Difference between revisions of "Part:BBa K4905006"

Revision as of 13:02, 11 October 2023

Elastin-Like Polypeptide Triblock with Leucine Zippers <head>

</header>

Sequence and Features

Molecular weight: 105.1 kDa

Codon optimized for E.coli BL21 cells.

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal EcoRI site found at 2023
Illegal EcoRI site found at 3949
Illegal XbaI site found at 140
Illegal XbaI site found at 2066
12
INCOMPATIBLE WITH RFC[12]
Illegal EcoRI site found at 2023
Illegal EcoRI site found at 3949
Illegal NheI site found at 4077
21
INCOMPATIBLE WITH RFC[21]
Illegal EcoRI site found at 2023
Illegal EcoRI site found at 3949
Illegal XhoI site found at 2040
Illegal XhoI site found at 3966
23
INCOMPATIBLE WITH RFC[23]
Illegal EcoRI site found at 2023
Illegal EcoRI site found at 3949
Illegal XbaI site found at 140
Illegal XbaI site found at 2066
25
INCOMPATIBLE WITH RFC[25]
Illegal EcoRI site found at 2023
Illegal EcoRI site found at 3949
Illegal XbaI site found at 140
Illegal XbaI site found at 2066
Illegal NgoMIV site found at 197
Illegal NgoMIV site found at 377
Illegal NgoMIV site found at 467
Illegal NgoMIV site found at 647
Illegal NgoMIV site found at 2123
Illegal NgoMIV site found at 2303
Illegal NgoMIV site found at 2393
1000
COMPATIBLE WITH RFC[1000]

Information

This part is made up of the basic parts: Leucine zipper Z1 (BBa_K4905004), Leucine zipper Z2 (BBa_K4905005), and two times Elastin-Like Polypeptide (ELP) sequence A_[60]I_[60] (BBa_K4905001]). This results in the sequence Z1-I_[60]-A_[120]-I_[60]-Z2. With A_[5] the sequence (VPGAG_[3]VPGGG_[2]).

Figure 1: Video with one of the MD simulations at 25°C. The red residues represent the Leucine zipper, the blue residues are the hydrophobic part, and the light-blue residues are the hydrophilic part.

This construct was used by the TU Eindhoven 2023 team to form a hydrogel extracellular as well as intracellular in E.coli BL21 cells. The goal of this was to stop bacteria from dividing, however many more applications can be thought of for this part. Inspiration for this part was taken from Gradišar et al.^[1] where they used Leucine zippers form heterodimers. We wanted to use these zippers in a similar way that Fernández‐Colino et al.^[2] used leucine zippers together with Elastin-Like Polypeptides (ELPs) to form a reversible, injectable hydrogel. Furthermore, we noticed that Contreras-Llano et al.^[3] were able to form a hydrogel inside of cells, however not with a protein based approach. We wanted to combine these ideas and form a protein based-hydrogel inside of E.coli and use these bacteria for therapeutic applications. However, with the hydrogel itself, many more applications can be thought of that we wanted to explore as much as possible possible so that other teams can make use of it in the future too. Below you can read more about the experiments we did to characterize this part and about the molecular dynamics simulations that we did.

A schematic overview of what the part looks like is shown in figure 2. It is also easy to ligate other pieces of sequences together to make different constructs, with either different lengths of ELPs, different amino acids or other gBlocks at their end.

Figure 2 | Schematic overview of the sequence of this construct. (VPGAG_[3]VPGGG_[2]) is from now on referred to as A_[5] and VPGIG will be referred to as I. This created the final name Z1-I_[60]A_[120]I_[60]

General applications

ELPs are protein polymers derived from human tropoelastin. One of their key features is that they exhibit a phase separation that is often reversible whereby samples remain soluble below their transition temperature (T_t) but form coacervates above T_t^[4]. They have many possible applications in purification, sensing, activation, and nano assembly. Furthermore, they are non-immunogenic, substrates for proteolytic biodegradation, and can be decorated with pharmacologically active peptides, proteins, and small molecules. Recombinant synthesis additionally allows precise control over ELP architecture and molecular weight, resulting in protein polymers with uniform physicochemical properties suited to the design of multifunctional biologics. As such, ELPs have been employed for various uses including as anti-cancer agents, ocular drug delivery vehicles, and protein trafficking modulators^[4].

Currently ELPs are also being explored in the formation of hydrogels and tissue engineering applications. We are the first iGEM team to use ELPs for the purpose of formation of a hydrogel and have made many different parts while investigating the possibilities. This part turned out to be the best. The reversible behaviour of ELPs is even suitable for injectable hydrogels for example^[2]. We think this is a part that can have many applications inside as well as outside of cells and we hope that many other iGEM teams will make use of it in the future.

