Rs1800431

From Bioinformatikpedia
Revision as of 14:36, 29 September 2011 by Link (talk | contribs) (Created page with "== General Information == {| border="1" style="text-align:center; border-spacing:0;" |SNP-id |rs1800431 |- |Codon |436 |- |Mutation Codon |Ile -> Val |- |Mutation Triplt |ATA -…")
(diff) ← Older revision | Latest revision (diff) | Newer revision → (diff)

General Information

SNP-id rs1800431
Codon 436
Mutation Codon Ile -> Val
Mutation Triplt ATA -> GTA

Back to [Sequence-based mutation analysis]

Sequence-based Mutation Analysis

Pysicochemical Properties

First of all, we explored the amino acid properties and compared them for the original and the mutated amino acid. Therefore we created the possible effect that the mutation could have on the protein.

Ile Val consequences
aliphatic, hydrophobic, neutral aliphatic, hydrophobic, neutral In this case, the pysicochemical properties are equal. Furthermore, they almost agree in their size. Therefore, we suggest, that there is no big effect on the 3D structure of the protein and therefore, also no big effect on the protein function.


Back to [Sequence-based mutation analysis]


Visualization of the Mutation

In the next step, we created the visualization of the mutation with PyMol. Therefore we created a picture of the original amino acid (Figure 1), of the new mutated amino acid (Figure 2) and finally of both together in one picture whereas the mutation is white colored (Figure 3). The following pictures display the mutation from Isoleucine to Valine. Isoleucine is longer than Valine and one part spreads in the other direction than this of Valine. Valine is small and forks at its end. All in all, the differences of both amino acids is not so drastic and therefore the protein will probably not have a structural and functional change.

picture original amino acid picture mutated amino acid combined picture
Figure 1: Amino acid Isoleucine
Figure 2: Amino acid Valine
Figure 3: Picture which visualize the mutation


Back to [Sequence-based mutation analysis]


Substitution Matrices Values

Afterwards, we looked at the values of the substitution matrices PAM1, PAM250 and BLOSSUM62. Therefore we looked detailed at the three values: the value for according amino acid substitution, the most frequent value for the substitution of the examined amino acid and the rarest substitution.

In this case, the substitution of Isoleucine to Valine has very high values that agree with the most frequent value for all three substitution matrices. Therefore, according to all matrices a mutation at this position will probably not cause structural changes which can affect functional changes.

PAM 1 Pam 250 BLOSOUM 62
value amino acid most frequent substitution rarest substitution value amino acid most frequent substitution rarest substitution value amino acid most frequent substitution rarest substitution
33 33 (Val) 0 (Gly, Pro, Trp) 9 9 (Val) 1 (Trp) 3 3 (Val) -4 (Gly)


Back to [Sequence-based mutation analysis]


PSSM Analysis

Besides, we looked additional at the position specific scoring matrix (PSSM) for our sequence. In contrast to PAM and BLOSOUM, the PSSM contains a specific substitution rate for each position in the sequence. Therefore, the PSSM is more position specific than PAM or BLOSOUM. We extracted the substitution value for the underlying mutation, the value for the most frequent substitution and the rarest substitution.

In this case the substitution rate for Asparagine to Aspartic acid at this position is in the middle between the value for the rarest substitution and the value for the most frequent substitution. Therefore, the value from PSSM is not really significant and we are not able to determine effects on the protein.

PSSM
value amino acid most frequent substitution rarest substitution
-4 3 -6

Conservation Analysis with Multiple Alignments

As a next step we created a multiple alignment which contains the HEXA sequence and 9 other mammalian homologous sequences from [UniProt]. Afterwards we looked at the position of the different mutations and looked at the conservation level on this position. The regarded mutation is presented by the second colored column on Figure 4. Here we can see, that almost all other mammalians have another amino acid on this position. Therefore, the mutation on this position is not highly conserved and a mutation there will cause probably no huge structural and functional changes for the protein.

Figure 4: Mutation in the multiple alignment


Back to [Sequence-based mutation analysis]


Secondary Structure Mutation Analysis

As a next step we compared the different results of the secondary structure prediction tools JPred and PsiPred. Afterwards we can examine in which secondary structure element and where therein the mutation takes place. This can give an overview of how drastic the mutation can be. In this case both tools agree and predict at the position of the mutation a coil. This has as a result, that the mutation at this position would not destroy or split a secondary structure element. It will probably only changes the coil between two secondary structure elements, but this can sometimes also cause a change of the the following secondary structure. We think that a drastic change of the protein structure and its function is unlikely because the mutation does not affect a secondary structure element. The change of the coil will probably only take places between two secondary structure elements which will probably not changes the protein.

