|Mutation Codon||Phe -> Ser|
|Mutation Triplet||TTC -> TCC|
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.
|polar, tiny, hydrophilic, neutral||aliphatic, hydrophobic, neutral||Ile is much bigger than Ser and also is branched, because it is an aliphatic amino acid. Therefore the structure of both amino acids is really different and Ile is to big for the position where Ser was. Therefore, there has to be a big change in the 3D structure of the protein and the protein probably will loose its function.|
Visualisation of the Mutation
In the next step, we created the visualization of the muation with PyMol. Therefore we created a picture for the original amino acid, for the new mutated amino acid and finally for both together in one picture whereas the mutation is white colored. The following pictures display that the mutated amino acid Serine looks very different to Phenylalanine. Phenylalanine has a huge aromatical ring. Contrary, Serine is very smaller and differs a little bit in the orientation. This shows that the amino acids have huge structural differences which will probably cause drastical effects on protein structure and function.
|picture original aa||picture mutated aa||combined picture|
Subsitution 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 accoding 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 Phenylalanine to Serine has low values that are nearer to the values for the rarest subsitution for all three matrices. Therefore, an exchange at this position is very unlikely and a mutation there will almost certainly cause structural changes which can affect functional changes.
|PAM 1||Pam 250||BLOSOUM 62|
|value aa||most frequent substitution||rarest substitution||value aa||most frequent substitution||rarest substitution||value aa||most frequent substitution||rarest substitution|
|2||28 (Tyr)||0 (Asp, Cys, Glu, Lys, Pro, Val)||2||20 (Tyr)||1 (Arg, Asp, Cys, Gln, Glu, Gly, Lys, Pro)||-2||3 (Tyr)||-4 (Pro)|
Besides, we looked additional at the position specific scoring matrix (PSSM) for ouer 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 Phenylalanine to Serine at this position is very low and near the value for the rarest substitution. This means a substitution at this positin is likely very uncommon which indicates that substitutions have bad effects as a consequence. Therefore, we conclude that this mutation will probably cause protein structure changes as well as functional changes.
|value aa||most frequent substitution||rarest substitution|
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 first colored column. Here we can see, that the all other mammalians have on this position the amino acid Phenylalanine. Therefore, the mutation on this position is highly conserved and a mutation there will cause probably huge structural and functional changes for the protein.
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 drastical the mutation can be. In this case both tools agree and predict at the position of the mutation a coil. This has 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 drastical change of the protein structure and its function is unlikly because the mutation does not affect a secondary struture element. The change of the coil will probably only take places between two secondary structure elements which will probably not change.
JPred: ...EEEECCCCCEEEEEECCCCCCCHHHHHHHHHHHHHHCCCEEEEEEECCCCCCCCC... PsiPred: ...EEECCCCCCCCCCCCCCCCCCCHHHHHHHHHHHHHCCCCEEEEEECCCCCCCEEC...
Comparison with the real Structure:
Afterwards we also visualize the position of the muation (red) in the real 3D-structure of PDB and compare it with the predicted secondary structure. The visualisation 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 mutationposition almost agree with the position of the predicted secondary structure and is within a coil. Like explained above this means a mutation will probably not destroy a secondary structure element which affects no drastical 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 extrem changes.
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 Serine which is the real mutation in this case. SNAP has a result that the exhange from Phenylalanine to Serine at this position is non-neutral with a high accuracy. This means that this certain mutation on this position cause very likely structural and functional changes of the protein.
|Substitution||Prediction||Reliability Index||Expected Accuracy|
A detailed list of all possible substitutions can be found [here]
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.
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.
Finally, we also regarded the PolyPhen2 prediction for this muation. This prediction visualizes have strongly demaging the mutation probably will be. Therefore it gives the result for two possible cases: HumDiv and HumVar. HumDiv is a prefered model for evaluation rare allels, dense mapping of regions identified by genome-wide assiociation studies and analysis of neutral selection. In contrast, HumVar is a prefered model for diagnostic of Mendelian diseases which require distinguishing mutations with drastic effects from all remaining human variations including abundant mildly deleterious allels. 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 probably damaging. This means that the mutation is not neutral and will probably destroy the structure and the function of the protein.