Construct design

Thean construct consists of ELPs and two different leucine zippers that have affinity for each other. In general, ELPs have hydrophilic and hydrophobic domains that exhibit reversible phase separation behavior that is temperature-dependent. They are made from a repeating VPGXG sequence, with X replaced by specific amino acids. This results in specific properties of the ELPs, especially related to the T_t at which the ELPs will interact with each other on the hydrophobic sites^[2]. When the temperature is below T_t, the water molecules surrounding the hydrophobic parts will go into the bulk water phase which gains the solvent entropy. This makes it possible to form interactions with other ELP molecules^[3].

As shown in figure 3, this construct has a hydrophilic region in the middle (A_[120]) and a hydrophobic region on each side of it (I_[60]). At the ends the leucine zippers Z1 and Z2 are located for stronger interactions between the ELPs. Leucine zippers consist of a repeating unit that forms an alpha helix. Two leucine zippers together form ion pairs between the helices, which causes association^[4]. These stronger and reversible interactions make them useful in the formation of a hydrogel at a specific T_t. In the end, the hydrogel is formed with electrostatic and hydrophobic interactions between the ELPs.

**Figure 3 |** Schematic representation of the composite part, an ELP with leucine zippers on the ends. It has a triblock ELP sequence that can form a thermoresponsive hydrogel.

The goal of our project specifically was to use this part to stop the cell from dividing and to co-express a therapeutic protein (Interleukin 10).

Another part that is often mentioned on this page is BBa_K4905008. This part was often used as a control. The ELP domains of the protein is the same as for the part on this page, however, part BBa_K4905008 has two Leucine zippers that are not complementary, which means that the interactions between the proteins will be weaker and this way, it could be studied what the effect of only the ELP triblocks was. This control part will be reffered to as Z2-A120-Z2 on the rest of this page. Lastly, another protein that can be seen a lot on this page is abbreviated as A120, which is the same sequence as the part on this page, but without the Leucine zippers. So it is part BBa_K4905001 ligated to itself. This protein also serves as a control group in many experiments.

Results

Protein expression and purification

This part was expressed in BL21 E.coli. The full plasmid that the DNA was cloned into can be seen below in figure 4. To check its expression we ran an SDS-PAGE gel of which the results can be seen in figure 5. The bands on the gel showed up where we expected, so this means that protein expression was succesfull.

**Figure 4 |** Full plasmid containing the sequence for this part in a pET24(+) vector that was transformed into *E.coli* BL21 cells for protein expression.

**Figure 5 |** SDS page gel run with this part. Samples were taken after 12, 20 and 36 hours of inducing protein expression. The band can be seen at the expected weight of 105.1 kDa.

Characterization

Hydrogel formation

First, we wanted to find out if we could use our part to form a hydrogel. All gels were formed by resuspending the ELPs in MilliQ (MQ) at different weight/volume%(w/v%). The leucine zipper-containing ELPs were expected to instantaneously form a gel upon increasing the temperature above T_t.

We saw that for both Z1-A120-Z2 and Z2-A120-Z2, a viscous fluid formed upon dissolving the ELPs in MQ water at 4°C. Subsequently, after warming the solution to room temperature, the liquid became very turbid and a gel started to form. Images of the gels can be seen in figure 6.

Figure 6 | Hydrogel solutions at 5 and 10 (w/v%). It can be seen that their solubility changes when the transition temperature is surpassed. Above this temperature a hydrogel is formed and the substance in the tube becomes cloudy and gel-like (w/v%).

The construct containing Z1-A120-Z2 formed a gel at 5% and 10% (w/v%) as described above. Upon cooling the gel down to 4°C again, the gel remained intact. As a control, Z2-A120-Z2 ( BBa_K4905008) was also dissolved to form solutions of 5% and 10% (w/v%). This construct too, was able to form a gel instantly upon heating to RT. However, in contrast to the leucine zipper-ELPs, the gel disassembled again when the solution was cooled to 4°C. This indicates that the latter of the two constructs shows reversibility, which is expected when the ELPs are brought below their T_t, while the former construct does not show this reversibility.

Transition temperature determination with UV-Vis

The transition temperatures (T_ts) Z1-A_[120]-Z2 and control Z2-A_[120]-Z2 were determined with the use of UV-visible spectroscopy. Solutions of 5 and 20 μM were made in MQ water. Absorbances were measured at 350 nm. T_ts were determined to be 23.5°C and 21°C for both constructs, respectively (Figure 7).

When the control protein Z2-A_[120]-Z2 crosses the T_t at 21°C, aggregates form leading to a sharp increase in absorbance. The absorbance then drops, which might be explained by the non-complementary two zipper domains repelling each other, leading to disassembly of the aggregates. Upon further increasing the temperature, the hydrophobic interactions become stronger, which further increases the absorbance, until finally even the hydrophilic block becomes insoluble above 50°C.

