference in height of the educt-peak and the product-peak which can be
+                showing the difference in height of the educt-peak and the product-peak which can be
-                                    used to estimate the yield of our Sortase&nbsp;A7M.
+                used to estimate the yield of our Sortase&nbsp;A7M.
-                                </p>
+            </p>
-                            </div>
+        </div>
-                            <p>In <b>Fig. 22</b> the 621.56 peak can be assigned to the TAMRA-LEPTGGGβA and the 528.85
+        <p>In <b>Fig. 22</b> the 621.56 peak can be assigned to the TAMRA-LEPTGGGβA and the 528.85
-                                to
+            to
-                                the TAMRA-LPETG. The count ratios of the two molecules mentioned show an excess of the
+            the TAMRA-LPETG. The count ratios of the two molecules mentioned show an excess of the
-                                product. </p>
+            product. </p>
-                            <h2>Is Sortase A7M able to attach cargo to P22 coat protein?</h2>
+        <h2>Is Sortase A7M able to attach cargo to P22 coat protein?</h2>
-                            <p>
+        <p>
-                                We performed the linking reaction with CP-LPETGG and GGGG-mCherry as substrates and
+            We performed the linking reaction with CP-LPETGG and GGGG-mCherry as substrates and
-                                applied them to an SDS-PAGE.
+            applied them to an SDS-PAGE.
-                                We saw products at the expected size (28&nbsp;kDa + 49&nbsp;kDa = 77&nbsp;kDa) thus the
+            We saw products at the expected size (28&nbsp;kDa + 49&nbsp;kDa = 77&nbsp;kDa) thus the
-                                requirement is fulfilled. However,
+            requirement is fulfilled. However,
-                                a lot of additional bands appeared that we did not expect. These bands also appeared
+            a lot of additional bands appeared that we did not expect. These bands also appeared
-                                when only Sortase&nbsp;A7M and CP were mixed.
+            when only Sortase&nbsp;A7M and CP were mixed.
-                            </p>
+        </p>
-                            <img class="img-fluid center"
+        <img class="img-fluid center" src="https://2019.igem.org/wiki/images/1/1d/T--TU_Darmstadt--EnzymeSubstrate1.png"
-                                src="https://2019.igem.org/wiki/images/1/1d/T--TU_Darmstadt--EnzymeSubstrate1.png"
+            style="max-width:50%" />
-                                style="max-width:50%" />
+        </a>
-                            </a>
-                            <img class="img-fluid center"
+        <img class="img-fluid center"
-                                src="https://2019.igem.org/wiki/images/7/76/T--TU_Darmstadt--Sortase7Mdiffprot.png"
+            src="https://2019.igem.org/wiki/images/7/76/T--TU_Darmstadt--Sortase7Mdiffprot.png" style="max-width:50%" />
-                                style="max-width:50%" />
+        </a>
-                            </a>
-                            <p>
+        <p>
-                                <b>
+            <b>
-                                    Figure 23: </b>
+                Figure 23: </b>
-                                <p><b>a)</b> Sortase&nbsp;A7M band is at expected height (17.85&nbsp;kDa).
+            <p><b>a)</b> Sortase&nbsp;A7M band is at expected height (17.85&nbsp;kDa).
-                                    The two negative controls containing only GGGG-mCherry (28&nbsp;kDa)
+                The two negative controls containing only GGGG-mCherry (28&nbsp;kDa)
-                                    and CP-LPETGG (49&nbsp;kDa) at the expected respective heights. <b>b)</b> Shown are
+                and CP-LPETGG (49&nbsp;kDa) at the expected respective heights. <b>b)</b> Shown are
-                                    sfGFP-SP and
+                sfGFP-SP and
-                                    CP-LPETGG each incubated with both Sortase&nbsp;A7M and Sortase&nbsp;A5M.
+                CP-LPETGG each incubated with both Sortase&nbsp;A7M and Sortase&nbsp;A5M.
-                                    Both gels display multimers when coat and a sortase variant are in a sample
+                Both gels display multimers when coat and a sortase variant are in a sample
-                                    together.
+                together.
-                                </p>
+            </p>
-                                <p>
+            <p>
-                                    To investigate this issue, we had a look at the
+                To investigate this issue, we had a look at the
-                                    literature and found a matching description in the publication of Patterson
+                literature and found a matching description in the publication of Patterson
-                                    et al.. They performed a similar experiment with P22 capsid proteins and observed
+                et al.. They performed a similar experiment with P22 capsid proteins and observed
-                                    the same multimers in their SDS-PAGEs
+                the same multimers in their SDS-PAGEs
-                                    </sup>
+                </sup>
-                                    . Comparing both SDS-PAGEs, we came to the following assumption:
+                . Comparing both SDS-PAGEs, we came to the following assumption:
-                                </p>
+            </p>
-                                <p>
+            <p>
-                                    Because of the promiscuity of Sortase&nbsp;A7M to accept primary amines as
+                Because of the promiscuity of Sortase&nbsp;A7M to accept primary amines as
-                                    substrates, as we discussed previously, the formation of CP multimers occurs,
+                substrates, as we discussed previously, the formation of CP multimers occurs,
-                                    unspecifically catalyzed by Sortase&nbsp;A7M.
+                unspecifically catalyzed by Sortase&nbsp;A7M.
-                                </p>
+            </p>
-                                <p>
+            <p>
-                                    Parallel to these experiments, we successfully modified the exterior of
+                Parallel to these experiments, we successfully modified the exterior of
-                                    pre-assembled VLPs <i>in vitro</i> (<a
+                pre-assembled VLPs <i>in vitro</i> (<a href="https://2019.igem.org/Team:TU_Darmstadt/Project/P22_VLP"
-                                        href="https://2019.igem.org/Team:TU_Darmstadt/Project/P22_VLP"
+                    target="_blank">VLP assembly</a>). These modified VLPs were homogenous and
-                                        target="_blank">VLP assembly</a>). These modified VLPs were homogenous and
+                overall correctly assembled. <b>Therefore, we conclude that the described multimer
-                                    overall correctly assembled. <b>Therefore, we conclude that the described multimer
+                    problem only occurs when Sortase&nbsp;A7M encounters free CP.</b>
-                                        problem only occurs when Sortase&nbsp;A7M encounters free CP.</b>
+            </p>
-                                </p>
-                                <h2>Does methionine affect Sortase linking?</h2>
+            <h2>Does methionine affect Sortase linking?</h2>
-                                <p>
+            <p>
-                                    Sortase A7M preferably attaches N-terminal poly-G to C-terminal LPETGG. However,
+                Sortase A7M preferably attaches N-terminal poly-G to C-terminal LPETGG. However,
-                                    the first amino acid of a protein is methionine (to be specific, formylmethionine in
+                the first amino acid of a protein is methionine (to be specific, formylmethionine in
-                                    bacteria).
+                bacteria).
-                                    For our constructs that possess N-terminal polyG-tags, we have to ask ourselves the
+                For our constructs that possess N-terminal polyG-tags, we have to ask ourselves the
-                                    question: If the initial
+                question: If the initial
-                                    methionines are not cleaved off after the proteins have been produced, will this
+                methionines are not cleaved off after the proteins have been produced, will this
-                                    interfere with the Sortase reaction?
+                interfere with the Sortase reaction?
-                                </p>
+            </p>
-                                <p>
-                                    To investigate this, we cloned and purified another protein: TVMVsite-GGGG-mCherry.
-                                    This protein can be treated with TVMV-protease,
-                                    leading to *GGGG-mCherry. This *GGGG-mCherry was then compared to (M)GGGG-mCherry we
-                                    used in all previous assays.
-                                </p>
-                                <p>
+            <p>
-                                    To investigate this, we cloned and purified two other proteins:
+                To investigate this, we cloned and purified two other proteins:
-                                    <b>TVMVsite-GGGG-mCherry</b> and <b>TEVsite-GGGG-sfGFP</b>.
+                <b>TVMVsite-GGGG-mCherry</b> and <b>TEVsite-GGGG-sfGFP</b>.
-                                    Then we treated these proteins with the respective proteases, resulting in
+                Then we treated these proteins with the respective proteases, resulting in
-                                    *GGGG-mCherry and *GGGG-sfGFP.
+                *GGGG-mCherry and *GGGG-sfGFP.
-                                    Following this *GGGG-mCherry was then compared to (M)GGGG-mCherry which we used in
+                Following this *GGGG-mCherry was then compared to (M)GGGG-mCherry which we used in
-                                    all previous assays.
+                all previous assays.
-                                    Assays were also conducted on <b>Fig.&nbsp;24</b> the processed *GGGG-sfGFP
+                Assays were also conducted on <b>Fig.&nbsp;24</b> the processed *GGGG-sfGFP
-                                    substrate.
+                substrate.
-                                    <b>Fig.&nbsp;24</b> confirmed our assumptions that the unprocessed substrate cannot
+                <b>Fig.&nbsp;24</b> confirmed our assumptions that the unprocessed substrate cannot
-                                    be linked to the sorting motif via Sortase&nbsp;A7M.
+                be linked to the sorting motif via Sortase&nbsp;A7M.
-                                    Subsequently, *GGGG-sfGFP (after protease digest) demonstrate successful linkage via
+                Subsequently, *GGGG-sfGFP (after protease digest) demonstrate successful linkage via
-                                    sortase-mediated ligation.
+                sortase-mediated ligation.
-                                </p>
+            </p>
-                                <p>
+            <p>
-                                    Due to these findings we modified our VLPs with
