Sequence-based mutation analysis GLA
by Benjamin Drexler and Fabian Grandke
Contents
Introduction
Selected Mutations
We randomly selected ten annotated point mutations of the human gene GLA and they were chosen out of a pool of mutations that consist of two subsets. The first subset contains mutations that are present in HGMD and these mutations were already gathered in the task 4 Mapping SNPs. The second subset are mutations that are present in dbSNP, but not included in HGMD. This was only the case for three mutations.
Mutations at the amino acid position between 1 and 31 were not included in the selection process, because they are part of the signal peptide (see UniProt entry) and they are not present in the reference structure (PDB ID 1R47).
Number | AA-Position | Codon change | Amino acid change | Visualization |
---|---|---|---|---|
1 | 42 | ATG-ACG | Met -> Thr | |
2 | 65 | AGT-ACG | Ser -> Thr | |
3 | 117 | ATT-AGT | Ile -> Ser | |
4 | 143 | cGCA-ACA | Ala -> Thr | |
5 | 186 | CAC-CGC | His -> Arg | |
6 | 205 | gCCT-ACT | Pro -> Thr | |
7 | 244 | gGAC-CAC | Asp -> His | |
8 | 283 | CAG-CCG | Gln -> Pro | |
9 | 321 | tCAG-TAG | Gln -> Glu | |
10 | 363 | TATa-TAA | Tyr -> Cys |
The visualization was done by using PyMol and the mutagensis of the residue was performed according to this tutorial. The residue of the wildtype is colored green and the mutated residue is colored red.
Mutation Analysis
Physicochemical Properties and Changes
Substitution Matrices
In this section, we take a look at substitution matrices to evaluate whether the introduced substitution of the mutation is favorable in a biological context. For this, we use two different kinds of substitution matrices. First, Blocks of Amino Acid Substitution Matrix (BLOSUM) is a evidence based matrix which is calculated of alignments between proteins <ref name=blosum>en.wikipedia.org/wiki/BLOSUM</ref>. Second, Point Accepted Mutation or Percent Accepeted Mutation (PAM) is a set of matrices that is derived of from the amino acid substitutions between closely related proteins <ref name=pam>en.wikipedia.org/wiki/Point_accepted_mutation</ref>. In general, a high value in a substitution matrix indicates a more likely substitution.
Number | Substitution | BLOSUM62 | PAM1 | PAM250 | ||||||
---|---|---|---|---|---|---|---|---|---|---|
Mutation | Best1 | Worst2 | Mutation | Best | Worst | Mutation | Best | Worst | ||
1 | Met -> Thr | -1 | 2 | -3 | 2 | 8 | 0 | 1 | 3 | 0 |
2 | Ser -> Thr | 1 | 1 | -3 | 38 | 38 | 0 | 9 | 9 | 3 |
3 | Ile -> Ser | -2 | 2 | -4 | 1 | 33 | 0 | 3 | 9 | 1 |
4 | Ala -> Thr | -1 | 1 | -3 | 32 | 35 | 0 | 11 | 12 | 2 |
5 | His -> Arg | 0 | 2 | -3 | 8 | 20 | 0 | 5 | 7 | 2 |
6 | Pro -> Thr | 1 | 1 | -4 | 4 | 13 | 0 | 5 | 7 | 1 |
7 | Asp -> His | -1 | 2 | -4 | 4 | 53 | 0 | 6 | 10 | 1 |
8 | Gln -> Pro | -1 | 2 | -3 | 6 | 27 | 0 | 4 | 7 | 1 |
9 | Gln -> Glu | 2 | 2 | -3 | 27 | 27 | 0 | 7 | 7 | 1 |
10 | Arg -> Cys | -3 | 2 | -3 | 1 | 19 | 0 | 2 | 9 | 1 |
1 Best is the highest value in the regarding column/row except for the self-substitution (e.g. Met -> Met).
2 Worst is the lowest value in the regarding column/row.
The following coloring scheme was applied:
- green: the substitution value of the mutation is closer to the best value than to the worst value
- red: the substitution value of the mutation is closer to the worst value than to the best value
- gray: the substitution value of the mutation has the same absolute difference to both values
The following substitution matrices were used:
PSSM
In this section, we use a point specific scoring matrix (PSSM) to evaluate how the conservation of the wildtype and mutant residue is in related proteins. For this, we used PSI-BLAST with the following command:
blastpgp -i GLA.fasta -j 5 -d /data/blast/nr/nr -e 10E-6 -Q psiblast.mat -o psiblast.out
The relevant values are listed in the following table. The full PSSM of the certain positions is provided on this page.