Z1-A_[120]-Z2 shows a different temperature dependent behavior compared to Z2-A_[120]-Z2, in that it shows a smaller increase in absorbance when it crosses T_t of 23.5°C. We think the interaction between Z1 and Z2 stabilizes the aggregates, forming into a branched network of ELPs, even at low concentrations of 5 μM.

Figure 7 | UV-Vis spectra for constructs Z1-A120-Z2 and Z2-A120-Z2 measured at 350 nm. The solution was heated at a rate of 0.5 degrees Celcius per minute. It can be seen that the transition temperature lays at 23.5 and 21 degrees Celcius for the two proteins respectively.

Absorbances were also measured upon cooling the solution back down. Once the solution goes below T_t, the absorbances of returned back to base levels, indicating that the ELPs can be reversibly precipitated and resuspended again (Figure 8) at these low concentrations.

Figure 8 | UV-Vis spectra for constructs Z1-A120-Z2 and Z2-A120-Z2 measured at 350 nm. The solution was cooled at a rate of 2 degrees celcius per minute. It can be seen that the transition temperature lays around 23.5 and 21 degrees Celcius for the two proteins respectively.

Dye release from hydrogel

To determine the relative ability for the diffusion of small molecules through our hydrogels, we made gels at two different weight-to-volume percentages containing the fluorescent dye rhodamine B. Finding out whether small molecules can gradually diffuse out of the hydrogel might be interesting for future applications, a drug could by incorporated for instance.

First, a calibration curve was made to determine at which concentration rhodamine B should be added to the gel. Figure 9 shows a linear relationship up to 26 μM of Rhodamine B. We decided to make the gels with a Rhodamine B concentration of 104 μM, since the amount of dye diffusing out of the gels was expected to be far lower than this amount. A 5% gel as well as a 10% gel were tested. Our hypothesis was that diffusion through the 5% gel would be faster than when a higher weight percentage of the ELPs was used,

Figure 9: Left: Calibration curve for Rhodamine B, a linear relationship can be seen up to 26 μM of Rhodamine B, with equation: y = 2*106x + 963.16 (R² = 0.993) Right: The hydrogels made with Rhodamine B at a concentration of 104 μM.

The gels were made at a volume of 100 μL. They were then brought to RT and washed with 1.5 mL of warm MQ. Then, 1 mL of warm MQ was pipetted on top of the gels, and they were incubated for up to 8 hours at RT, with samples of 100 μL being taken every 2 hours. After measuring the fluorescence intensity, the values were corrected for the reduction in volume upon taking the samples. From figure 10, it becomes clear that the dye diffuses faster from the 5% gel than from the 10% gel. However, more experiments are needed to truly find the rate of diffusion, it is hard to draw a conclusion now with only few data points. The faster diffusion rate of the 5% gel is in line with our hypothesis that a lower weight percentage gel is able to release small molecules faster than a 10% gel.

Figure 10 | Fluorescence intensity measured from the supernatant on top of the gel over a period of time.

CCK8

We wanted to find a way that we could measure the amount of cells that we can stop from dividing with our hydrogel and whether they would stay alive. We came across Cell counting kit 8 (CCK8). This is an assay that is commonly used to determine the number of alive cells in a sample. The kit uses a highly water-soluble tetrazolium salt, WST-8, which produces a formazan dye upon reduction in the presence of an electron mediator. The amount of formazan generated by dehydrogenases is directly in proportion to the number of living cells. Yang et al. (2021) have demonstrated that this assay is also suitable for the detection of living E. coli^[5].

We used this assay to test whether our hydrogelated cells would be more resistant to several conditions, including incubation with ampicillin and freeze-drying. Our biggest goal was to find out if cells would stop dividing, but stay functional with our hydrogel. To test this, we came up with the idea of combining the CCK8 assay described above with the antibiotic ampicillin. Ampicillin prevents the synthesis of the bacterium’s cell wall. It does this by binding to enzymes necessary for the formation of the cell wall ^[6]. This means it only kills bacteria when they divide. Our hypothesis is that if our hydrogel prevents the bacteria from dividing, they should be affected less by the presence of this antibiotic, and therefore show a higher survival rate than bacteria that do not contain the hydrogel since these will still divide and be killed by the antibiotic. A schematic representation of the experiment can be seen below in figure 11.

Figure 11: A schematic representation of the CCK8 assay protocol. Samples were taken and incubated with or without ampicillin. After 1 hour, the cell pellets were collected and resuspended in PBS. Then the samples were incubated again with the substrate WST-8. The amount of produced fluorescent product that is measured via the _OD450 with a plate reader is directly proportional to the amount of cells in the sample.

For this assay, it was nessecary to measure the OD₄₅₀ with a plate reader. This can directly be related to the OD₆₀₀^[5]. For this relationship, the calibration curve in figure 12 was made.

Figure 12: Calibration curve relating the OD₆₀₀ to the OD₄₅₀.