+                Due to these findings we modified our VLPs with
-                                    <a href="https://2019.igem.org/Team:TU_Darmstadt/Project/VLP_Modification"
+                <a href="https://2019.igem.org/Team:TU_Darmstadt/Project/VLP_Modification"
-                                        target="_blank">*GGGG-sfGFP</a>.
+                    target="_blank">*GGGG-sfGFP</a>.
-                                </p>
+            </p>
-                                <img class="img-fluid center"
+            <img class="img-fluid center"
-                                    src="https://2019.igem.org/wiki/images/d/d4/T--TU_Darmstadt--comparison_cleaved_uncleaved_sfGFP.png"
+                src="https://2019.igem.org/wiki/images/d/d4/T--TU_Darmstadt--comparison_cleaved_uncleaved_sfGFP.png"
-                                    style="max-width:70%" />
+                style="max-width:70%" />
-                                </a>
+            </a>
-                                <div class="caption">
+            <div class="caption">
-                                    <p>
+                <p>
-                                        <b>
+                    <b>
-                                            Figure 24 :
+                        Figure 24 :
-                                        </b>
+                    </b>
-                                        Sortase-mediated ligation of TAMRA-LPETG and GGGG-sfGFP (with TEV
+                    Sortase-mediated ligation of TAMRA-LPETG and GGGG-sfGFP (with TEV
-                                cleavage site) one cut with TEV protease and one not. The sample with the unprocessed
+                    cleavage site) one cut with TEV protease and one not. The sample with the unprocessed
-                                substrate shows no increase in RFU. In contrast the processed substrate shows a clear
+                    substrate shows no increase in RFU. In contrast the processed substrate shows a clear
-                                increase in ΔRFU. After 90 min the reverse reaction begins.The ΔRFU refers to the
+                    increase in ΔRFU. After 90 min the reverse reaction begins.The ΔRFU refers to the
-                                respective negative control without each sortase at 514 nm. The mean ΔRFU value of the
+                    respective negative control without each sortase at 514 nm. The mean ΔRFU value of the
-                                duplicates was normalized to zero for better visualization.
+                    duplicates was normalized to zero for better visualization.
-                                    </p>
+                </p>
-                                </div>
+            </div>
-                                <p>
+            <p>
-                                    We performed FRET-assays with TAMRA-LPETG and either of the following reaction
+                We performed FRET-assays with TAMRA-LPETG and either of the following reaction
-                                    partners:
+                partners:
-                                </p>
+            </p>
-                                <ul>
+            <ul>
-                                    <li>(M)GGGG-mCherry, a protein sample that might still carry an N-terminal
+                <li>(M)GGGG-mCherry, a protein sample that might still carry an N-terminal
-                                        methionine</li>
+                    methionine</li>
-                                    <li>*GGGG-mCherry that does not carry any additional N-terminal residue</li>
+                <li>*GGGG-mCherry that does not carry any additional N-terminal residue</li>
-                                </ul>
+            </ul>
-                                <p>
+            <p>
-                                    Before the FRET-assay was started, we adjusted the mCherry-concentrations of both
+                Before the FRET-assay was started, we adjusted the mCherry-concentrations of both
-                                    fluorescent protein solutions to the same level. To do so, we diluted them until
+                fluorescent protein solutions to the same level. To do so, we diluted them until
-                                    both showed the same fluorescence at 610 nm.
+                both showed the same fluorescence at 610 nm.
-                                </p>
+            </p>
-                                <img class="img-fluid center"
+            <img class="img-fluid center" src="https://2019.igem.org/wiki/images/5/5d/T--TU_Darmstadt--Hannah1.png"
-                                    src="https://2019.igem.org/wiki/images/5/5d/T--TU_Darmstadt--Hannah1.png"
+                style="max-width:60%" />
-                                    style="max-width:60%" />
+            </a>
-                                </a>
-                                <img class="img-fluid center"
+            <img class="img-fluid center" src="https://2019.igem.org/wiki/images/1/17/T--TU_Darmstadt--Hannah2.jpeg"
-                                    src="https://2019.igem.org/wiki/images/1/17/T--TU_Darmstadt--Hannah2.jpeg"
+                style="max-width:60%" />
-                                    style="max-width:60%" />
+            </a>
-                                </a>
-                                <p>
+            <p>
-                                    <b>
+                <b>
-                                        Figure 25:
+                    Figure 25:
-                                    </b>
+                </b>
-                                    FRET of the sortase reaction connecting TAMRA-LPETG and GGGG-sfGFP mediated by Sortase A7M. The
+                FRET of the sortase reaction connecting TAMRA-LPETG and GGGG-sfGFP mediated by Sortase A7M. The
-                                    concentration of the Sortase A7M and TAMRA-LPETG was kept at the same level why the
+                concentration of the Sortase A7M and TAMRA-LPETG was kept at the same level why the
-                                    concentration of sfGFP was either 10,7&nbsp;µM or 1,4&nbsp;µM. The graphs show that the reverse
+                concentration of sfGFP was either 10,7&nbsp;µM or 1,4&nbsp;µM. The graphs show that the reverse
-                                    reaction happens earlier if the GGGG-substrate concentration is lower. The ΔRFU refers to the
+                reaction happens earlier if the GGGG-substrate concentration is lower. The ΔRFU refers to the
-                                    respective negative control without each sortase at 514 nm. The mean ΔRFU value of the
+                respective negative control without each sortase at 514 nm. The mean ΔRFU value of the
-                                    duplicates was normalized to zero for better visualization.
+                duplicates was normalized to zero for better visualization.
-                                </p>
+            </p>
-                                <p>
+            <p>
-                                    Strikingly, only the (M)GGGG-mCherry construct showed a clear decrease in delta RFU
+                Strikingly, only the (M)GGGG-mCherry construct showed a clear decrease in delta RFU
-                                    after the maximum delta RFU was reached (at about 160 min).
+                after the maximum delta RFU was reached (at about 160 min).
-                                </p>
+            </p>
-                                <img class="img-fluid center"
+            <img class="img-fluid center" src="https://2019.igem.org/wiki/images/f/f4/T--TU_Darmstadt--Hannah3.png"
-                                    src="https://2019.igem.org/wiki/images/f/f4/T--TU_Darmstadt--Hannah3.png"
+                style="max-width:70%" />
-                                    style="max-width:70%" />
+            </a>
-                                </a>
+            <div class="caption">
-                                <div class="caption">
+                <p>
-                                    <p>
+                    <b>
-                                        <b>
+                        Figure 26 :
-                                            Figure 26 :
+                    </b>
-                                        </b>
+                    Sortase-mediated ligation of TAMRA-LPETG and GGGG-mCherry one cut with TVMV protease and one
-                                        Sortase-mediated ligation of TAMRA-LPETG and GGGG-mCherry one cut with TVMV protease and one
+                    with a methionine infront of the GGGG-tag. As visible the reverse reaction happens earlier if
-                        with a methionine infront of the GGGG-tag. As visible the reverse reaction happens earlier if
+                    the methionine is not cleaved of the GGGG-tag. The ΔRFU is referring to the negative controls
-                        the methionine is not cleaved of the GGGG-tag. The ΔRFU is referring to the negative controls
+                    without Sortase&nbsp;A7M at 570 nm. The mean ΔRFU value of the tripicates was normalized to zero
-                        without Sortase&nbsp;A7M at 570 nm. The mean ΔRFU value of the tripicates was normalized to zero
+                    for better visualization.
-                        for better visualization.
+                </p>
-                                    </p>
+            </div>
-                                </div>
-                                <p>
+            <p>
-                                    We assume the following: Although we adjusted the overall mCherry concentration by
+                We assume the following: Although we adjusted the overall mCherry concentration by
-                                    fluorescence, we cannot determine the absolute amount of <b>M</b>GGGG-mCherry in the
+                fluorescence, we cannot determine the absolute amount of <b>M</b>GGGG-mCherry in the
-                                    (M)GGGG-mCherry sample. However, if this amount was relatively high, the
+                (M)GGGG-mCherry sample. However, if this amount was relatively high, the
-                                    <b>effective substrate concentration</b> that could enter the sortase reaction would
+                <b>effective substrate concentration</b> that could enter the sortase reaction would
-                                    be low. That is because MGGGG is a worse sortase substrate than GGGG – if any at
+                be low. That is because MGGGG is a worse sortase substrate than GGGG – if any at
-                                    all. If we furthermore consider that a low substrate concentration correlates with a
+                all. If we furthermore consider that a low substrate concentration correlates with a
-                                    faster reverse reaction, we can explain the observed decrease in delta RFU for the
+                faster reverse reaction, we can explain the observed decrease in delta RFU for the
-                                    (M)GGGG-mCherry sample that contrasts the delta RFU trend of the *GGGG-mCherry
+                (M)GGGG-mCherry sample that contrasts the delta RFU trend of the *GGGG-mCherry
-                                    sample.
+                sample.
-                                </p>
+            </p>
-                                <p>
+            <p>
-                                    On this basis we can assume that a certain, yet unknown portion of the