JPred:
...HHHHHHHHCCCCEEECCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCEEEE...
PsiPred:
...HHHHHHHHCCCEEEECCCCCCCCCCCCCCCCCCCCCCCCCCCCCHHHHCCCCCE...

Comparison with the real Structure:

Afterwards we also visualize the position of the mutation (red) in the real 3D-structure of PDB and compare it with the predicted secondary structure. The visualization can therefore like above the predicted secondary structure display if the mutation is in a secondary structure element or in some other regions.

Here in this case the mutation position almost agree with the position of the predicted secondary structure and is within a coil, which can be seen on Figure 5 and Figure 6. Like explained above this means a mutation will probably not destroy a secondary structure element which affects no drastic structural change. Otherwise it can cause a change of the position of the two nearest secondary structure element which can has a functional loose as a consequence. We think that a structural change is unlikely, because it is not within a secondary structure element and will therefore not cause extreme changes of the protein.

Figure 5: Mutation at position 436
Figure 6: Mutation at position 436 - detailed view


Back to [Sequence-based mutation analysis]


SNAP Prediction

Next, we looked at the result of the SNAP prediction. For this prediction we took the amino acid of the certain position and checked every possible amino acid mutation. Afterwards we extract the result for Aspartic acid which is the real mutation in this case. SNAP has as a result that the exchange from Asparagine to Aspartic acid at this position is neutral with an average accuracy of only 53%. The low accuracy is critical for this prediction, but we suppose that it is right and the mutation at this position is neutral which means it likely causes no structural and functional changes of the protein.

Substitution Prediction Reliability Index Expected Accuracy
V Neutral 0 53%

A detailed list of all possible substitutions can be found here]


Back to [Sequence-based mutation analysis]


SIFT Prediction

Next, we used SIFT Prediction which displays if a mutation is neutral or not. Therefore, it first shows a row which contains a score for the particular mutation position to a certain amino acid. The amino acid which are not tolerated at this position are colored red. Besides, it also constructs a table which lists the amino acids that are predicted as tolerated and not-tolerated (Figure 8).

In this case, there are only two substitutions that are not tolerated: Tryptophan and Cysteine. The substitution to Valine is tolerated at this position. This means that this mutation at this position is probably neutral and will not cause any structural and function changes of the protein.

SIFT Matrix:
Each entry contains the score at a particular position (row) for an amino acid substitution (column). Substitutions predicted to be intolerant are highlighted in red.

Figure 7: Legend
Figure 8: SIFT Table
Threshold for intolerance is 0.05.
Amino acid color code: nonpolar, uncharged polar, basic, acidic.
Capital letters indicate amino acids appearing in the alignment, lower case letters result from prediction.




Predict Not ToleratedPositionSeq RepPredict Tolerated
wc436I1.00fYHMpIVgLnTQDRASEK




Back to [Sequence-based mutation analysis]


PolyPhen2 Prediction

Finally, we also regarded the PolyPhen2 prediction for this mutation. This prediction visualizes have strongly damaging the mutation probably will be. Therefore it gives the result for two possible cases: HumDiv and HumVar. HumDiv is the preferred model for evaluation rare alleles, dense mapping of regions identified by genome-wide association studies and analysis of neutral selection. In contrast, HumVar is the preferred model for diagnostic of Mendelian diseases which require distinguishing mutations with drastic effects from all remaining human variations including abundant mildly deleterious alleles. We decided to look at both possible models, which are agrees in the most cases.

In this case both models predict that the mutation is benign (Figure 9 and Figure 10). This means that the mutation is neutral and will probably not damage the structure and the function of the protein.

Figure 9: HumDiv prediction
Figure 10: HumVar prediction

Back to [Sequence-based mutation analysis]


Structure-based Mutation Analysis

Mapping onto Crystal Structure

Figure 11: Visualization of the mutation and important functional sites
Color declaration:
* red: position of mutation
* green: position of active side
* yellow: position of glycolysation
* cyan: position of Cysteine

First of all, we colored the important residues and also the mutated residue in the crystal structure, to see if the mutation is near of far away from the functional residues. As you can see on Figure 11, the mutation is located at the begin of a helix but far away from all the functional residues. It is possible, that a mutation at this position damage the helix, but it is just the begin of the helix and therefore this should not have that much effects on the structure. Therefore, we do not know in which way this mutation affects the global structure of the protein.

Back to [Structure-based mutation analysis]


SCWRL Prediction

Because the mapping analysis does not give a good explanation why the mutation causes damages on the protein, we decided to analyse this mutation in more detail. Therefore, we looked for the structure of the original amino acid and the structure of the amino acid after the mutation event and compared them in size and orientation. For this purpose we used SCWRL.

picture original amino acid picture mutated amino acid combined picture
Figure 12: Amino acid Isoleucine
Figure 13: Amino acid Valine
Figure 14: Picture which visualize the mutation

The amino acids (Figure 12, Figure 13, Figure 14) do not change much in size, but the branches of them are very different. Therefore it is possible that the mutation cause additional or missing H-bonds.