Ampicillin resistance

Three different conditions were tested over a period of time. Namely; no (protein) expression, ELPs with no cross linkers (A120), and ELPs with Leucine zippers (Z1-A120-Z2). Samples were taken at 12h, 20h, and 36h of protein expression. Before incubation ampicillin, samples were diluted to at a baseline of OD₆₀₀ = 0.5. They were then either incubated with ampicillin (1 mg/mL) or without for 1h at 37°C. The results are summarized in Table 1 and Figure 13.

WST-8 conversion Amp/No amp	No expression	A120	Z1-A120-Z2
12h	0.71 ± 0.05	1.02 ± 1.00	3.05 ± 0.33
20h	0.60 ± 0.21	5.81 ± 0.22	20.85 ± 4.14
36h	0.28 ± 0.08	6.79 ± 0.17	17.05 ± 2.13

Table 1: Measured WST-8 conversion for samples with no expression, A120 and Z1-A120-Z2 at different timepoints.

Ampicillin-treated E. coli that were not induced to express proteins, and which were treated with ampicillin, showed at most a WST-8 conversion of 0.71% ± 0.05% compared to cells which were not treated, as measured by absorbance at 450 nm. In contrast, E. coli where expression of A120 was induced, showed a maximum conversion of 6.80% ± 0.17% after 36h of protein expression. This is likely due to the fact that this construct is also able to form a gel, although less strong compared to the Z1-A120-Z2 construct, leading to an attenuation in cell division. Finally, Z1-A120-Z2 was expressed for the three-time points. An increase was observed in the conversion of substrate, most notably after 20h of expression (20.86% ± 4.14%). We had expected to see a higher percentage after 36h of expression, since at this time point, more bacteria should be gelated and thus unable to divide. The standard deviation of the samples is quite high, with this small number of measurements and large deviation, it is hard to tell whether there is really a difference between these two time points. Overall, a larger sample size could help to reduce the variability of this assay in future tests. Other explanations could be that some of the cells were over-gelated, which might have led to a premature death unrelated to the incubation with ampicillin. Additionally, cell death could also have occurred due to nutrients in the culturing medium running out.

For comparison, OD₆₀₀ of all samples was also measured after incubation with and without ampicillin. When the samples were incubated with ampicillin, they all decreased in OD₆₀₀. The samples incubated without ampicillin in which ELPs were expressed show a relatively stable OD₆₀₀ compared to the sample in which no expression was induced. This was the only sample in which a substantial increase in OD₆₀₀ could be observed.

Figure 13 | Left) WST-8 conversion of bacteria treated with ampicillin versus untreated bacteria. right) corresponding OD₆₀₀ values. Measurements were done at different timepoints (12h, 20h, and 36h). It can be seen that when the samples were incubated with ampicillin, they all decreased in OD₆₀₀. The samples incubated without ampicillin in which ELPs were expressed show a relatively stable OD₆₀₀ compared to the sample in which no expression was induced.

The OD₆₀₀ was also measured without addition of ampicillin over time for each of the three samples (No expression, A120 and Z1-A120-Z2). After 20h of expression, these were diluted to OD = 0.1 and then measured every 30 minutes. From figure 14, it becomes clear that when no expression of ELPs was induced, the bacteria divided more rapidly than when expression was induced. These findings are in line with the results of the ampicillin treatment. It seems that the presence of the ELPs does not altogether inhibit division, but it does slow it down, which seems to be the reason why they are killed slower upon the addition of ampicillin. However, it is hard to draw a conclusion as to whether the attenuation of bacterial division is driven by the formation of a gel inside the cytosol, or simply because of the crowded environment inside the cell, caused by the presence of a high amount of protein. Therefore, additional controls need to be added in future experiments, like samples in which a mono-or diblock ELP is expressed, in order to account for molecular crowding. Furthermore, a sample where a completely different protein is also interesting, to see what the effect of protein expression is on cell division.

Figure 14 | measurements of the three samples over a period of time. Protein expression in two of the three samples was induced for 20h, after which the samples were diluted to OD = 0.1 and measured every 30 min. Expression of gel-forming ELPs greatly reduces the rate of replication compared to cells without expression.

These results were compared to the classic colony counting method. From each sample, a diltion of 10⁵ was made and then plated out on an LB-agar plate. The plates were incubated at 37°C overnight. Images of the plates were taken (Figure 15) and the number of colonies was counted using the colony counter tool from ImageJ. The results of which are summarized in Figure 15 As expected, the wild-type E. coli treated with ampicillin did not form any colonies, whereas untreated bacteria formed several hundreds of colonies. Samples containing A120 and Z1-A120-Z2 both showed a significantly smaller amount of colonies.

Figure 15 | Photos of all LB-agar plates after incubation at 37°C overnight, including a a graph and table in which the amount of colonies is summarized. It can be seen that the bacteria which do not contain the ELPs form a greater amount of colonies than when expression of these proteins was induced.

