Difference between revisions of "C264W"
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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 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. |
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[[File:BCKDHA_mut5.png|thumb|center| Figure 1: Structure of BCKDHA with highlighted amino acids]] |
[[File:BCKDHA_mut5.png|thumb|center| Figure 1: Structure of BCKDHA with highlighted amino acids]] |
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| + | The position of this mutation is relatively close by the active sites in the protein. With the knowledge that the compact amino acid cysteine is replaced by a very bulky tryptophan the fact, that this mutation could disturb the binding of ligands should not be excluded. |
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=== Side chain properties === |
=== Side chain properties === |
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|[[File:Mutation5_BCKDHAMinimise.png|thumb|Figure 3: Structure of tryptophan on position 264 in the mutated protein]] |
|[[File:Mutation5_BCKDHAMinimise.png|thumb|Figure 3: Structure of tryptophan on position 264 in the mutated protein]] |
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| + | Looking at figures 2 and 3 it is obvious that this substitution is harmful to the protein's function. This mutation has a huge impact on the structure and physiochemical properties of the amino acid at this position. A small, sulphur-containing amino acid is replaced by an aromatic amino acid, which occupies a lot more space. The hydrophobicity and polarity remain the same, nevertheless are the amino acids very different and this mutation will destroy the protein's function. |
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=== Hydrogen Bonding network === |
=== Hydrogen Bonding network === |
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| − | The total energy of the mutated structure is a |
+ | The total energy of the mutated structure is a lot higher than the energy of the wildtype protein structure. This indicates that this mutation has an enormous effect on the stability of the protein. As changing the stability of the protein goes along with the function of the protein, this mutation might very likely affect the protein's function. |
===minimise Energy Comparison=== |
===minimise Energy Comparison=== |
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| − | + | Minimise calculated again an much lower energy for the mutated structure than for the wildtype structure. |
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===gromacs Energy comparison=== |
===gromacs Energy comparison=== |
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| − | + | Interestingly, the calculated energy for the mutated structure is higher than for the wildtype structure, leading to the assumption that the mutated structure is more instabile than the wildtype structure. |
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=== Conclusion === |
=== Conclusion === |
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| + | This time the calculations of the different tools disagree completely. While foldX again predicted a higher energy (as for all the other mutations under consideration) and the minimise energy is still lower than for the wildtype structure, Gromacs now calculated a very high energy for the mutated structure compared to the wildtype structure. As a higher energy usually indicates a protein that is more instabile that a protein with lower energy, one could easily argue that this mutation is delerious. This statement does not agree with the calculations from foldX and minimise. The structural differences of the amino acids however are a strong indication for the deleterious effect of this mutation, that leads to instability of the protein structure and therefore affects the protein's function. |
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return to [[Structure-based_mutation_analysis_BCKDHA| Structure-based mutation analysis]] |
return to [[Structure-based_mutation_analysis_BCKDHA| Structure-based mutation analysis]] |
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Latest revision as of 20:47, 11 August 2011
Contents
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.
The position of this mutation is relatively close by the active sites in the protein. With the knowledge that the compact amino acid cysteine is replaced by a very bulky tryptophan the fact, that this mutation could disturb the binding of ligands should not be excluded.
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.
Looking at figures 2 and 3 it is obvious that this substitution is harmful to the protein's function. This mutation has a huge impact on the structure and physiochemical properties of the amino acid at this position. A small, sulphur-containing amino acid is replaced by an aromatic amino acid, which occupies a lot more space. The hydrophobicity and polarity remain the same, nevertheless are the amino acids very different and this mutation will destroy 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.
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 lot higher than the energy of the wildtype protein structure. This indicates that this mutation has an enormous effect on the stability of the protein. As changing the stability of the protein goes along with the function of the protein, this mutation might very likely affect the protein's function.
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% |
Minimise calculated again an much lower energy for the mutated structure than for the wildtype structure.
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% |
Interestingly, the calculated energy for the mutated structure is higher than for the wildtype structure, leading to the assumption that the mutated structure is more instabile than the wildtype structure.
Conclusion
This time the calculations of the different tools disagree completely. While foldX again predicted a higher energy (as for all the other mutations under consideration) and the minimise energy is still lower than for the wildtype structure, Gromacs now calculated a very high energy for the mutated structure compared to the wildtype structure. As a higher energy usually indicates a protein that is more instabile that a protein with lower energy, one could easily argue that this mutation is delerious. This statement does not agree with the calculations from foldX and minimise. The structural differences of the amino acids however are a strong indication for the deleterious effect of this mutation, that leads to instability of the protein structure and therefore affects the protein's function.
return to Structure-based mutation analysis
