Part:BBa_K4905006

Elastin-Like Polypeptide Triblock with Leucine Zippers

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 the sequence (VPGAG(3)VPGGG(2)) and I the sequence (VPGIG). The numbers indicate the number of repeats of these sequences. This construct was used by the TU Eindhoven 2023 team to form a hydrogel outside as well as inside E.coli BL21 cells. A schematic overview of this is shown in figure 1.

Figure 1 | Schematic overview of the sequence of this construct. VPGAG(3)VPGGG(2) is from now on referred to as A and VPGIG is referred to as I.

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 Tt but form coacervates above Tt. 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 modulators³.

Construct design

The 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 transition temperature Tt at which the ELPs will interact with each other on the hydrophobic sites². When the temperature is below Tt, 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 molecules³.

As shown in figure 2, this construct has a hydrophilic region in the middle (A[120]) and a hydrophobic region on each side of it (I[60]). On 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 association¹. These stronger and reversible interactions make them useful in the formation of a hydrogel at a specific Tt. In the end, the hydrogel is formed with electrostatic and hydrophobic interactions between the ELPs.

Figure 2 | Schematic representation of the composite part, an ELP with leucine zippers on the ends

As soon as the hydrogel is made inside E.coli BL21 cells, the cells are prevented from dividing. However, the cells remain functional. So they can still be used to express therapeutic agents, like Interleukin 10 in the TU Eindhoven 2023 teams project.

Sequence and Features

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]

Results

Protein expression and purification

The protein has an expected molecular weight of 105.1 kDa Organic solvent extraction, ITC

SDS-page

Figure 3 | SDS page from this construct

Figure 4 | SDS page from this construct

13. Z1-I60-A120-I60-Z2
14. Z2-I60-A120-I60-Z2
15. Z2-I60-A100-I60-Z2

Characterization

Massspec

Hydrogel formation

To see if a hydrogel could form, 10 wt% ELP was dissolved in cold MQ and left at room temperature to warm up and form a gel. This gel could be dissolved again when put at 4 °C overnight.

Figure 5 | A ten percent hydrogel was formed inside of a mass spectrometry vial. ELP constructs were dissolved in MQ at 4 °C and left at room temperature as soon as they dissolved. Within minutes, a hydrogel started to form.

Transition temperature determination

To determine the transition temperature of the ELP constructs, different solutions of the proteins were made in PBS. Using a UV-VIS spectrometer, the absorbance of light at 350 and 600 nm was measured to find the temperature at which phase separation happens. First, the samples were heated up to find the temperature where the hydrogel forms. Later, the samples were cooled again to show their reversible behavior. Two transition temperatures can be seen, the first is where the hydrophobic parts aggregate and the hydrogel forms, the second transition temperature is where the hydrophilic blocks also collapse. Two different constructs are plotted together, Z1-I60-A120-I60-Z2 has complementary leucine zippers and Z2-I60-A120-I60-Z2 has two leucine zippers that are the same, so it acts as a control group. It can be seen that their transition temperatures differ. The transition temperature of Z2-I6-A120-I60-Z2 is about 17 °C and Z1-I60-A120-I60-Z2 has a transition temperature of around 20 °C. It can also be seen hat the Z1-I60-A120-I60-Z2 construct only exhibits one transition temperature, although two were expected, this might be because of the zippers already bringing the hydrophilic domains together which loses the second transition temperature.

Figure 6 | Temperature dependent behaviour of the Z1-I60-A120-I60-Z2 and Z2-I60-A120-I60-Z2 ELP constructs at a concentration of 5 uM measured at 350 nm. The temperature was varied with 0.5 °C per minute. The transition temperature of Z2-I60-A120-I60-Z2 is about 17 °C and Z1-A120-Z2 has a transition temperature of around 20 °C.

Figure 7 | Temperature dependent behaviour of the Z1-I60-A120-I60-Z2 and Z2-I60-A120-I60-Z2 ELP constructs at 20 uM and 5 uM concentrations measured at 600 nm. The temperature was varied with 0.5 °C per minute. The transition temperature of Z2-I60-A120-I60-Z2 is about 17 °C and Z1-I60-A120-I60-Z2 has a transition temperature of around 20 °C.

Figure 8 | Temperature dependent behaviour of the Z1-I60-A120-I60-Z2 and Z2-I60-A120-I60-Z2 ELP constructs at 20 uM and 5 uM concentrations measured at 350 nm. The temperature was varied with 2 °C per minute. The transition temperature of Z2-I60-A120-I60-Z2 is about 17 °C and Z1-A120-Z2 has a transition temperature of around 20 °C.

