F409C
Contents
Structure-based Mutation Analysis
Mapping onto crystal structure
Figure 1 shows the protein structure and figure 2 the protein surface with highlighted amino acids. The mutation position is colored violet, the thiamine pyrophosphate binding sites are orange, and metal binding sites are yellow.
Figure 1 shows, that this mutation is not nearby any active site. But when looking at figure 2 it is clear that this mutation takes place on the surface of the protein. So the biophysical properties of the mutated amino acid have to be considered when someone wants to understand the damaging effect of this mutation (e.g. a hydrophobic amino acid on the protein's surface would reduce the protein's stability).
Side chain properties
Figures 3 and 4 show the superposition of the mutated amino acid with the wildtype. The pictures showing the wildtype structure display the unmutated residue in bold and vice versa.
This substitution leads to totally different amino acids concerning structure and physiochemical properties. A bulky aromatic residue is substituted by a small, polar, sulphur-containing amino acid. These drastic changes might very likely change the protein's structure and therefore affect its function.
Hydrogen Bonding network
Hydrogen bonds are interactions between an hydrogen atom and an electronegative atom. Electronegative atoms which often take part in hydrogen bonds are oxygen, nitrogen and fluorine (not present in amino acid side chains). They serve as a hydrogen bond acceptor, whereas a hydrogen bond donor is a electronegative atom bonded to a hydrogen atom. Hydrogen bonds are essential for the three-dimensional structures of proteins. They play a important role in the formation of helices and beta-sheets and cause proteins to fold into a specific structure.
Showing hydrogen bonds with Pymol: A -> find -> polar contacts -> within selection The respective amino acids were colored by element, s.t. oxygen is red, nitrogen is blue, hydrogen is white and sulfur is yellow.
The following figures show the hydrogen bonds between the wildtype residue and its environment compared to the formation of hydrogen bonds when the corresponding residue is mutated.
The phenylalanine side chain in the wildtype protein does not participate in any hydrogen bonds (see Figure 5). The mutation to serine doesn't introduce new hydrogen bonding donors or acceptors, therefore the mutation has no effect on the hydrogen bonding network (see Figure 6).
foldX Energy Comparison
We used the foldX tool to compare the energy of the wildtype protein and the mutated structure. The following table shows the calculated energy values as well as the percentage of difference, to compare the energy calculations with other tools:
| Energy | wildtype energy | total energy of mutated protein | difference |
|---|---|---|---|
| absolute | 401.00 | 433.33 | 32.33 |
| relative | 100% | 108% | 8% |
The total energy of the mutated structure is a little bit higher than the energy of the wildtype protein structure. As protein energies should be low for a stable protein, the increasing energy leads to the assumption that this mutation might be damaging for the protein structure.
minimise Energy Comparison
Next we used the minimise tool to compare the energy of the wildtype protein and the mutated structure. The following table shows the calculated energy values as well as the percentage of difference, to compare the energy calculations with other tools:
| Energy | wildtype energy | total energy of mutated protein | difference |
|---|---|---|---|
| absolute | -2485.452755 | -4358.528143 | -1873.075388 |
| relative | 100% | 57% | 43% |
The mutated structure has an energy that is only 57% of the energy of the wildtype structure. This energy difference might be due to different protein structures after the mutation took place.
gromacs Energy comparison
The Gromacs energy comparison was conducted using the AMBER03 force field. The following table shows the calculated energies for the wildtype protein structure.
| Energy | Average | Err.Est | RMSD | Tot-Drift (kJ/mol) |
|---|---|---|---|---|
| Bond | 3072.83 | 2200 | -nan | -13100.2 |
| Angle | 3616.97 | 230 | -nan | -1295.57 |
| Potential | 2.67001e+07 | 2.6e+07 | -nan | -1.60382e+08 |
Here are the results for the mutated protein structure.
| Energy | Average | Err.Est | RMSD | Tot-Drift (kJ/mol) |
|---|---|---|---|---|
| Bond | 2341.69 | 1600 | 6048.14 | -9087.07 |
| Angle | 3597.89 | 240 | 594.267 | -1309.54 |
| Potential | 4.68e+06 | 4.7e+06 | 7.12e+07 | -2.85e+07 |
As we want to compare the Gromacs energies with the other tools, we calculate the ratio of difference considering the potential energy:
| Energy | wildtype energy | total energy of mutated protein | difference |
|---|---|---|---|
| absolute | 2.67001e+07 | 4.68e+06 | -22020100 |
| relative | 100% | 17% | 83% |
The Gromacs energy for the mutated structure is very low, it is less than a fifth of the wildtype energy. This leads to the assumption that this mutation causes a major effect on the protein structure and stability.
Conclusion
For this mutation all energy calculation tools agree that there is a structural difference in the mutated structure. Just the degree to which the mutated structure varies from the wildtype structure differs between these tools. While the predicted energy difference is very small for foldX, it is extremely high for Gromacs. The comparison with the structure and biophyiscal properties of the amino acids would lead to the result that the mutation from phenylalanine to cysteine is deleterious, because the structure and properties are very different. The local hydrogen bonding network however does not seem to change much.
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