Using the water soluble chlorophyll binding protein(6GIX) for purification in an oil based product forced the team to understand the system and how we can mitigate the stress placed on the protein. To understand the stress placed on 6GIX the team aimed to observe the nanoscale dynamics of the protein within different systems. These observations are incredibly difficult and expensive to obtain within the laboratory, thus forcing us to look towards in silico measurements and observations instead. Through the use of molecular dynamic simulations and a supercomputer iGEM Calgary was able to look deeper into our proteins nanoscale struggles.

Questions

To guide our Molecular dynamic modelling we generated the four questions that would have the largest impact on our project.

Question 1: What are the dynamics of the protein in an aqueous environment?

Question 2: Would the individual monomers of the 6GIX protein aggregate to form the tetramer structure?

Question 3: How will the protein hold up in a non-aqueous environment?

Question 4: How comfortable is 6GIX in the emulsion on the nanoscale?

All simulations were conducted in GROMACS 18.1 on the cpu2019 partition of the ARC computing cluster at the University of Calgary. Along with utilizing the same hardware over all simulations a similar model generating method was used.
Step 1. Converting Structure files into GROMACS files. The first step is to take the pdb files generated from structure prediction modelling or provided by the protein data bank. Then after converting the previous file to a .gro file we place it in a theoretical cube with the dimensions set to fit around the protein with at least a nanometer of padding in all dimensions.

Step 2. Solvate the Box. Now that the protein is in a theoretical box we can systematically place solvent molecules to fill it. In the case of two solvents they are completed one at a time.

Step 3. Run Energy Minimization. Now that the box has been solvated we will now run energy minimization to ensure a stable system for the future steps. This is done through a relaxation stage known as energy minimization to ensure no catastrophic issues with the system. This step also makes sure that solvents are realistically aligned for the next steps. This step usually takes 2 hours

Step 4. Running Isothermal-Isochoric Equilibration. This step looks at ensuring that the solvent and our protein are stable together at the temperature we are looking to simulate. This step usually takes 4 hours

Step 5. Running Isothermal-Isobaric Equilibration. This step is much like the previous step but it accounts for the pressure and density instead of temperature. This step usually takes 4 hours

Step 6. Atom by Atom Molecular Dynamics. Now that the system has been equilibrated we will run molecular dynamics. This simulates the atom by atom movement of the solvated box for a given amount of time. This step will generate a file containing the trajectories for every atom of the simulation. Due to the extreme computational requirements of this step, molecular dynamics are computed for nanoseconds at a time. This step takes 23 hours per nanosecond.

Step 7.Visualize the Trajectory. For visualizing the dynamics of the protein we have to take the large numeric files and make them understandable to our wetlab. To make these visualizations our team used VMD and Pymol. For other teams using this strategy, Pymol is easier to install and the more user friendly option. Though it being harder to use and install VMD also is very powerful for visualization and has many other applications.

The models generated were able to answer the key questions our wetlab had about the nanoscale properties of the protein.

Question 1. The tetramer structure was maintained and the protein was stable throughout the nanosecond simulation. [simulation] This enabled our team to understand the stability of our protein in water and the stability of the tetramer structure. Along with the gained understanding this model is also the easiest to accomplish, allowing for a teams drylab to gain familiarity with the software.

Question 2. This model showed that the protein monomers long range forces did form a tetramer when close but was less likely to form long range without chlorophyll. [model] This model ensured the teams use of the protein was founded as it would not immediately aggregate, thereby inhibiting chlorophyll binding. This model was extremely complex and required over 80 hours of computing time. For this reason we recommend that this model be completed after gaining experience with the software for others attempting it.

Question 3. From this model we discovered that when placed in a nonpolar solvent the 6GIX protein is ripped apart from its tetramer structure and the monomers are observed to denature. [model here] This demonstrated for our wetlab how problematic nonpolar solvent can be to 6GIX. This model really put how important it was for the team to ensure we protect the proteins from any unnecessary contact with the canola oil.

Question 4. The bisolvent system simulation showed that the 6GIX protein was comfortable in the emulsion at the nanoscale. This means that for our system the 6GIX protein should be comfortable within the emulsion bubbles. [model here] From the emulsion bubble model our teams plan to use the emulsion system for chlorophyll degradation was verified. All together these models assisted the team in understanding the response a protein may have to its given environment. These knowledge then integrated into every facet of the project design. With a nanoscale understanding of our system iGEM Calgary was able to design and carryout yOIL in such a way to get the maximum out of our protein.

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