	No expression		A120		Z1-A120-Z2
	Amp	No Amp	Amp	No Amp	Amp	No Amp
12h	0	614	0	4	2	16
20h	0	597	0	166	21	242
36h	0	1610	35	169	142	297

Table 2 | Table with the amount of colonies on each plate

Microscopy (FRAP)

Characterization of the ability of our part to form a gell inside the cell with the use of microscopy required us to design a model protein which could interact with the hydrogel, and which we could visualize under the microscope. We decided to use VPGIG_[60]-mNeonGreen (part BBa_K4905016), since the hydrophobic ELP part of the protein is able to make hydrophobic interactions with the gel. Because of these interactions, we expected that the proteins would al localize in the same places in the cell. We cloned the gene fragment into a pBad vector under the control of an arabinose promotor, to allow for orthogonality between this vector and the pET24a(+) vector encoding the zipper-ELP construct. Success of cloning was verified by a double digestion of the plasmids as well as sequencing.

However, to our surprise, the sequencing results indicated that instead of a VGPIG_[60] fragment, the mNeonGreen was attached to a VPGAG_[3]VPGGG_[2][12] fragment, which is hydrophilic, relative to the VPGIG_[60] fragment. This is likely due to a labeling mistake by a previous user of the plasmid. Due to a lack of time, we decided to continue using this construct in our microscopy experiments as a model protein to monitor the diffusion of proteins within the gelated bacteria.

Figure 15 shows two samples before and after photobleaching. Following bleach of E.coli which co-expressed Z1-A120-Z2 and and [A3G2]₁₂-mNeonGreen, a gradual recovery of fluorescence could be observed up to 30 seconds post-bleach (Fig. 15A, Fig. 15B). The control, containing only [A3G2]₁₂-mNeonGreen, showed a virtually instantaneous recovery, as evidenced by Fig 16B, Fig 16C.

Figure 16: A) sample containing Z1-A120-Z2 and [A3G2]₁₂-mNeonGreen. B) Control containing only [A3G2]₁₂-mNeonGreen. Both samples were first measured at three timepoints (1s each) pre-bleach, then bleached for 5 seconds and finally measured for another 30 timepoints (1s each). The ROI that was bleached is indicated with the red circles.

The raw data was corrected for photobleaching during post-bleach image acquisition and normalized to the intensity pre-bleach. The data was then fitted with equation 1, Where I(t) stands for the normalized fluorescence intensity at time t, A is the mobile fraction, t is time and τ is the time constant^[7]. This was done for each of the measurements, resulting in four graphs for the co-expressed sample and two graphs for the control. Using the equations, the recovery half-life (τ_1/2) was calculated, using t_1/2 = ln(2)*τ for each data set which is summarized in Figure 15. This experiment shows that proteins are distributed throughoit the whole cell and that the diffusion rate becomes lower when our ELP constructs are expressed. It is impossible to exactly determine the intracellular concentration of ELPs, and thus to make a comparison with the gels made extracellularly. However, it would be worthwhile doing these FRAP measurements on more samples in the future where, just as was done for the CCK8 assays, protein expression is induced for a series of different timepoints to see the effect on diffusion.

I(t) = A(1-e^(-t/τ))+c (1)

Measurement	τ_1/2 (s)
1	11.9
2	7.1
3	10.9
4	8.4
Average + stdev	9.6 ± 1.9

Figure 17: A) table with the average recovery half-lives. B) Fluorescence recovery of samples induced to express Z1-A120-Z2 and [A3G2]₁₂-mNeonGreen, nornalized to the pre-bleach intensity. All samples show a gradual recovery post-bleach with a mean recovery half-life of 9.6 ± 1.9 s. C) Fluorescence recovery of bacteria induced to express only [A3G2]₁₂-mNeonGreen, normalized to the pre-bleach intensity. The was almost instantaneous.

To further characterize the gelated versus non-gelated bacteria, we used a DNA staining, Hoechst. We saw that a large number of gelated bacteria showed a clustering of DNA in the center of the cell, whereas for the control sample, the DNA was more homogenously spread throughout the cell. This might indicate that the DNA in gelated cells is not as accessible to replication machinery and that therefore division of the cells is stopped or slowed down.

Figure 18: A) bacteria gelated with Z1-A120-Z2 stained with Hoechst. The DNA is densely clustered in the center of the cells. B) non-gelated cells stained with Hoechst. The DNA is homogenously spread throughout the cell.

Molecular Dynamics simulation

Introduction

Molecular dynamics (MD) simulations are commonly used to see how proteins or other molecules move over time. This can contribute to the understanding of molecular systems, which cannot be seen with the eye or even with the best microscopes. In our application of forming a hydrogel with Elastin-Like Polypeptides (ELPs), it can be useful to see how the ELPs will behave at different temperatures. Above a certain transition temperature (T_t), there is a disruption in the thermal energy of the water molecules around the ELPs, which causes the water molecules to move away from the ELPs. This makes room for interactions between the ELPs. It has been found that there will be conformational changes in another ELP sequence when the temperature rises above T_t, which makes them more ordered and increases the hydrophobicity^[8].