+                On this basis we can assume that a certain, yet unknown portion of the
-                                    (M)GGGG-mCherry sample still carries an N-terminal methionine.
+                (M)GGGG-mCherry sample still carries an N-terminal methionine.
-                                </p>
+            </p>
-                                <p>
+            <p>
-                                    These FRET-assays let us assume that methionine disturbs or at least interferes with
+                These FRET-assays let us assume that methionine disturbs or at least interferes with
-                                    the sortase reaction mechanism. Indeed, our <b>modeling</b> suggests that
+                the sortase reaction mechanism. Indeed, our <b>modeling</b> suggests that
-                                    <b>methionine affects the interaction of polyG</b> and the flexible loop near the
+                <b>methionine affects the interaction of polyG</b> and the flexible loop near the
-                                    active site of Sortase A7M.<a href="https://2019.igem.org/Team:TU_Darmstadt/Model"
+                active site of Sortase A7M.<a href="https://2019.igem.org/Team:TU_Darmstadt/Model" target="_blank">
-                                        target="_blank"> Click here</a> if you want to know more about our modeling
+                    Click here</a> if you want to know more about our modeling
-                                    results!
+                results!
-                                </p>
+            </p>
-                                <p>
+            <p>
-                                    This strengthens our hypothesis: If there is any amino acid in front of the
+                This strengthens our hypothesis: If there is any amino acid in front of the
-                                    poly-glycine sequence, substrate binding to Sortase A7M is negatively influenced.
+                poly-glycine sequence, substrate binding to Sortase A7M is negatively influenced.
-                                </p>
+            </p>
-                                <h1>Modeling</h1>
+            <h1>Modeling</h1>
-                                <h3>Introduction</h3>
+            <h3>Introduction</h3>
-                                <p>
+            <p>
-                                    In synthetic biology, theoretical models are often used to gain insights, predict
+                In synthetic biology, theoretical models are often used to gain insights, predict
-                                    and
+                and
-                                    improve
+                improve
-                                    experiments. In our project we are modifying Virus-like particles (VLPs) by
+                experiments. In our project we are modifying Virus-like particles (VLPs) by
-                                    attaching
+                attaching
-                                    proteins to the
+                proteins to the
-                                    surface of the P22 capsid
+                surface of the P22 capsid
-                                    <!-- Link zum Background oder Project overview  --> through a linker. The linking is
+                <!-- Link zum Background oder Project overview  --> through a linker. The linking is
-                                    catalyzed using
+                catalyzed using
-                                    the enzyme Sortase A7M, which is a calcium-independent mutant of the wild type
+                the enzyme Sortase A7M, which is a calcium-independent mutant of the wild type
-                                    Sortase A
+                Sortase A
-                                    <!-- Link zum Sortase Background --> from <i>Staphylococcus aureus</i>. We performed
+                <!-- Link zum Sortase Background --> from <i>Staphylococcus aureus</i>. We performed
-                                    modeling to predict the unknown structure of the
+                modeling to predict the unknown structure of the
-                                    Sortase A7M, to improve the linker between proteins and therefore optimizing the
+                Sortase A7M, to improve the linker between proteins and therefore optimizing the
-                                    modification
+                modification
-                                    efficiency. <br>
+                efficiency. <br>
-                                    Here <i>Rosetta Comparative Modeling</i> was performed to predict the structure of
+                Here <i>Rosetta Comparative Modeling</i> was performed to predict the structure of
-                                    Sortase A7M. The generated structure was characterized in terms of the molecular
+                Sortase A7M. The generated structure was characterized in terms of the molecular
-                                    mechanisms and binding affinities of ligand peptides using molecular dynamics
+                mechanisms and binding affinities of ligand peptides using molecular dynamics
-                                    and docking simulations.
+                and docking simulations.
-                                </p>
+            </p>
-                                <h2>Structure determination</h2>
+            <h2>Structure determination</h2>
-                                <p>
+            <p>
-                                    <i>In silico</i> modeling and simulation of proteins requires a 3D structure,
+                <i>In silico</i> modeling and simulation of proteins requires a 3D structure,
-                                    which can be
+                which can be
-                                    obtained from the <a href="https://www.rcsb.org/" target="_blank">RCSB Protein
+                obtained from the <a href="https://www.rcsb.org/" target="_blank">RCSB Protein
-                                        Data
+                    Data
-                                        Bank</a>. However, if no 3D structures are annotated, as it is the case with
+                    Bank</a>. However, if no 3D structures are annotated, as it is the case with
-                                    sortase
+                sortase
-                                    A7M, the structure has to be determined by other means. The structure prediction
+                A7M, the structure has to be determined by other means. The structure prediction
-                                    of sortase A7M was done using two different approaches.
+                of sortase A7M was done using two different approaches.
-                                </p>
+            </p>
-                                <h2>RosettaCM</h2>
+            <h2>RosettaCM</h2>
-                                <h3>Results</h3>
+            <h3>Results</h3>
-                                <p>
+            <p>
-                                    The simulation yielded 15,000 structures which have been compared using the
+                The simulation of our Sortase A7M yielded 15,000 structures which have been compared using the
-                                    Rosetta scoring functions (talaris2013).
+                Rosetta scoring functions (talaris2013).
-                                    <!-- scoring -->
+                <!-- scoring -->
-                                    From the 15,000 structures generated, we inspected the ten best scoring
+                From the 15,000 structures generated, we inspected the ten best scoring
-                                    structures.
+                structures.
-                                </p>
+            </p>
-                                <p>
+            <p>
-                                    As can be seen in <b>Fig. 27</b> 27, the most prominent differences can
+                As can be seen in <b>Fig. 27</b>, the most prominent differences can
-                                    be found in the regions close to the N- and C-terminus. As
+                be found in the regions close to the N- and C-terminus. As
-                                    fluctuations in those
+                fluctuations in those
-                                    regions are not untypical, we decided to use the best scoring
+                regions are not untypical, we decided to use the best scoring
-                                    structure, candidate S_14771 (<b>Fig.</b> 28), as the input for the
+                structure, candidate S_14771 (<b>Fig.</b> 28), as the input for the
-                                    simulations to follow.
+                simulations to follow.
-                                </p>
+            </p>
-                                <img class="img-fluid center"
+            <img class="img-fluid center"
-                                    src="https://2019.igem.org/wiki/images/4/40/T--TU_Darmstadt--top10_corporate.png"
+                src="https://2019.igem.org/wiki/images/4/40/T--TU_Darmstadt--top10_corporate.png"
-                                    style="max-width:40%" />
+                style="max-width:40%" />
-                                </a>
+            </a>
-                                <div class="caption">
+            <div class="caption">
-                                    <p>
+                <p>
-                                        <b>
+                    <b>
-                                            Figure 27 :
+                        Figure 27 :
-                                        </b>
+                    </b>
-                                        The structural alignment of the ten best scoring
+                    The structural alignment of the ten best scoring
-                                        sortase structures
+                    sortase structures
-                                        displaying minor differences with the exception of the C- and
+                    displaying minor differences with the exception of the C- and
-                                        N-terminal
+                    N-terminal
-                                        regions. N- and C-terminal regions tend to show strong
+                    regions. N- and C-terminal regions tend to show strong
-                                        fluctuations, thus it is
+                    fluctuations, thus it is
-                                        unsurprising to find the terminal regions to be unaligned
+                    unsurprising to find the terminal regions to be unaligned
-                                    </p>
+                </p>
-                                </div>
+            </div>
-                                <img class="img-fluid center"
+            <img class="img-fluid center" src="https://2019.igem.org/wiki/images/b/b3/T--TU_Darmstadt--s14771.gif"
-                                    src="https://2019.igem.org/wiki/images/b/b3/T--TU_Darmstadt--s14771.gif"
+                style="max-width:40%" />
-                                    style="max-width:40%" />
+            </a>
-                                </a>
+            <div class="caption">
-                                <div class="caption">
+                <p>
-                                    <p>
+                    <b>
-                                        <b>
+                        Figure 28 :
-                                            Figure 28 :
+                    </b>
-                                        </b>
