C264W

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 tyrosine on position 82 in the wildtype protein

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 cysteine on position 264 in the wildtype protein

Figure 3: Structure of tryptophan on position 264 in the mutated protein

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 cysteine on pos 264 in the wild type structure

Figure 5: Hydrogen Bonds for tryptophan on pos 264 in the mutated type structure

Although the amino acids cysteine and tryptophan have very different structures and chemical properties, no change in the hydrogen bonding network occurs (compare Figure 4 and 5).

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	488.43	87.43
relative	100%	121%	21%

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	-3745.313620	-1259.860865
relative	100%	66%	34%

The mutated structure has an energy that is much smaller than the wildtype

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	3186.75	2300	-nan	-13603.2
Angle	3831.06	370	-nan	-2070.89
Potential	3.41e+07	3.3e+07	-nan	-2.03e+08

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	3.41e+07	7399900
relative	100%	127%	27%

The calculated energy for the mutated structure is higher than for the wildtype structure.

Conclusion

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