It is interesting to see how this applies to our own ELP. The MD simulation will give a better understanding of the differences in the structure below and above the T_t and it will help with understanding how the ELP actually looks like. We will analyze different properties of the proteins, like the secondary structure formation, compactness, and the surface area that is accessible for the solvent, to see how the conformation differs at specific temperatures below and above the T_t.

Methods

MD simulations are typically computationally expensive. This made it necessary to shorten our ELP sequence to make the computational time manageable. We chose to shorten the hydrophobic parts to I_[9] instead of I_[60] and to shorten the hydrophilic part to A_[50] instead of A_[120] or A_[100], as shown in Figure 19. The leucine zippers stay the same. In this length reduction, the proportions are not the same as in the original ELP. However, we wanted to make sure that the hydrophobic parts were not shorter than the leucine zippers. This maintains a greater distance between the Leucine zippers and the hydrophilic part, and it prevents unrealistic folding. The hydrophilic part is kept long since this part gives the ELP its length and it might be interesting to see how this part behaves during the folding. The choices made for this might affect the outcome, but the combination of regions is still the same and the difference between the temperatures can still be compared.

Figure 19: ELP construct used for the MD simulation. In this construct, the hydrophobic VPGIG parts are repeated 10 times instead of 60 times and the hydrophilic part in the middle is repeated 10 times instead of 24 or 20 times. At the ends, the two different leucine zippers are located, which remain the same.

A generalized born implicit solvent is used to fold the protein. In an implicit solvent, the solvent (in this case water) molecules are not individually modeled, but with an approximation of the mean force from the solvent on the protein. Different theories can be used for this, and we chose to use the generalized born model. Implicit solvents are faster than an explicit solvent with simulated water molecules and more detailed than a simulation in vacuum^[10]. The MD simulation is performed at temperatures 3 °C, 15 °C, 25 °C, and 37 °C, since the transition temperature was found to be 20 °C in the conducted UV-VIS experiments. The simulations are run in triplet to reduce the chance of coincidence in the results. This is necessary, since the initial velocities are chosen by the NAMD software and thus differ every time. Different velocities can cause differences in the final stable conformations, which can affect the results of the analytical methods.

Just like in López Barreiro et al., first energy minimalization is done for 20 000 time steps. Then, Langevin dynamics is used with Generalized Born implicit solvent. The simulation step was 2 fs with a simulation time of 50 ns. The cutoff distance is 18 Å, the switch distance 16 Å^[11].

Analysis methods

The results are analyzed with Root Mean Square Distance (RMSD), Radius of Gyration and relative shape anisotropy, Ratio of Secondary Structures, and Solvent Accessible Surface Area (SASA) to show the different behaviors of the ELP at different temperatures. They are calculated with the MDTraj Python library. For the analysis, the analysis methods are calculated for the 1000 frames of the last 5 ns, which has a stable conformation at all temperatures. Even when the conformation is stable, there are still some small differences. These differences are taken into account with this distribution of the results of the last 5 ns.

Results

In Figure 20, a video is shown from one of the simulations at 25°C. Not all videos are included, since the process is comparable for the other simulations, especially when comparing them by eye. Some frames of those simulations can be found in the file below. When comparing the simulations at the four temperatures, they all start with folding at the ends of the ELP. This was expected since those parts are the most hydrophobic. Some of the simulations also show the folding of the ELP in the middle at the more hydrophilic part. However, there is no clear relationship between temperature and this folding behavior, so it is probably caused by luck in the simulation.

Figure 20: Video with one of the MD simulations at 25°C. The red residues represent the Leucine zipper, the blue residues are the hydrophobic part, and the light-blue residues are the hydrophilic part.

RMSD

RMSD plots are made for each of the three simulations at the four temperatures. They are made for the complete 50 ns, but also for the last 5 ns to show how stable the conformation is related to frame 9000 (45 ns). This is important since there can still be a lot of changes in the structure while the moved distance of the atoms stays the same. Since there are a lot of figures made, they are not visualized here. However, they can be found on the wiki of the TU Eindhoven 2023 team.

For all of the measurements, the equilibrium seems to be reached. When looking at the plots of the last 5 ns, most RMSD plots have a variability of a maximum of 1 nm. Only the third simulation at 37 °C has a slightly larger difference. An RMSD of 0 describes a stable conformation, but there are always some small deviations. So it can be said that an RMSD of 0.25 nm has a high stability, but also 0.2-0.5 nm will give a high stability^[12]. With this information and when looking at the course of the plots, it can still be concluded that the conformations are relatively stable since the RMSD is still very small.

