Difference between revisions of "I326T"

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=== Conclusion ===
 
=== Conclusion ===
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The overall trend of the calculated energies stays the same. FoldX calculated an energy difference of 7% between the wildtype and the mutated protein structure and both the minimise and the Gromacs energies are a lot lower for the mutated protein structures than for the wildtype structures. Looking at the structure and the biophysical properties of the mutating residues, which lead to the formation of hydrogen bonds, we can see that isoleucine and threonine are quite similar concerning these aspects. The same structure and the hydrogen bond network that didn't change could be an explanation for repeating expected energy values.
   
 
return to [[Structure-based_mutation_analysis_BCKDHA| Structure-based mutation analysis]]
 
return to [[Structure-based_mutation_analysis_BCKDHA| Structure-based mutation analysis]]

Latest revision as of 21:55, 11 August 2011

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

Looking at figure 1 it is not obvious why this mutation should be damaging, as it is not nearby the active center. The mutation does also not take place on the surface of the protein. So we have to take a look at the side chain properties to understand the damaging effect of this mutation.

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 isoleucine on position 326 in the wildtype protein
Figure 3: Structure of threonine on position 326 in the mutated protein

This substitution does not change very much in the amino acid structure but in the biochemical properties of the amino acids. Isoleucine is aliphatic and hydrophobic and therefore well suited for being in the inside of the protein. The mutation to Threonine introduces a hydroxylic, polar amino acids, which might cause instability of the 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 isoleucine on pos 326 in the wild type structure
Figure 5: Hydrogen Bonds for threonine on pos 326 in the mutated type structure

The mutation from isoleucine to threonine doesn't have an influence on the hydrogen bonding network, although the oxygen atom of threonine could serve as an additional hydrogen bond donor (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 401.00 432.94 31.94
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 -4317.105618 -1831.652863
relative 100% 57% 43%

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 3214.03 2300 7364.47 -13490.1
Angle 3738.44 310 698.943 -1792.01
Potential 7.29e+06 6.9e+06 8.86e+07 -4.38e+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 7.29e+06 -19410100
relative 100% 27% 73%

The calculated energy for the mutated structure is much smaller than for the wildtype structure.

Conclusion

The overall trend of the calculated energies stays the same. FoldX calculated an energy difference of 7% between the wildtype and the mutated protein structure and both the minimise and the Gromacs energies are a lot lower for the mutated protein structures than for the wildtype structures. Looking at the structure and the biophysical properties of the mutating residues, which lead to the formation of hydrogen bonds, we can see that isoleucine and threonine are quite similar concerning these aspects. The same structure and the hydrogen bond network that didn't change could be an explanation for repeating expected energy values.

return to Structure-based mutation analysis