Figure 9 | Temperature dependent behaviour of the Z1-I60-A120-I60-Z2 and Z2-I60-A120-I60-Z2 ELP constructs at 20 uM and 5 uM concentrations measured at 600 nm. The temperature was varied with 2 °C per minute. The transition temperature of Z2-I60-A120-I60-Z2 is about 17 °C and Z1-I60-A120-I60-Z2 has a transition temperature of around 20 °C.

Dye release from hydrogel

Inhibition of bacterial growth

To follow the growth inhibition of the bacteria because of the hydrogel, a calorimetric assay was conducted with CCK-8. This type of assay can be used to detect the concentration of live bacteria in a sample and relies on the reaction between CCK-8 and dehydrogenase, which results in the formation of orange-yellow formazan. The concentration of live bacteria is proportional to the absorbance value of formazan measured at 450 nm. According to literature, the OD600 is proportional to the number of bacteria in the sample, and the relation between the OD600 and OD450 measured in samples containing CCK-8 has been shown to have a linear relationship. Based on this information, a standard curve was made that relates the OD600 to the OD450. This curve was used in further experiments to determine the number of live (and over-time thus dividing) bacteria in each sample.

Figure 10 | Standard curve relating the OD600 and OD450 of bacterial samples containing CCK-8. Imaging experiments.

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.

In our application of forming a hydrogel with Elastin-Like Polypeptides (ELPs), it can be useful to see how our ELP will behave at different temperatures. Above a certain transition temperature (Tt), there is a thermal disruption of the water molecules which makes interactions between the ELPs possible. In literature, it is found that there will be conformational changes in another ELP sequence when the temperature rises above Tt, which makes them more ordered and increases the hydrophobicity (Li et al., 2014).

It is interesting to see how this applies to our own ELP. This will help us to understand the mechanism behind the hydrogel formation and to see what it actually looks like. We will analyze different properties of the proteins to see how the conformation differs at specific temperatures below and above the Tt.

MD simulations are typically computational expensive. This made it necessary to shorten our ELP sequence to make the computational time tolerable. We chose to shorten the hydrophobic parts to I[10] instead of I[60] and to shorten the hydrophilic part to A[10] instead of A[120], as shown in figure 1. 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 and that the hydrophilic part was not too long for the computational expenses. The choices made for this might affect the outcome, but all parts of the ELP are still present and the same molecule is used in the different simulations that are compared. It is expected that it will especially affect the hydrophilic part, which might fold differently in the original ELP due to the reduction in length. However, this hydrophilic part is not very important in the interactions between the ELPs, so the results are expected to still represent the original ELP.

Methods

As the first step, a generalized born implicit solvent is used to fold the protein. In an implicit solvent, the solvent is modeled with an approximation of the mean force exerted by the external media on the molecule. It is faster than an explicit solvent with simulated water molecules and more detailed than a simulation in a vacuum (Kleinjung & Fraternali, 2014). The MD simulation is performed at temperatures 276 K, 288 K, 298 K, and 310 K since the transition temperature is found to be 293 K with our lab work. Simulations at 288 K and 298 K are runned in triplets 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 different results for the final stable conformations.

The results are analyzed with Root Mean Square Distance (RMSD), Radius of Gyration (Rg), the ratio of secondary structures, and Solvent Accessible Surface Area (SASA) to show the different behavior of the ELP at the temperatures. They are calculated with the MDTraj Python library. For the analyzation, the analyzation methods are calculated for the 1000 frames of the last 0.5 ns, which has a stable conformation at all temperatures. This gives more results and takes the small conformation differences into account.

RMSD

RMSD can be used to measure the difference in position between the backbone of a protein and its initial structural conformation. During the folding process, the RMSD curve rises until the point where the folding stops and the conformation is stable (Aier et al., 2016). So with the RMSD curve, we can see if the folding is finished and from what time the further analysis can start. In figure ..., the complete curves and the curves of the last 0.5 ns are shown. The complete curves of the different temperatures show that the folding stabilized at all temperatures for at least the last 0.5 ns. The last 0.5 ns is plotted separately, to see how much movement there still is in the stable conformation.

(figure with temperatures, left the full plot, right the last 0.5 ns) Radius of Gyration

Secondary structures

SASA

Conclusion

With the results from the different analysis methods, we can conclude that ...

References

[1] Alber, T. (1992). Structure of the leucine zipper. Current Opinion in Genetics and Development, 2, 205–210 [2] Christensen, T., Hassouneh, W., Trabbic-Carlson, K., & Chilkoti, A. (2023). Predicting Transition Temperatures of Elastin-Like Polypeptide Fusion Proteins. https://doi.org/10.1021/bm400167h [3] Despanie, J., Dhandhukia, J. P., Hamm-Alvarez, S. F., & MacKay, J. A. (2016). Elastin-like polypeptides: Therapeutic applications for an emerging class of nanomedicines. Journal of Controlled Release, 240, 93–108. https://doi.org/10.1016/j.jconrel.2015.11.010