Team:Duke/Model/Trials
Thermostability Engineering Trials of Monomeric GRFT (3ll2)
B Factor Modeling
The B Factor of a protein residue is a measure of displacement due to temperature dependent vibrations and thermal motion of atoms in sidechain. Often, these values are used to identify amino acids which hinder protein thermostability. Using Yasara, we were able to calculate the B values of each residue and modeled the B values of monomeric Griffithsin using PyMOL pictured below. In the image below, red corresponds to residues with B factors above 17 Å^2, yellow corresponds to residues with B factors above 15 Å^2, and residues shaded green and blue represent stable amino acids left unaltered in subsequent steps.
Through these calculations, we discovered, as expected, that only certain amino acids (SER, ARG, and GLY) in loops had B factors above 15 Å^2. Now that we discovered where the protein lacked in stability, we decided to run Yasara's position scan function to predict how we could alter these amino acids and quantify the improvement of thermostability.
Position Scan Profiling
The Yasara position scan function calculates the change in stability (ΔΔG) for each selected residue and the 19 other amino acids in the position of that residue. This function also shows how different noncovalent interactions are altered once each amino acid is altered and calculates the overall change in stability of the entire molecule based on the changed topology of the protein. Initially, we only used this function on the S, R, G amino acids which had a high B factor, but realized there were additional amino acids which could also have very negative ΔΔG values. We expanded the use of this function to all amino acids not in the binding loops of monomeric GRFT.
Trends: Mutating Serines to Leucines and charged amino acids to Phenylalanine had a very negative ΔΔG values. This suggests the protein's stability could be greatly improved with the addition of hydrophobic residues.
**Additionally, only residues that stabilized the protein more than 1.0 kcal/mol were used in the next step, since the Position Scan function has a margin of error of .50 kcal/mol. **
Mutate Multiple Residues
Through the mutate multiple residue function in Yasara, we were able to use the data collected through the B factor and position scan calculations to build thermoengineered monomeric Griffithsin variants. For this step, we wanted to maximizing the thermostability of the protein while minimizing the number of changes made on the protein. We also wanted to remove all of the methionines from the molecule to assist in the purification of our protein, even if that meant slightly destabilizing our protein. The ΔG of folding was used to rank protein variants by their thermostability
Trial 1: Mutated top 2 amino acids SER2 and ASP72 Stability (ΔG): -5.603 kcal/mol.
Trial 2: Mutated top 5 amino acids Stability (ΔG): -11.402 kcal/mol.
Trial 3: Mutated top 10 amino acids Stability (ΔG): -20.816 kcal/mol.
Trial 4: Mutate every unstable amino acid (38 residues) with DDG>1.0 kJ/mol Stability (ΔΔG): -40.319 kcal/mol.
Trial 5: Mutated top 15 Amino acids Stability (ΔG): -36.83 kcal/mol.
Original 3ll2 Monomeric GRFT structure with a ΔG of 8.44 kcal/mol.
Thermoengineered 3ll2 Monomeric GRFT structure with a predicted ΔG of -36.83 kcal/mol. The altered amino acids are pictured in red.
The two structures pictured above were calculated to have a RMSD of .45 Å between each other, suggesting extreme structural similarity.
Thermostability Engineering Trials of Dimeric GRFT (2HYQ)
B Factor Modeling
The same B factor calculations and modeling was ran for the dimeric form of GRFT. In the PyMOL movie below, red corresponds to residues with B factors above 30 Å^2, yellow corresponds to residues with B factors above 24 Å^2, and residues shaded green and blue represent stable amino acids left unaltered in subsequent steps.
Position Scan Profiling
Similar to the position scanning of monomeric GRFT, the residues of dimeric GRFT were checked to see if their ΔΔG were negative and could contribute to a more stable protein. Unlike Monomeric GRFT, we needed to take greater account of electrostatic interactions and disulfide bridges connecting the two GRFT subunits. Trends: Aspartate and Serine in loops were very unstable and the protein's stability was significantly increased after these residues were changed to asparagine and phenylalanine respectively.This suggests the protein's stability could be greatly improved with the addition of hydrophobic residues and removal of negatively charged residues.
Mutate Multiple Residues
Similar to the previous construct, the data used from position scan and B factor calculations were used to run trials on the thermostability of the dimeric GRFT. However, in these trials, we had to make the same changes on both subunits to retain the homodimeric nature of the protein, match the binding topology between subunits, and reduce confounding variables when analyzing the thermostability and biological activity of the protein.
Original 2HYQ homodimeric GRFT structure with a ΔG of 21.1 kcal/mol.
Thermoengineered 2HYQ homodimeric GRFT structure with a predicted ΔG of -17.93 kcal/mol. The altered amino acids are pictured in red.
The two structures pictured above were calculated to have a RMSD of .2945 Å between each other, suggesting extreme structural similarity.
