M82L

Structure-based Mutation Analysis

Mapping onto crystal structure

Figure 1 shows the protein structure 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: Structure of BCKDHA with highlighted amino acids

The substitution of methionine on position 82 with leucine does not hinder ligand binding in the protein's active center. Therefore there must be other reasons why this mutation might be harmful for the protein's function.

Side chain properties

Figures 2 and 3 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.

Figure 2: Structure of methionine on position 82 in the wildtype protein

Figure 3: Structure of Leucine on position 82 in the mutated protein

Comparing figure 2 and 3 the structures of considerated amino acids are quite similar. Both amino acids are hydrophobic, but methionine contains a sulfur-atom which is then lost after the mutation. This could have an effect on the protein's 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.

Figure 4: Hydrogen Bonds for methionine on pos 82 in the wild type structure

Figure 5: Hydrogen Bonds for leucine on pos 82 in the mutated type structure

Comparing the figures 2 and 3 for the wildtype and the mutated amino acid on position 82, no change in the hydrogen bonding network can be observed. This is due to the similar physiochemical properties of these two amino acids. No atom which could serve as additional hydrogen-bond donor or acceptor was introduced or removed.

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	437.88	36.88
relative	100%	109%	9%

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	-4253.174790	-1767.722015
relative	100%	58%	42%

The mutated structure has an energy which is only about half of the energy of the wildtype structure. This is an extreme change in energy which might be accompanied by the instability of the mutated protein.

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	2518.71	1700	6337.97	-10023.3
Angle	3642.41	270	638.624	-1479.34
Potential	5.16e+06	5.1e+06	7.47e+07	-3.13e+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	5.16e+06	-21540100
relative	100%	19%	81%

The energy difference calculated by Gromacs is even higher than the calculations by minimise. These enormous energy changes are due to totally different protein structures, caused by the mutation of one amino acid. This mutation lowers the protein's energy and might therefore be to destabilizing and affect the protein's function.

Conclusion

All energy calculations show a significant divergence of the mutated protein structure from the wildtype-structure which indicate that this mutation leads to an instable protein. These calculations could be affirmed as the biophysical properties of the corresponding amino acids are quite similar and the hydrogen bonding network didn't change.