Difference between revisions of "Y166N"

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(Structure-based Mutation Analysis)
(Structure-based Mutation Analysis)
 
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=== Mapping onto crystal structure ===
 
=== 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 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_mut3.png|thumb|center| Figure 1: Structure of tyrosine on position 82 in the wildtype protein]]
 
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{|class="centered
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|[[File:BCKDHA_mut3.png|thumb|center| Figure 1: Structure of BCKDHA with highlighted amino acids]]
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|[[File:BCKDHA_mut3_surface.png|thumb|center| Figure 2: Surface of BCKDHA with highlighted amino acids]]
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|}
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Looking at figure 1 it is obvius that the mutation does not interfere with the active sites of the protein. But figure 2 shows that the mutated amino acid is on the surface of the protein and therefore we have to take a look at the biophysical properties of the mutated and the wildtype sidechains.
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=== Side chain properties ===
 
=== 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.
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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.
   
   
 
{|class="centered"
 
{|class="centered"
|[[File:Wildtype3_BCKDHAMinimise.png|thumb|Figure 2: Structure of tyrosine on position 166 in the wildtype protein]]
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|[[File:Wildtype3_BCKDHAMinimise.png|thumb|Figure 3: Structure of tyrosine on position 166 in the wildtype protein]]
|[[File:Mutation3_BCKDHAMinimise.png|thumb|Figure 3: Structure of asparagine on position 166 in the mutated protein]]
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|[[File:Mutation3_BCKDHAMinimise.png|thumb|Figure 4: Structure of asparagine on position 166 in the mutated protein]]
 
|}
 
|}
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The substitution if tyrosine by asparagine leads to a mutation of an hydrophobic aromatic residue to a small polar amino acid. These differences in the amino acids' properties are likely to change the proteins' structure as this residue is located in a helix and therefore this mutation is very likely to affect the protein's function.
   
 
=== Hydrogen Bonding network ===
 
=== Hydrogen Bonding network ===
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|}
   
The mutated structure has an energy that is much smaller than the wildtype
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The mutated structure has an energy that is much smaller than the wildtype, which is only about half of the wildtype energy. This indicates a change in protein structure and stability after the introduction of this mutation.
   
 
===gromacs Energy comparison===
 
===gromacs Energy comparison===
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The energy of the mutated structure is about 30% of the wildtype structure. This is still a huge difference, but compared to the other energies calculated by Gromacs for other mutations, the difference is not that high. Still this decrease in energy is the result of a very different protein structure which could cause an instable protein. Therefore this mutation might effect the stability and the function of the protein.
The calculated energy for the mutated structure is much smaller than for the wildtype structure.
 
   
 
=== Conclusion ===
 
=== Conclusion ===
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All energy calculations show a significant divergence from the wildtype-structure energy. This indicates that the mutation led to an instable protein. These calculations agree with the observations made based on the biophysical properties of the amino acids and the local hydrogen bonding network. The biophyiscal properties changed drastically and different hydrogen bonds were formed. This could be the reason for the instability of the mutated protein structure.
 
 
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 21:23, 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
Figure 2: Surface of BCKDHA with highlighted amino acids

Looking at figure 1 it is obvius that the mutation does not interfere with the active sites of the protein. But figure 2 shows that the mutated amino acid is on the surface of the protein and therefore we have to take a look at the biophysical properties of the mutated and the wildtype sidechains.

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 tyrosine on position 166 in the wildtype protein
Figure 4: Structure of asparagine on position 166 in the mutated protein

The substitution if tyrosine by asparagine leads to a mutation of an hydrophobic aromatic residue to a small polar amino acid. These differences in the amino acids' properties are likely to change the proteins' structure as this residue is located in a helix and therefore this mutation is very likely to affect 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.

Figure 4: Hydrogen Bonds for tyrosine on pos 166 in the wild type structure
Figure 5: Hydrogen Bonds for asparagine on pos 166 in the mutated type structure

Although tyrosine and asparagine both could play a role in the hydrogen bonding network, no hydrogen bond is formed for position 166 (see Figure 4 and 5). Therefore this substitution has no influence on the hydrogen bonding network of the protein.

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 401.00 432.24 31.24
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 -4354.495238 -1869.042483
relative 100% 57% 43%

The mutated structure has an energy that is much smaller than the wildtype, which is only about half of the wildtype energy. This indicates a change in protein structure and stability after the introduction of this mutation.

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 3029.19 2200 -nan -12529.5
Angle 3654.58 280 -nan -1486.71
Potential 7.95e+06 7.8e+06 -nan -4.67e+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 7.95e+06 -21540100
relative 100% 29% 71%

The energy of the mutated structure is about 30% of the wildtype structure. This is still a huge difference, but compared to the other energies calculated by Gromacs for other mutations, the difference is not that high. Still this decrease in energy is the result of a very different protein structure which could cause an instable protein. Therefore this mutation might effect the stability and the function of the protein.

Conclusion

All energy calculations show a significant divergence from the wildtype-structure energy. This indicates that the mutation led to an instable protein. These calculations agree with the observations made based on the biophysical properties of the amino acids and the local hydrogen bonding network. The biophyiscal properties changed drastically and different hydrogen bonds were formed. This could be the reason for the instability of the mutated protein structure. return to Structure-based mutation analysis