|Mutation Codon||Leu -> Arg|
|Mutation Triplet||CTT -> CGT|
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.
|aliphatic, hydrophobic, neutral||positive charged, polar, hydrophilic||Leucine is smaller and without a positive charge. Therefore, Arg is too big for the position of Leu, therefore, the change of Leu with Arg has to cause changes in the 3D structure of the protein. Furthermore, Leu is a hydrophobic amino acid, whereas Arg is hydrophilic. This is the complete contrary and therefore we suggest, that the protein will not function any longer.|
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 Arginine has a longer chain than Leucine. Contrary Leucine comes to a fork at the end of its rest. This shows that the amino acids have some structural differences that are not drastical, but can be essential.
|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 Leucine to Arginine has very low values that are near the values for the rarest subsitution for PAM1 and PAM250. Furthermore, the value for the most frequent substitution differs also a lot from the value for this certain mutation for both PAMs. Contrary for BLOSUM62 the value for the amino acid subsitution Leucine to Arginine is average. This means the most frequent subsitution value is as far as the rarerest subsitution from the the underlying value. The difference between the two PAMs and BLOSUM62 can be ascribed to the different preparations of these two kind of substitutions matrices. The PAM-matrices is an evolutionary model whereas BLOSUM is based on protein famalies. Therefore probably this mutation is evolutionary not that unlikely whereas within a protein family it is unusual.
|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|
|1||22 (Ile)||0 (Asp, Cys)||4||20 (Met)||2 (Cys)||-2||0 (Phe)||-4 (Asp, Gly)|
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. Here we can see, that the most other mammalians have on this Position a Leucine. Only three mammalians differ and have on this position an Isoleucine. Therefore, the mutation on this position has probably a structural and functional change as a result.
Secondary Structure Mutation Analysis
As a next step we compared the different results of the secondary structure prediction tools JPred and PsiPred. In this case both tools agree and predict a sheet. This shows th
JPred: CCHHHHHHHHHHHHHCCCCCCCEEEEEEEEEECCCEEEEECCCEEEEECCCCCCC... PsiPred: CHHHHHHHHHHHHHHHCCCCCCCCCCCCEEEECCCEEEEECCCEEEEECCCCCCC...
Comparison with the real Structure:
Afterwards we looked also at the 3D-structure of our protein and colored the position of this muation red. The visualisation can therefore display if the mutation is in a secondary structure element or in some other regions. This can give also give an overview how drastical the mutation could be. Here in this case the mutation is at the end of a beta sheet. This means a mutation would not destroy the whole beta sheet what could hase as a result that the structural change is not as drastical. Otherwise it can cause a change of the further secondary structure element which can also have functional loose as a consequence.
|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.