+                    Sortase A7M candidate S_14771 created through RosettaCM.
-                                        Sortase A7M candidate S_14771 created through RosettaCM.
+                </p>
-                                    </p>
+            </div>
-                                </div>
-                                <p> In order to evaluate the secondary structure of the Sortase A7M
+            <p> In order to evaluate the secondary structure of the Sortase A7M
-                                    candidate S_14771 a Ramachandran plot has been created and compared to
+                candidate S_14771 a Ramachandran plot has been created and compared to
-                                    the five sortases used as input for the comparitive modeling.
+                the five sortases used as input for the comparitive modeling.
-                                </p>
+            </p>
-                                <img class="img-fluid center"
+            <img class="img-fluid center"
-                                    src="https://2019.igem.org/wiki/images/2/28/T--TU_Darmstadt--ramachandran_s14711.png"
+                src="https://2019.igem.org/wiki/images/2/28/T--TU_Darmstadt--ramachandran_s14711.png"
-                                    style="max-width:40%" />
+                style="max-width:40%" />
-                                </a>
+            </a>
-                                <img class="img-fluid center"
+            <img class="img-fluid center"
-                                    src="https://2019.igem.org/wiki/images/e/ee/T--TU_Darmstadt--ramachandran_five_sortases.png"
+                src="https://2019.igem.org/wiki/images/e/ee/T--TU_Darmstadt--ramachandran_five_sortases.png"
-                                    style="max-width:40%" />
+                style="max-width:40%" />
-                                </a>
+            </a>
-                                <img class="img-fluid center"
+            <img class="img-fluid center"
-                                    src="https://2019.igem.org/wiki/images/7/73/T--TU_Darmstadt--Comp_Ramachandran.PNG"
+                src="https://2019.igem.org/wiki/images/7/73/T--TU_Darmstadt--Comp_Ramachandran.PNG"
-                                    style="max-width:40%" />
+                style="max-width:40%" />
-                                </a>
+            </a>
-                                <p> <b>Figure 29</b>: The Ramachandran plot of randomly sampled proteins
+            <p> <b>Figure 29</b>: The Ramachandran plot of randomly sampled proteins
-                                    and the input structures of the <i>comparative modeling</i> show
+                and the input structures of the <i>comparative modeling</i> show
-                                    similar secondary structures. Secondary structure analysis of both
+                similar secondary structures. Secondary structure analysis of both
-                                    sortase candidates reveals absence
+                sortase candidates reveals absence
-                                    of secondary structures for the ML candidate. This is not the case
+                of secondary structures for the ML candidate. This is not the case
-                                    with candidate S_14771 as the Ramachandran plot shows all relevant
+                with candidate S_14771 as the Ramachandran plot shows all relevant
-                                    structures.
+                structures.
-                                </p>
+            </p>
-                                <p>
+            <p>
-                                    The Ramachandran plot (<b>Fig.&nbsp;29</b>) showing &alpha;-helices and
+                The Ramachandran plot (<b>Fig.&nbsp;29</b>) showing &alpha;-helices and
-                                    &beta;-sheets is a
+                &beta;-sheets is a
-                                    strong indicator of a successful structure determination, as those
+                strong indicator of a successful structure determination, as those
-                                    secondary
+                secondary
-                                    structures are crucial for the functionality of sortases.
+                structures are crucial for the functionality of sortases.
-                                </p>
+            </p>
-                                <h3>Conclusion</h3>
+            <h3>Conclusion</h3>
-                                <p>
+            <p>
-                                    We used monte-carlo simulations
+                We used monte-carlo simulations
-                                    to determine the structure of the mutated transpeptidase Sortase A7M.
+                to determine the structure of the mutated transpeptidase Sortase A7M.
-                                    As a first investigation of the secondary structure a Ramachandran plot of
+                As a first investigation of the secondary structure a Ramachandran plot of
-                                    candidate <i>S_14771</i> was made. The plot showing secondary structures was a first
+                candidate <i>S_14771</i> was made. The plot showing secondary structures was a first
-                                    indicator
+                indicator
-                                    of successful structure prediction.
+                of successful structure prediction.
-                                    The approach, using <i>Rosetta Comparative Modeling</i>, yielded
+                The approach, using <i>Rosetta Comparative Modeling</i>, yielded
 ,000
-                                    structures scored with the talaris2013 scoring function. The ten
+                structures scored with the talaris2013 scoring function. The ten
-                                    best structures
+                best structures
-                                    were aligned and exhibited almost identical secondary structures
+                were aligned and exhibited almost identical secondary structures
-                                    (<b>Fig. 27</b>).
+                (<b>Fig. 27</b>).
-                                    The greatest structural differences are present in the N- and
+                The greatest structural differences are present in the N- and
-                                    C-terminal regions. Since terminal regions tend to fluctuate more strongly than
+                C-terminal regions. Since terminal regions tend to fluctuate more strongly than
-                                    non-terminal segments of the protein, we deemed those fluctuations
+                non-terminal segments of the protein, we deemed those fluctuations
-                                    non-relevant for the proteins functionality.
+                non-relevant for the proteins functionality.
-                                    <br>
+                <br>
-                                    Being the best scoring candidate, structure S_14771 was analyzed
+                Being the best scoring candidate, structure S_14771 was analyzed
-                                    structurally using a Ramachandran plot (<b>Fig. 29</b>). The plot shows all the
+                structurally using a Ramachandran plot (<b>Fig. 29</b>). The plot shows all the
-                                    relevant and typical structures sortases exhibit and serves as an indicator for
+                relevant and typical structures sortases exhibit and serves as an indicator for
-                                    a successful structure prediction.
+                a successful structure prediction.
-                                    <br>
+                <br>
-                                    In the steps to follow, a molecular dynamics (MD)
+                In the steps to follow, a molecular dynamics (MD)
-                                    simulation was performed on both structures.
+                simulation was performed on both structures.
-                                </p>
+            </p>
-                                <h2>Molecular dynamics</h2>
+            <h2>Molecular dynamics</h2>
-                                <h3>Results</h3>
+            <h3>Results</h3>
-                                <p>
+            <p>
-                                    The first possible indicators of a stable protein structure are converging
+                The first possible indicators of a stable protein structure are converging
-                                    root-mean-square deviation (RMSD),
+                root-mean-square deviation (RMSD),
-                                    small root-mean-square
+                small root-mean-square
-                                    fluctuation (RMSF) values
+                fluctuation (RMSF) values
-                                    as well as converging radii of gyration. Using the Python software package and
+                as well as converging radii of gyration. Using the Python software package and
-                                    the module Biotite we calculated
+                the module Biotite we calculated
-                                    these quantities and plotted the result for candidate S_14771.
+                these quantities and plotted the result for candidate S_14771.
-                                </p>
+            </p>
-                                <img class="img-fluid center"
+            <img class="img-fluid center" src="https://2019.igem.org/wiki/images/4/4f/T--TU_Darmstadt--rmsd_s14771.png"
-                                    src="https://2019.igem.org/wiki/images/4/4f/T--TU_Darmstadt--rmsd_s14771.png"
+                style="max-width:40%" />
-                                    style="max-width:40%" />
+            </a>
-                                </a>
+            <div class="caption">
-                                <div class="caption">
+                <p>
-                                    <p>
+                    <b>
-                                        <b>
+                        Figure 30 :
-                                            Figure 30 :
+                    </b>
-                                        </b>
+                    The RMSD is one of three main indicators of a stable
-                                        The RMSD is one of three main indicators of a stable
+                    protein structure of the MD simulation of
-                                        protein structure of the MD simulation of
+                    S_14771 over the period of 200,000 ps. As time progressed the RMSD
-                                        S_14771 over the period of 200,000 ps. As time progressed the RMSD
+                    increased with a smaller slope.
-                                        increased with a smaller slope.
+                    The value stabilizes at a time of 110,000 ps and fluctuated around the
-                                        The value stabilizes at a time of 110,000 ps and fluctuated around the
+                    value of 6 &#8491;.
-                                        value of 6 &#8491;.
+                </p>
-                                    </p>
+            </div>
-                                </div>
-                                <img class="img-fluid center"
+            <img class="img-fluid center"
-                                    src="https://2019.igem.org/wiki/images/9/94/T--TU_Darmstadt--gyration_s14771.png"