Number | Substitution | PSSM | |||
---|---|---|---|---|---|
Mutation | Wildtype | Best1 | Worst2 | ||
1 | Met -> Thr | -3 | 9 | 9 | -6 |
2 | Ser -> Thr | 3 | 3 | 5 | -3 |
3 | Ile -> Ser | -3 | 4 | 5 | -6 |
4 | Ala -> Thr | -1 | 1 | 2 | -5 |
5 | His -> Arg | 1 | 0 | 2 | -5 |
6 | Pro -> Thr | -2 | 2 | 3 | -5 |
7 | Asp -> His | 1 | 4 | 4 | -5 |
8 | Gln -> Pro | -5 | 3 | 8 | -5 |
9 | Gln -> Glu | 1 | 7 | 7 | -5 |
10 | Arg -> Cys | -1 | 2 | 3 | -4 |
1 Best is the highest value in the regarding row.
2 Worst is the lowest value in the regarding row.
The following coloring scheme was applied:
- green: the substitution value of the mutation is closer to the best value than to the worst value
- red: the substitution value of the mutation is closer to the worst value than to the best value
- gray: the substitution value of the mutation has the same absolute difference to both values
Multiple Sequence Alignment
We take a look at a multiple sequence alignment (MSA) to evaluate the conservation of the residues which are affected by one of the mutations. We used BLAST to get the sequences for the MSA. A table of the sequences is provided on this page. Afterwards we created two MSAs with the locally installed T-Coffee.
- MSA with 100 sequences: see here
- MSA with 25 sequences: see figure 1
We used the conservation index according to Livingstone C.D. and Barton G.J.<ref name=livingstone>Livingstone C.D. and Barton G.J. (1993), "Protein Sequence Alignments: A Strategy for the Hierarchical Analysis of Residue Conservation.", CABIOS Vol. 9 No. 6 (745-756)), PubMed</ref> to determine whether a residue is conserved or not. The conservation index was calculated by JalView and ranges from 0 to 11.
Number | Position | Conservation (100 sequences) | Conservation (25 sequences) |
---|---|---|---|
1 | 42 | 10 | 11 |
2 | 65 | 8 | 11 |
3 | 117 | 9 | 9 |
4 | 143 | 10 | 8 |
5 | 186 | 4 | 2 |
6 | 205 | 10 | 11 |
7 | 244 | 10 | 11 |
8 | 283 | 11 | 11 |
9 | 321 | 11 | 11 |
10 | 363 | 3 | 5 |
Secondary Structure
We examined the potential influence of the mutation on the secondary structure of the α-galactosidase. For this, we used two programs that predict the secondary structure, i.e. PSIPRED and JPred3. Please see task 2 - sequence-based predictions for further explanations of the programs.
We performed one run with the wildtype sequence of α-galactosidase and afterwards ten runs with an isolated mutation, so that the concurrence of two or more mutations at the same time do not influence the prediction. Even though this would be unlikely, since the distance between each pair of mutations should be large enough. The results are listed in the table below. As it can be seen, none of the mutations seem to influence the secondary structure atleast in the prediction.
Number | Substitution | UniProt | PSIPRED | PSIPRED | ||
---|---|---|---|---|---|---|
Wildtype | Mutation | Wildtype | Mutation | |||
1 | Met -> Thr | Beta strand | Coil | Coil | Coil | Coil |
2 | Ser -> Thr | Coil | Coil | Coil | Helix | Helix |
3 | Ile -> Ser | Helix | Coil | Coil | Helix | Helix |
4 | Ala -> Thr | Coil | Coil | Coil | Coil | Coil |
5 | His -> Arg | Helix | Helix | Helix | Helix | Helix |
6 | Pro -> Thr | Helix | Coil | Coil | Coil | Coil |
7 | Asp -> His | Helix | Helix | Helix | Helix | Helix |
8 | Gln -> Pro | Helix | Helix | Helix | Helix | Helix |
9 | Gln -> Glu | Coil | Coil | Coil | Coil | Coil |
10 | Arg -> Cys | Beta strand | Beta strand | Beta strand | Beta strand | Beta strand |
Programs
SNAP
SIFT
PolyPhen
Discussion
References
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