Secondary structure

In Figures 21a, b, and c, the boxplots for the secondary structure analysis of the individual simulations are shown. This shows some variability between the simulations of a single temperature, but also overlapping deviations. This happens especially for the α-helix and β-sheet plots. The simulation results are taken together for each temperature to make them easier to analyze, which is shown in Figure 21d, e, and f.

Figure 21: Secondary structure analysis with a, b, and c for respectively the coils, α-helices, and β-sheets three individual simulations for each of the four temperatures and d, e, and f the overall results with the different simulations per temperature taken together.

Overall, almost no secondary structures seem to be formed. The rate of the coils is much higher than the rates for alpha-helices and beta-sheets. It is possible that more secondary structures make the ELP more stable, but that it is stuck in a local minimum. This can be proved with more simulations and some other simulation techniques.

When comparing the boxplots of the different temperatures, there does not seem to be a convincing difference in the secondary structure formation. There are some small differences in the medians and distributions, but there is no clear relation between those differences. We can thus conclude that there is no difference in the secondary structure between the temperatures.

Radius of gyration

The radius of gyration results of the individual simulations are shown in Figure 22a. The individual simulation results of 3 and 37°C show large differences in the results. This can be caused by the different velocities that are chosen for the single simulations. Since the individual simulations of 3 and 37°C differ a lot, the total boxplot in Figure 22b shows a large distribution of the results.

Figure 22: Radius of gyration results with a three individual simulations for each of the four temperatures and b the overall results with the different simulations per temperature taken together.

There is a clear difference between the radius of gyration at 15 °C and 25 °C, which are just below and just above the transition temperature. When looking at the median at 37 °C, also these results show a lower radius of gyration than 15 °C. At 3 °C, the radius of gyration is for two of the simulations very low compared to the other temperatures. When looking at the final conformations of the simulations, this can be caused by the shape of the stable configurations. When the shape is more cylindrical instead of spherical, which seems especially the case for the simulations at 15°C, the radius of gyration will increase. As mentioned in the methods section, we also computed the relative shape anisotropy.

Figure 23: Relative shape anisotropy results with a three individual simulations for each of the four temperatures and b the overall results with the different simulations per temperature taken together.

In Figure 23a, the relative shape anisotropy is shown for the individual simulations. There is clearly a difference in the shape between the different simulations, also in those with the same temperature. For example 25°C, which has a clear variability between the simulations. Some of the differences were already expected after the analysis of the radius of gyration. The second simulation at 3°C is clearly less spherical than the other two simulations at that temperature. Overall, when looking at Figure 23b, the shape of the ELP at 15°C is less spherical than at 25°C. Because of the large variabilities between the measurements at 3 and 37°C, it cannot be concluded how this is affected at lower and higher temperatures.

This analysis suggests that there is a difference in the compactness of the ELP below and above the T_t. Below the T_t, the ELP is less compact. At a temperature above the T_t, the ELP will become more hydrophobic with fewer solvent molecules around it. This makes the folded ELP more compact, but also more likely to induce interactions with other ELPs. However, for harder conclusions with the role of solvent molecules included, explicit simulations are needed.

SASA

In Figure 24a, the individual results show some deviations between the simulations at specific temperatures, especially at 3 and 37 °C. When taking the individual simulations together for each temperature in Figure 24b, some small deviations are shown. SASA at 25 °C seems to be lower than SASA at 15 °C, while the median SASA at 3 °C is also lower than the rest of the temperatures, but with a large deviation. The results for 37 °C also have a large deviation, with a median higher than the median of 15 °C. When comparing the results with the relative shape anisotropy, there is a relationship between more spherical simulation results and a lower SASA. There seems to be a smaller difference than the difference found in the radius of gyration and the anisotropy, which might be because ends that stick out probably have more effect on SASA than the relative shape anisotropy.

Figure 24: SASA results with a three individual simulations for each of the four temperatures and b the overall results with the different simulations per temperature taken together.

Conclusion

The MD simulation of the folding of a shortened version of our ELP taught us that an increase in temperature above the T_t results in a more compact and more spherical ELP due to increased hydrophobicity. This causes more and stronger interactions between the ELPs, which is an important driver of the hydrogel formation^[2].

The greatest difference in results is found for the radius of gyration and relative shape anisotropy between 15 and 25°C. SASA also shows a difference, but this is less convincing. There is no difference found in the secondary structure between the different temperatures. When comparing the triplicate simulations, there are large differences for especially 3°C but also 37°C. This makes these results less reliable, but there still seems to be a difference in properties between 15 and 37°C. In further research, explicit solvent simulations can be used with more simulated temperatures. This would give more details for better and possibly more convincing results. Also, more extensive research can be done on the interactions between the ELPs at different temperatures and conditions.