+                src="https://2019.igem.org/wiki/images/9/94/T--TU_Darmstadt--gyration_s14771.png"
-                                    style="max-width:40%" />
+                style="max-width:40%" />
-                                </a>
+            </a>
-                                <div class="caption">
+            <div class="caption">
-                                    <p>
+                <p>
-                                        <b>
+                    <b>
-                                            Figure 31 :
+                        Figure 31 :
-                                        </b>
+                    </b>
-                                        The prominent fluctuations of the residues from ranges
+                    The prominent fluctuations of the residues from ranges
 to 115 might
-                                        indicate a binding site or another form of functional structure. The
+                    indicate a binding site or another form of functional structure. The
-                                        radius of gyration, just as
+                    radius of gyration, just as
-                                        the RMSD <b>Fig. 30</b>, stabilizes around a simulation time of of 110,000 ps
+                    the RMSD <b>Fig. 30</b>, stabilizes around a simulation time of of 110,000 ps
-                                        and converges towards a value of
+                    and converges towards a value of
 .7 &#8491;.
-                                    </p>
+                </p>
-                                </div>
+            </div>
-                                <img class="img-fluid center"
+            <img class="img-fluid center" src="https://2019.igem.org/wiki/images/f/f4/T--TU_Darmstadt--rmsf_s14771.png"
-                                    src="https://2019.igem.org/wiki/images/f/f4/T--TU_Darmstadt--rmsf_s14771.png"
+                style="max-width:40%" />
-                                    style="max-width:40%" />
+            </a>
-                                </a>
+            <div class="caption">
-                                <div class="caption">
+                <p>
-                                    <p>
+                    <b>
-                                        <b>
+                        Figure 32 :
-                                            Figure 32 :
+                    </b>
-                                        </b>
+                    The fluctuations
-                                        The fluctuations
+                    (RMSF) of most residues appear insignificant compared to the first, the
-                                        (RMSF) of most residues appear insignificant compared to the first, the
+                    last residues and
-                                        last residues and
+                    the residues close to residue 110 . Typically the N- and C-terminus tend
-                                        the residues close to residue 110 . Typically the N- and C-terminus tend
+                    to fluctuate more intensively due to the lack of
-                                        to fluctuate more intensively due to the lack of
+                    stabilizing structures. The prominent fluctuations in the range of
-                                        stabilizing structures. The prominent fluctuations in the range of
+                    residue 105 to 115
-                                        residue 105 to 115
+                    can indicate a binding site or another form of functional structure.
-                                        can indicate a binding site or another form of functional structure.
+                </p>
-                                    </p>
+            </div>
-                                </div>
-                                <p>
+            <p>
-                                    Typical RMSDs and radii of gyration converge towards a value dependent on the
+                Typical RMSDs and radii of gyration converge towards a value dependent on the
-                                    size of the
+                size of the
-                                    protein. Convergence of those quantities can be interpreted as a stable state of
+                protein. Convergence of those quantities can be interpreted as a stable state of
-                                    the protein
+                the protein
-                                    structure. As it can be seen in <b>Fig. 30</b> and <b>Fig. 31</b> both the RMSD and
+                structure. As it can be seen in <b>Fig. 30</b> and <b>Fig. 31</b> both the RMSD and
-                                    the radius of
+                the radius of
-                                    gyration
+                gyration
-                                    stabilize at the same time as the simulation reaches 110,000 ps (110 ns),
+                stabilize at the same time as the simulation reaches 110,000 ps (110 ns),
-                                    suggesting a now
+                suggesting a now
-                                    stabilized structure of candidate S_14771 solvated in water. Another indicator
+                stabilized structure of candidate S_14771 solvated in water. Another indicator
-                                    of a
+                of a
-                                    functional protein is the RMSF. Instead of being averaged over all atoms, the
+                functional protein is the RMSF. Instead of being averaged over all atoms, the
-                                    RMSF is
+                RMSF is
-                                    averaged over time with respect to each amino acid. It provides insights in both
+                averaged over time with respect to each amino acid. It provides insights in both
-                                    protein
+                protein
-                                    stability and functionality. Fig <b>Fig. 32</b> reveals the RMSF of residues 105 to
+                stability and functionality. Fig <b>Fig. 32</b> reveals the RMSF of residues 105 to
 to
-                                    be
+                be
-                                    significantly higher than that of other residues. This hints at the presence of
+                significantly higher than that of other residues. This hints at the presence of
-                                    a
+                a
-                                    functional unit along these residues. As commented on in the section
+                functional unit along these residues. As commented on in the section
-                                    describing our structure prediction approaches, the N-
+                describing our structure prediction approaches, the N-
-                                    and C-terminal regions tend to fluctuate more strongly as a result of the
+                and C-terminal regions tend to fluctuate more strongly as a result of the
-                                    absence of
+                absence of
-                                    stabilizing structures.
+                stabilizing structures.
-                                </p>
+            </p>
-                                <p>
+            <p>
-                                    To further analyse the potential binding site, we performed a
+                To further analyse the potential binding site, we performed a
-                                    <i>Principle Component Analysis</i>.
+                <i>Principle Component Analysis</i>.
-                                </p>
+            </p>
-                                <h3>Principle component analysis</h3>
+            <h3>Principle component analysis</h3>
-                                <p>
+            <p>
-                                    To analyze our system further Principle Component Analysis (PCA) was performed
+                To analyze our system further Principle Component Analysis (PCA) was performed
-                                    using GROMACS. By applying PCA to a protein it is possible to gain insights into the
+                using GROMACS. By applying PCA to a protein it is possible to gain insights into the
-                                    relevant
+                relevant
-                                    vibrational motions and thereby the physical mechanism of the protein.
+                vibrational motions and thereby the physical mechanism of the protein.
-                                </p>
+            </p>
-                                <img class="img-fluid center"
+            <img class="img-fluid center" src="https://2019.igem.org/wiki/images/d/db/T--TU_Darmstadt--modes_s14771.gif"
-                                    src="https://2019.igem.org/wiki/images/d/db/T--TU_Darmstadt--modes_s14771.gif"
+                style="width:40%">
-                                    style="width:40%">
+            <p><b>Animation 33: </b> A Principle Component Analysis of a fast (blue) and a
-                                <p><b>Animation 33: </b> A Principle Component Analysis of a fast (blue) and a
+                slow (red) mode showing the most prominent movements of the C&alpha;-chain
-                                    slow (red) mode showing the most prominent movements of the C&alpha;-chain
+                of candidate S_14771. Both modes show movement of the &beta;6&#47;&beta;7
-                                    of candidate S_14771. Both modes show movement of the &beta;6&#47;&beta;7
+                loop consisting of residues 105 to 115 towards the active site . Thus we can
-                                    loop consisting of residues 105 to 115 towards the active site . Thus we can
+                assume that the closing &beta;6&#47;&beta;7 loop is involved in the reaction
-                                    assume that the closing &beta;6&#47;&beta;7 loop is involved in the reaction
+                mechanism.
-                                    mechanism.
+            </p>
-                                </p>
-                                <p>
+            <p>
-                                    The results from the Principle Component Analysis of candidate S_14771
+                The results from the Principle Component Analysis of candidate S_14771
-                                    (<b>Amination 33</b>) show a movement of the residues 105 to 115 towards the active
+                (<b>Amination 33</b>) show a movement of the residues 105 to 115 towards the active
-                                    site, supporting our theory that residues 105 to 115 are important for the
+                site, supporting our theory that residues 105 to 115 are important for the
-                                    reaction mechanism. Since the most relevant eigenvector (red), which shows the most
+                reaction mechanism. Since the most relevant eigenvector (red), which shows the most
-                                    relevant
+                relevant
-                                    movement of the sortase, moves further towards the active site, it is possible
+                movement of the sortase, moves further towards the active site, it is possible
-                                    that the &beta;6&#47;&beta;7 loop either closes the binding site of the ligand
+                that the &beta;6&#47;&beta;7 loop either closes the binding site of the ligand
