Q125E

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

Figure 2: Surface of BCKDHA with highlighted amino acids

As one can see from figure 1, the mutation of glutamine on position 125 to glutamic acid does not interfere with any active site. But looking at figure 2 it is clear that this mutation changes an amino acid on the surface of the protein. This could be problematic if the biochemical properties of the mutated amino acid would not agree with the biophysical environmental conditions of the protein's surface. Therefore we have to take a look at the side chain properties.

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.

Figure 3: Structure of glutamine on position 125 in the wildtype protein

Figure 4: Structure of glutamic acid on position 125 in the mutated protein

As one can see from figures 3 and 4 the structures of glutamine and glutamic acid are very similar. The biochemical properties of glutamine and glutamic acid are also quite similar as both are polar amino acids, only glutamic acids has an additional negative charge, which should not be perturbing on the protein's surface. This leads to the assumption that this mutation might to be very harmful.

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 5: Hydrogen Bonds for glutamine on pos 125 in the wild type structure

Figure 6: Hydrogen Bonds for glutamic acid on pos 125 in the mutated type structure

The substitution from glutamine to glutamic acid changes the side chain properties completely. A NH2 group is substituted by a negatively charged oxygen. The NH2 which served in the wildtype structure as a hydrogen bond acceptor (see Figure 5) is not present any more, so the hydrogen bonding network changed for this substitution (compare Figure 5 and 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	431.77	30.77
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	-4080.989512	-1595.536757
relative	100%	60%	40%

The energy difference of the mutated protein and the wildtype structure calculated by minimise quite high. The mutated structure has an energy that is much smaller than the wildtype, which could be explained by a damaging effect of the mutation that leads a protein that is not stable any more.

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	2519.85	1700	6351.32	-10027.5
Angle	3626.21	260	618.433	-1418.24
Potential	5.23e+06	5.2e+06	7.5e+07	-3.17e+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	5.23e+06	--21470100
relative	100%	19%	81%

Gromacs calculates an even smaller energy for the mutated structure than minimise. The mutated structure has only about 20% of the energy of the wildtype structure, which is an enormous difference. This energy difference indicates that the stability of the protein changed, which causes an effect on the protein's function.

Conclusion

All energy calculations predicted a totally different protein structure for the mutated protein. These differences in energy resulted from different protein stabilities. The differences in stability can also be seen from looking at the hydrogen bonding network, where different hydrogen bonds are formed after the mutation, although the biochemical properties of the amino acids are quite similar. return to Structure-based mutation analysis