References

[1] Gradišar, H., & Jerala, R. (2010). De novodesign of orthogonal peptide pairs forming parallel coiled-coil heterodimers. Journal of Peptide Science, 17(2), 100–106. https://doi.org/10.1002/psc.1331

[2] Fernández‐Colino, A., Arias, F. J., Alonso, M., & Rodríguez-Cabello, J. C. (2015). Amphiphilic Elastin-Like Block Co-Recombinamers Containing Leucine Zippers: Cooperative Interplay between Both Domains Results in Injectable and Stable Hydrogels. Biomacromolecules, 16(10), 3389–3398. https://doi.org/10.1021/acs.biomac.5b01103

[3] L. E. Contreras-Llano et al., “Engineering Cyborg Bacteria Through Intracellular Hydrogelation,” Adv. Sci., Mar. 2023, doi: 10.1002/ADVS.202204175.

[4] Alber, T. (1992). Structure of the leucine zipper. Current Opinion in Genetics and Development, 2, 205–210

[5] X. Yang, Y. Zhong, D. Wang, and Z. Lu, “A simple colorimetric method for viable bacteria detection based on cell counting Kit-8,” Anal. Methods, vol. 13, no. 43, pp. 5211–5215, Nov. 2021, doi: 10.1039/D1AY01624E

[6] E. L. Foltz, J. W. West, I. H. Breslow, and H. Wallick, “Clinical pharmacology of pivampicillin.,” Antimicrob. Agents Chemother., vol. 10, no. 3, pp. 442–454, 1970, doi: 10.47363/jprsr/2022(3)129.

[7] Poudyal, R. R., Guth-Metzler, R., Veenis, A. J., Frankel, E. A., Keating, C. D., & Bevilacqua, P. C. (2019). Template-directed RNA polymerization and enhanced ribozyme catalysis inside membraneless compartments formed by coacervates. Nature Communications, 10(1). https://doi.org/10.1038/s41467-019-08353-4

[8] N. K. Li, F. G. Quiroz, C. K. Hall, A. Chilkoti, and Y. G. Yingling, “Molecular description of the lcst behavior of an elastin-like polypeptide,” Biomacromolecules, vol. 15, no. 10, pp. 3522–3530, Oct. 2014, doi: 10.1021/BM500658W/SUPPL_FILE/BM500658W_SI_001.PDF.

[9] J. Kleinjung and F. Fraternali, “Design and application of implicit solvent models in biomolecular simulations,” Curr Opin Struct Biol, vol. 25, no. 100, p. 126, 2014, doi: 10.1016/J.SBI.2014.04.003.

[10]J. Iwata and T. Ando, “Molecular Dynamics Study on Behavior of Resist Molecules in UV-Nanoimprint Lithography Filling Process,” Nanomaterials, vol. 12, no. 15, Aug. 2022, doi: 10.3390/NANO12152554/S1.

[11] D. López Barreiro, A. Folch-Fortuny, I. Muntz, J. C. Thies, C. M. J. Sagt, and G. H. Koenderink, “Sequence Control of the Self-Assembly of Elastin-Like Polypeptides into Hydrogels with Bespoke Viscoelastic and Structural Properties,” Biomacromolecules, vol. 24, no. 1, pp. 489–501, Jan. 2023, doi: 10.1021/ACS.BIOMAC.2C01405/ASSET/IMAGES/LARGE/BM2C01405_0003.JPEG.

[12] M. Arnittali, A. N. Rissanou, and V. Harmandaris, “ScienceDirect Structure Of Biomolecules Through Molecular Dynamics Simulations,” 2019, doi: 10.1016/j.procs.2019.08.181.

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 <figcaption style="text-align:center; font-size: 12px;"><b>Figure 15 |</b> Photos of all LB-agar plates after incubation at 37°C overnight, including a a graph and table in which the amount of colonies is summarized. It can be seen that the bacteria which do not contain the ELPs form a greater amount of colonies than when expression of these proteins was induced.</figcaption><br>
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 <figcaption style="text-align:center; font-size: 12px;"><b>Table 2 |</b> Table with the amount of colonies on each plate</figcaption><br>
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 The raw data was corrected for photobleaching during post-bleach image acquisition and normalized to the intensity pre-bleach. The data was then fitted with equation 1, Where I(t) stands for the normalized fluorescence intensity at time t, A is the mobile fraction, t is time and τ is the time constant<sup>[7]</sup>. This was done for each of the measurements, resulting in four graphs for the co-expressed sample and two graphs for the control. Using the equations, the recovery half-life (τ<sub>1/2</sub>) was calculated, using t<sub>1/2</sub> = ln(2)*τ for each data set which is summarized in Figure 15. This experiment shows that proteins are distributed throughoit the whole cell and that the diffusion rate becomes lower when our ELP constructs are expressed. It is impossible to exactly determine the intracellular concentration of ELPs, and thus to make a comparison with the gels made extracellularly. However, it would be worthwhile doing these FRAP measurements on more samples in the future where, just as was done for the CCK8 assays, protein expression is induced for a series of different timepoints to see the effect on diffusion.
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