-                                    peptides or even transports one peptide towards the other.
+                peptides or even transports one peptide towards the other.
-                                </p>
+            </p>
-                                <h3>Conclusion</h3>
+            <h3>Conclusion</h3>
-                                <p>
+            <p>
-                                    Candidate S_14771 that was generated using <i>RosettaCM</i> appears to be a fitting
+                Candidate S_14771 that was generated using <i>RosettaCM</i> appears to be a fitting
-                                    candidate not
+                candidate not
-                                    only due to successful analyses, but also due to the residues of the active site
+                only due to successful analyses, but also due to the residues of the active site
-                                    being in close proximity.
+                being in close proximity.
-                                    Not the RMSF but also Principle Component Analysis
+                Not the RMSF but also Principle Component Analysis
-                                    indicate that residues 105 to 115 forming the &beta;6&#47;&beta;7 loop
+                indicate that residues 105 to 115 forming the &beta;6&#47;&beta;7 loop
-                                    are crucial for the molecular mechanism of our model.
+                are crucial for the molecular mechanism of our model.
-                                </p>
+            </p>
-                                <h2>Docking</h2>
+            <h2>Docking</h2>
-                                <p>
+            <p>
-                                    Now that the binding site of the Sortase had been found, the peptide ligand
+                Now that the binding site of the Sortase had been found, the peptide ligand
-                                    needed to be inserted into the binding site to create a peptide-protein complex.
+                needed to be inserted into the binding site to create a peptide-protein complex.
-                                    The procedure of choice
+                The procedure of choice
-                                    for the introduction of a ligand into the binding site of a protein is called
+                for the introduction of a ligand into the binding site of a protein is called
-                                    <i>docking</i>. In the
+                <i>docking</i>. In the
-                                    following sections, we will present the protocol and methods we used as well as
+                following sections, we will present the protocol and methods we used as well as
-                                    the results they yielded.
+                the results they yielded.
-                                </p>
+            </p>
-                                <h3>Results</h3>
+            <h3>Results</h3>
-                                <p>
+            <p>
-                                    For sequences MGGGGPPPPPP(M-polyG), GGGGPPPPPP(polyG) and PPPPPPLPETGG(LPETGG)
+                For sequences MGGGGPPPPPP(M-polyG), GGGGPPPPPP(polyG) and PPPPPPLPETGG(LPETGG)
 ,000 structures have been created and clustered.
-                                    After the clustering the sample consisted of 100 structures of docked complexes.
+                After the clustering the sample consisted of 100 structures of docked complexes.
-                                </p>
+            </p>
-                                <img class="img-fluid center"
+            <img class="img-fluid center" src="https://2019.igem.org/wiki/images/7/78/T--TU_Darmstadt--dock_lpetgg.png"
-                                    src="https://2019.igem.org/wiki/images/7/78/T--TU_Darmstadt--dock_lpetgg.png"
+                style="max-width:40%" />
-                                    style="max-width:40%" />
+            </a>
-                                </a>
+            <div class="caption">
-                                <div class="caption">
+                <p>
-                                    <p>
+                    <b>
-                                        <b>
+                        Figure 34 :
-                                            Figure 34 :
+                    </b>
-                                        </b>
+                    The three best scoring structures (total score, interface
-                                        The three best scoring structures (total score, interface
+                    score, reweighted score) of the LPETGG-tag are shown. Only two results are
-                                        score, reweighted score) of the LPETGG-tag are shown. Only two results are
+                    visible as the best reweighted score candidate is identical to the best
-                                        visible as the best reweighted score candidate is identical to the best
+                    interface score candidate. The reacting section of the LPETGG-tag namely
-                                        interface score candidate. The reacting section of the LPETGG-tag namely
+                    glycine is colored yellow as is the active site. The glycin of both ligand
-                                        glycine is colored yellow as is the active site. The glycin of both ligand
+                    peptides is facing the active site.
-                                        peptides is facing the active site.
+                </p>
-                                    </p>
+            </div>
-                                </div>
-                                <p>
+            <p>
-                                    Analysis of the scores has shown a similar score for all the three dockings. The
+                Analysis of the scores has shown a similar score for all the three dockings. The
-                                    best scoring results of the LPETGG docking show a tendency of the glycines to
+                best scoring results of the LPETGG docking show a tendency of the glycines to
-                                    face the active site while also being in close proximity to the active site.
+                face the active site while also being in close proximity to the active site.
-                                </p>
+            </p>
-                                <img class="img-fluid center"
+            <img class="img-fluid center" src="https://2019.igem.org/wiki/images/8/8d/T--TU_Darmstadt--dock_polyg.png"
-                                    src="https://2019.igem.org/wiki/images/8/8d/T--TU_Darmstadt--dock_polyg.png"
+                style="max-width:40%" />
-                                    style="max-width:40%" />
+            </a>
-                                </a>
+            <div class="caption">
-                                <div class="caption">
+                <p>
-                                    <p>
+                    <b>
-                                        <b>
+                        Figure 35:
-                                            Figure 35:
+                    </b>
-                                        </b>
+                    The three best scoring structures (total score,
-                                        The three best scoring structures (total score,
+                    interface score, reweighted score) of the polyG peptide are shown. Only
-                                        interface score, reweighted score) of the polyG peptide are shown. Only
+                    two results are visible as the best reweighted score candidate is
-                                        two results are visible as the best reweighted score candidate is
+                    identical to the best interface score candidate. Instead of facing the
-                                        identical to the best interface score candidate. Instead of facing the
+                    active site (yellow) the reacting glycines (yellow) appear to interact
-                                        active site (yellow) the reacting glycines (yellow) appear to interact
+                    with the &beta;6&#47;&beta;7 loop of the sortase.
-                                        with the &beta;6&#47;&beta;7 loop of the sortase.
+                </p>
-                                    </p>
+            </div>
-                                </div>
-                                <img class="img-fluid center"
+            <img class="img-fluid center" src="https://2019.igem.org/wiki/images/9/92/T--TU_Darmstadt--dock_mpolyg.png"
-                                    src="https://2019.igem.org/wiki/images/9/92/T--TU_Darmstadt--dock_mpolyg.png"
+                style="max-width:40%" />
-                                    style="max-width:40%" />
+            </a>
-                                </a>
+            <div class="caption">
-                                <div class="caption">
+                <p>
-                                    <p>
+                    <b>
-                                        <b>
+                        Figure 36:
-                                            Figure 36:
+                    </b>
-                                        </b>
+                    The three best scoring structures (total score,
-                                        The three best scoring structures (total score,
+                    interface score, reweighted score) of the poly-g peptide are shown. Only
-                                        interface score, reweighted score) of the poly-g peptide are shown. Only
+                    two results are visible as the best reweighted score candidate is
-                                        two results are visible as the best reweighted score candidate is
+                    identical to the best interface score candidate.
-                                        identical to the best interface score candidate.
+                    Concerning the M-poly-G peptide no uniform directional orientation can
-                                        Concerning the M-poly-G peptide no uniform directional orientation can
+                    be observed.
-                                        be observed.
+                    The structure with the best interface score (light blue) is oriendted
-                                        The structure with the best interface score (light blue) is oriendted
+                    towards the loop while the structure with the best total/reweighted
-                                        towards the loop while the structure with the best total/reweighted
+                    (dark blue) is oriented towards the &beta;-sheets.
-                                        (dark blue) is oriented towards the &beta;-sheets.
+                </p>
-                                    </p>
+            </div>
-                                </div>
-                                <p>
+            <p>
-                                    <b>Fig. 34</b> shows the docking result of the LPETGG peptide to
+                <b>Fig. 34</b> shows the docking result of the LPETGG peptide to
-                                    the sortase. The results shown are the best scoring structures of the clustering
+                the sortase. The results shown are the best scoring structures of the clustering
-                                    with respect to the total score, interface score and reweighted score. As the