Back to [Structure-based mutation analysis]


FoldX Energy Comparison

Next we use the FoldX energy tool to compare the energy values of the two different structures.

Original total energy Total energy for the mutated protein Strongest energy changes within the mutated protein
-154.17 -154.36 -

The energy values of the two structures are nearly equal. Therefore if the mutation damage the protein it is not because of a too rigid or too stable protein conformation. To have the possibility to compare these energy values with the values of the other analysis tools, we calculated a ratio between these energy values.

Ratio of the original structure Ratio of the mutated structure Difference
100 100.23 -0.23

The mutated structure has 0.23% more energy than the original structure. But this difference is nearly 0 and therefore it is not an explanation for a damaging mutation.

Back to [Structure-based mutation analysis]


Minimise Energy Comparison

Next we use the minimise energy tool to compare the energy values of the two different structures.

Comparing Energy:

Original total energy Total energy for the mutated protein
-9610.467157 -9618.062763

The energy of the mutated structure is a little bit lower than the original energy. It is hard to compare these values with the values calculated by FoldX and therefore we calculated a ratio between the energy values.

Ratio of the original structure Ratio of the mutated structure Difference
100 100.08 -0.08

The difference is only 0.08% which is nearly 0. This is consistent with the result of FoldX.

Comparing Structure:

This tool also gives as output a [PDB] file with the position of the original and the mutated amino acid.

picture original amino acid picture mutated amino acid combined picture
Figure 15: Amino acid Isoleucine
Figure 16: Amino acid Valine
Figure 17: Picture which visualize the mutation

The pictures (Figure 15, Figure 16, Figure 17) show nearly the same as the pictures created with SCWRL (Figure 12, Figure 13, Figure 14).

Visualization of H-bonds and Clashes:

To get more insight in the effects of the mutated amino acid on the structure, we also analysed the H-bonds and clashes of the Serine residue.

H-bonds of the original structure H-bonds near the mutation Clashes of the mutation
Figure 18: H-bonds of the original amino acid (colored in magenta)
Figure 19: H-bonds of the mutated amino acid (colored in red)
Figure 20: Possible clashes

Neither the original (Figure 18) nor the mutated amino acid (Figure 19) has any H-bonds with other residues or the backbone of the protein. Therefore if this mutation damage the protein, it can not be explained by a missing or additional H-bond. Furthermore, there are no clashes between the mutated amino acid and the rest of the protein, which can be seen on Figure 20.

Back to [Structure-based mutation analysis]


Gromacs Energy Comparison

Comparing Energy:

To analyse the energy values calculated by Gromacs, we used the AMBER99SB-ILDN force field.

Here are the values of the original structure:

Energy Average Err.Est. RMSD Tot-Drift
Bond 1091.57 270 -nan -1622.75
Angle 3326.81 62 -nan 404.076
Potential -61304.1 960 -nan -6402.44

Here you can see the values which gromacs calculated for the structure with the mutated amino acid:

Energy Average Err.Est. RMSD Tot-Drift
Bond 1063.64 320 -nan -2016.06
Angle 3196.68 49 -nan 295.91
Potential -48418.9 1000 -nan -7020.14

One difference between gromacs and the other tools we used is, that gromacs also calculated the energy for the bonds and the angles. To compare the energies between the different tools we only consider the potential energy in our analysis, because the potential energy is the energy of the complete protein. Therefore, we calculated the ratio between the energies only for the potential energy.

Ratio of the original structure Ratio of the mutated structure Difference
100 78.98 21.02

In contrast to the other tools the energy values from gromacs differ strongly.

Comparing Structure:

picture original amino acid picture mutated amino acid combined picture
Figure 21: Amino acid Isoleucine
Figure 22: Amino acid Valine
Figure 23: Picture which visualize the mutation

The pictures (Figure 21, Figure 22, Figure 23) are very similar to them of SCWRL and minimize. Therefore the amino acids differ in structure but the size of the amino acids is very similar.

Visualization of H-bonds and Clashes:

To get more insight in the effects of the mutated amino acid on the structure, we also analysed the H-bonds and clashes of the Serine residue.

H-bonds of the original structure H-bonds near the mutation Clashes of the mutation
Figure 24: H-bonds of the original amino acid (colored in magenta)
Figure 25: H-bonds of the mutated amino acid (colored in red)
Figure 26: Possible clashes

In both cases there are no H-bonds between the amino acid we looked at and the rest of the protein or the backbone (Figure 24, Figure 25). This result is consistent with the result of minimize. Furthermore, there are no clashes between the mutated amino acid and the rest of the protein (Figure 26).

Back to [Structure-based mutation analysis]