G249S

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

This mutation seems not to cause trouble with the active site. In the next section we will have a look at the side chain properties to understand why this mutation might be deleterious to 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 glycine on position 249 in the wildtype protein

Figure 3: Structure of serine on position 249 in the mutated protein

Comparing figures 2 and 3 there seems to be no big structural difference between glycine and serine. Both are quite small amino acids, but the substitution of the tiny, unpolar glycin with the polar hydroxylic serine could be damaging. Furthermore mutations of glycine are often not tolerated as the unique smallness of glycine is advantageous in many positions in the protein<ref> M.O. Dayhoff, R.M. Schwartz, B.C. Orcutt: A Model of Evolutionary Change in Proteins, Atlas of Protein Sequence and Structure, 1978</ref>.

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 glycine 249 in the wild type structure

Figure 5: Hydrogen Bonds for serine on pos 249 in the mutated type structure

Introducing a serine on position 249 leads to the formation of several additional hydrogen bonds (see Figure 5). Two of the newly established bonds are due to the new hydroxy group which is very likely to participate in hydrogen bonds. Another additional hydrogen bond is formed using the nitrogen atom as a hydrogen bond acceptor.

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	432.22	31.22
relative	100%	107%	7%

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	-4280.043000	-1794.590245
relative	100%	58%	42%

Minimise calculated a much smaller energy for the mutated structure than for the wildtype. This should be due to the introduced mutation which causes changes in the stability of the 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	2775.97	2000	6761.45	-11375.2
Angle	3682.24	300	670.885	-1625.24
Potential	5.96e+06	5.0e+06	8.02e+07	-3.61e+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	5.96e+06	-20740100
relative	100%	22%	78%

The Gromacs energy difference is again quite high.

Conclusion

All energy calculations, especially Gromacs and minimise, agree that the energy of the mutated structure changed drastically compared to the wildtype structure. Comparing these calculations with the observations made from the structure of the amino acids, the high difference is not explainable. Both amino acids are relatively small. But serine takes part in the formation of new hydrogen bonds and may therefore stabilize the protein wrongly. This could be reason for the high energy differences.

References

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