+                with respect to the total score, interface score and reweighted score. As the
-                                    best scoring structure is the same for the total score and the reweighted score
+                best scoring structure is the same for the total score and the reweighted score
-                                    only two peptides are shown. This also applies to <b>Fig. 35</b> and <b>Fig. 36</b>.
+                only two peptides are shown. This also applies to <b>Fig. 35</b> and <b>Fig. 36</b>.
-                                    For both
+                For both
-                                    results the reacting glycin residues (yellow) are facing the active site.
+                results the reacting glycin residues (yellow) are facing the active site.
-                                    Additionally, the same residues are in close proximity to the active site.
+                Additionally, the same residues are in close proximity to the active site.
-                                </p>
+            </p>
-                                <p>
+            <p>
-                                    The figures <b>Fig. 35</b> and <b>Fig. 36</b> show the docking of the both polyG and
+                The figures <b>Fig. 35</b> and <b>Fig. 36</b> show the docking of the both polyG and
-                                    M-polyG. While polyG
+                M-polyG. While polyG
-                                    results align well and seem to be interacting with the &beta;6&#47;&beta;7 loop
+                results align well and seem to be interacting with the &beta;6&#47;&beta;7 loop
-                                    rather than with the active site, this does not seem to be the case for M-polyG.
+                rather than with the active site, this does not seem to be the case for M-polyG.
-                                    Instead of both structures interacting with the &beta;6&#47;&beta;7 loop or
+                Instead of both structures interacting with the &beta;6&#47;&beta;7 loop or
-                                    active site one (best interaction score; dark blue) interacts with the
+                active site one (best interaction score; dark blue) interacts with the
-                                    &beta;6&#47;&beta;7 loop and the other (best reweighted/total score; light
+                &beta;6&#47;&beta;7 loop and the other (best reweighted/total score; light
-                                    blue-gray) appears to interact with the active site.
+                blue-gray) appears to interact with the active site.
-                                </p>
+            </p>
-                                <img class="img-fluid center"
+            <img class="img-fluid center"
-                                    src="https://2019.igem.org/wiki/images/7/76/T--TU_Darmstadt--dock_zoom_active.png"
+                src="https://2019.igem.org/wiki/images/7/76/T--TU_Darmstadt--dock_zoom_active.png"
-                                    style="max-width:40%" />
+                style="max-width:40%" />
-                                </a>
+            </a>
-                                <div class="caption">
+            <div class="caption">
-                                    <p>
+                <p>
-                                        <b>
+                    <b>
-                                            Figure 37:
+                        Figure 37:
-                                        </b>
+                    </b>
-                                        The close up of the M-polyG peptide (best
+                    The close up of the M-polyG peptide (best
-                                        total/reweighted score) indicates an interaction of methionine with
+                    total/reweighted score) indicates an interaction of methionine with
-                                        arginine<sub>139</sub> and cysteine<sub>126</sub>.
+                    arginine<sub>139</sub> and cysteine<sub>126</sub>.
-                                    </p>
+                </p>
-                                </div>
+            </div>
-                                <img class="img-fluid center"
+            <img class="img-fluid center"
-                                    src="https://2019.igem.org/wiki/images/4/48/T--TU_Darmstadt--dock_zoom_loop.png"
+                src="https://2019.igem.org/wiki/images/4/48/T--TU_Darmstadt--dock_zoom_loop.png"
-                                    style="max-width:40%" />
+                style="max-width:40%" />
-                                </a>
+            </a>
-                                <div class="caption">
+            <div class="caption">
-                                    <p>
+                <p>
-                                        <b>
+                    <b>
-                                            Figure 38:
+                        Figure 38:
-                                        </b>
+                    </b>
-                                        Methionine of the result with the best interface score
+                    Methionine of the result with the best interface score
-                                        interacted with the &beta;6&#47;&beta;7 loop rather than the active
+                    interacted with the &beta;6&#47;&beta;7 loop rather than the active
-                                        site. Still the reactive glycine residues appear to be bound to the
+                    site. Still the reactive glycine residues appear to be bound to the
-                                        &beta;6&#47;&beta;7 loop.
+                    &beta;6&#47;&beta;7 loop.
-                                    </p>
+                </p>
-                                </div>
+            </div>
-                                <p>
+            <p>
-                                    As can be seen in <b>Fig. 37</b> visualizing the result of the the docking
+                As can be seen in <b>Fig. 37</b> visualizing the result of the the docking
-                                    simulation
+                simulation
-                                    (total/reweighted score) suggests an interaction of methionine and two of the
+                (total/reweighted score) suggests an interaction of methionine and two of the
-                                    active sites namely arginine<sub>139</sub> and cysteine<sub>126</sub>.
+                active sites namely arginine<sub>139</sub> and cysteine<sub>126</sub>.
-                                    <b>Fig. 38</b> shows the interaction of M-polyG with the &beta;6&#47;&beta;7 loop.
+                <b>Fig. 38</b> shows the interaction of M-polyG with the &beta;6&#47;&beta;7 loop.
-                                    The glycines still interact with the &beta;6&#47;&beta;7 loop.
+                The glycines still interact with the &beta;6&#47;&beta;7 loop.
-                                    Instead of binding above the &beta;6&#47;&beta;7 loop, which is the case for
+                Instead of binding above the &beta;6&#47;&beta;7 loop, which is the case for
-                                    polyG as illustrated in <b>Fig. 36</b>,
+                polyG as illustrated in <b>Fig. 36</b>,
-                                    the interaction seems to be influenced by methionine. By interacting with the
+                the interaction seems to be influenced by methionine. By interacting with the
-                                    residues in the &beta;-helix
+                residues in the &beta;-helix
-                                    methionine could potentially hinder binding of glycine to the
+                methionine could potentially hinder binding of glycine to the
-                                    &beta;6&#47;&beta;7 loop by partial
+                &beta;6&#47;&beta;7 loop by partial
-                                    immobilization of the peptide. Overall peptide binding and orientation is less
+                immobilization of the peptide. Overall peptide binding and orientation is less
-                                    uniform compared
+                uniform compared
-                                    polyG without the leading methionine, which could be an indicator of lesser
+                polyG without the leading methionine, which could be an indicator of lesser
-                                    binding affinity of M-PolyG towards
+                binding affinity of M-PolyG towards
-                                    the &beta;6&#47;&beta;7 loop.
+                the &beta;6&#47;&beta;7 loop.
-                                </p>
+            </p>
-                                <h3>Conclusion</h3>
+            <h3>Conclusion</h3>
-                                <p>
+            <p>
-                                    To computationally investigate binding affinities of the polyG and M-polyG as
+                To computationally investigate binding affinities of the polyG and M-polyG as
-                                    well as the LPETGG tags we performed
+                well as the LPETGG tags we performed
-                                    docking simulations using the <i>Rosetta FlexPepDock</i> application. We used a
+                docking simulations using the <i>Rosetta FlexPepDock</i> application. We used a
-                                    modified version of the recommended
+                modified version of the recommended
-                                    protocol as the modified version was easier to automate and served our purpose
+                protocol as the modified version was easier to automate and served our purpose
-                                    better than the standard protocol.
+                better than the standard protocol.
-                                    From the calculated scores only, we could not see a difference in binding
+                From the calculated scores only, we could not see a difference in binding
-                                    affinities.
+                affinities.
-                                    Thus, we inspected the best scoring
+                Thus, we inspected the best scoring
-                                    structures regarding the total score, the interface score and the reweighted
+                structures regarding the total score, the interface score and the reweighted
-                                    score using PyMOL.
+                score using PyMOL.
-                                    Since the best structures with respect to total score and reweighted score were
+                Since the best structures with respect to total score and reweighted score were
-                                    the same for all simulations,
+                the same for all simulations,
-                                    only two structures have been inspected per run. A polyproline tag was appended
+                only two structures have been inspected per run. A polyproline tag was appended
-                                    to all the peptides to simulate
+                to all the peptides to simulate
-                                    the modification of the VLPs with a small peptide.
+                the modification of the VLPs with a small peptide.
-                                    <!-- GRoß helices etc erwähnen als begründung -->
+                <!-- GRoß helices etc erwähnen als begründung -->
-                                </p>
+            </p>
-                                <p>
+            <p>
-                                    As expected, the results showed that for LPETGG, the glycines of both peptides
+                As expected, the results showed that for LPETGG, the glycines of both peptides
-                                    oriented towards the active site.
+                oriented towards the active site.
-                                    This is unsurprising as peptides with the sequence LPXTGG are known to be
+                This is unsurprising as peptides with the sequence LPXTGG are known to be
-                                    substrate of the Sortase. It was more surprising to
+                substrate of the Sortase. It was more surprising to
-                                    see the polyG tag oriented away from the active site since polyG also is a known
+                see the polyG tag oriented away from the active site since polyG also is a known
-                                    substrate of the sortase. Both polyG peptides
+                substrate of the sortase. Both polyG peptides
-                                    were facing the &beta;6&#47;&beta;7 loop (residues 105 to 115) uniformly and
+                were facing the &beta;6&#47;&beta;7 loop (residues 105 to 115) uniformly and
-                                    appeared to be interacting with it. The M-polyG peptides did not
+                appeared to be interacting with it. The M-polyG peptides did not
-                                    show a uniform orientation or interaction scheme. On one hand the visualization
+                show a uniform orientation or interaction scheme. On one hand the visualization
-                                    of the best result concerning the total and reweighted
+                of the best result concerning the total and reweighted
-                                    score has shown interaction of methionine with the cysteine<sub>126</sub> and
+                score has shown interaction of methionine with the cysteine<sub>126</sub> and
-                                    arginine<sub>139</sub>, two residues of the active
+                arginine<sub>139</sub>, two residues of the active
-                                    site. On the other hand, the visualization of the best result with respect to
+                site. On the other hand, the visualization of the best result with respect to
-                                    the interface score shows the M-polyG facing the mobile &beta;6&#47;&beta;7
+                the interface score shows the M-polyG facing the mobile &beta;6&#47;&beta;7
-                                    loop.
+                loop.
-                                    In contrast to the polyG peptide the lacking the methionine, the M-polyG peptide
+                In contrast to the polyG peptide the lacking the methionine, the M-polyG peptide
-                                    is pulled down below the &beta;6&#47;&beta;7 loop by the methionine interacting
+                is pulled down below the &beta;6&#47;&beta;7 loop by the methionine interacting
-                                    with one of the &beta;-sheets leading to the active site. This is not the case
+                with one of the &beta;-sheets leading to the active site. This is not the case
-                                    with the polgG results, which lie aligned in one plane
+                with the polgG results, which lie aligned in one plane
-                                    with the &beta;6&#47;&beta;7 loop.
+                with the &beta;6&#47;&beta;7 loop.
-                                </p>
+            </p>
-                                <h2>Modeling Conclusion</h2>
+            <h2>Modeling Conclusion</h2>
-                                <p>
+            <p>
-                                    For our project it was key to understand and characterize Sortase A7M.
+                For our project it was key to understand and characterize Sortase A7M.
-                                    As there is no annotated 3D structure for this specific Sortase, an <i>in
+                As there is no annotated 3D structure for this specific Sortase, an <i>in
-                                        silico</i> structure determination
+                    silico</i> structure determination
-                                    was performed. Comparative modeling with Rosetta produced valid structures. We used
+                was performed. Comparative modeling with Rosetta produced valid structures. We used
-                                    the best structure, candidate S_14771, for extensive characterization.
+                the best structure, candidate S_14771, for extensive characterization.
-                                    We evaluated the model with regard to its secondary structure using Ramachandran
+                We evaluated the model with regard to its secondary structure using Ramachandran
-                                    plots. The Ramachandran plot suggested plausible secondary structures.
+                plots. The Ramachandran plot suggested plausible secondary structures.
-                                </p>
+            </p>
-                                <p>
+            <p>
-                                    Molecular Dynamics simulations were used to investigate stability and dynamic
+                Molecular Dynamics simulations were used to investigate stability and dynamic
-                                    properties of the candidate.
+                properties of the candidate.
-                                    The RMSD and radius of gyration stabilized over the course of the simulation, a
+                The RMSD and radius of gyration stabilized over the course of the simulation, a
-                                    first indicator of an equilibrated structure.
+                first indicator of an equilibrated structure.
-                                    Interestingly, RMSF analysis showed strond fluctuations of residues 105 to 115.
+                Interestingly, RMSF analysis showed strond fluctuations of residues 105 to 115.
-                                    We further investigated this by performing
+                We further investigated this by performing
-                                    Principle Component Analysis. Doing so, we extracted the principle movements of
+                Principle Component Analysis. Doing so, we extracted the principle movements of
-                                    the model. We could observe movement
+                the model. We could observe movement
-                                    of the &beta;6&#47;&beta;7 loop towards the active site, suggesing the presence
+                of the &beta;6&#47;&beta;7 loop towards the active site, suggesing the presence
-                                    of a binding site.
+                of a binding site.
-                                    Consequently, we performed docking simulations.
+                Consequently, we performed docking simulations.
-                                </p>
+            </p>
-                                <p>
+            <p>
-                                    FlexPepDock was used to conduct the docking simulations with target peptides.
+                FlexPepDock was used to conduct the docking simulations with target peptides.
-                                    Each run yielded 50,000 structures.
+                Each run yielded 50,000 structures.
-                                    In multiple steps we reduced the amount of complexes to 100 clusters with
+                In multiple steps we reduced the amount of complexes to 100 clusters with
-                                    respect to total, reweighted and interface score.
+                respect to total, reweighted and interface score.
-                                    We extracted the best scoring complexes and investigated interactions.
+                We extracted the best scoring complexes and investigated interactions.
-                                </p>
+            </p>
-                                <p>
+            <p>
-                                    For LPETGG we observed a uniform binding to the active site, fullfilling our
+                For LPETGG we observed a uniform binding to the active site, fullfilling our
-                                    expectation.
+                expectation.
-                                    Strikingly, polyG appeared to bind to the &beta;6&#47;&beta;7 loop in a uniform
+                Strikingly, polyG appeared to bind to the &beta;6&#47;&beta;7 loop in a uniform
-                                    manner.
+                manner.
-                                    As it is know from literature polyG is a functioning ligand of sortase.
+                As it is know from literature polyG is a functioning ligand of sortase.
-                                    Supported by literature and our data, <b>we postulate the following
+                Supported by literature and our data, <b>we postulate the following
-                                        mechanism</b>:
+                    mechanism</b>:
-                                    the &beta;6&#47;&beta;7 loop transports bound polyG towards the active site of
+                the &beta;6&#47;&beta;7 loop transports bound polyG towards the active site of
-                                    Sortase A7M, thereby lowering the activation energy of the linking reaction.
+                Sortase A7M, thereby lowering the activation energy of the linking reaction.
-                                </p>
+            </p>
-                                <p>
-                                    As the theory is neither backed up by nor contradicts experimental data, further
-                                    research is required.
-                                </p>
+            <p>
+                As the theory is neither backed up by nor contradicts experimental data, further
+                research is required.
+            </p>
+            <h2>References</h2>
+            <p>For our references, please have a look at our wiki.</p>
 </html>
@@ Line 1,746: / Line 1,674: @@
 <!-- -->
-<span class='h3bb'>Sequence and Features</span>
+<h2><span class='h3bb'>Sequence and Features</span></h2>
 <partinfo>BBa_K3187028 SequenceAndFeatures</partinfo>

Difference between revisions of "Part:BBa K3187028"

Revision as of 18:26, 21 October 2019

Profile

Structure

Usage and Biology

Transpeptidase: Sortase

Reaction

Sortase variants

Sortase A7M

Methods

Cloning

Expression and purification

SDS-Page

Flourescence Resonance Energy Transfer (FRET)

Mass Spectrometry

Enzyme-linked Immunosorbent Assay (ELISA)

Results

Characterization of Sortase A7M (and comparison to BBa_K3187016)

How do we measure if our purified sortases are active?

How do we measure sortase reaction kinetics

Development of a new FRET pair

Why are enzyme-substrate ratio and duration important parameters of the sortase reaction?

Who wins - Sortase A7M or Sortase A5M

What about other substrates?

Primary Amines

Yield

Is Sortase A7M able to attach cargo to P22 coat protein?

Does methionine affect Sortase linking?

Modeling

Introduction

Structure determination

RosettaCM

Results

Conclusion

Molecular dynamics

Results

Principle component analysis

Conclusion

Docking

Results

Conclusion

Modeling Conclusion

References

Sequence and Features