Canavan Task 6 - Sequence-based mutation analysis
Contents
Protocol
Further information can be found in the protocol.
Choosing mutations
The ten chosen mutations are listed below in <xr id="canavan_muts"/>.
<figtable id="canavan_muts">
Mutant | E285A | A305E | G123E | R71H | R71K | K213E | V278M | A93A | T277T | P280P |
Known effect | <= 1% activity left | 0% activity left | <= 25% | Canavan Disease | not disease related | Canavan Disease | not disease related | synon. SNPs - not disease related |
synon. SNPs - not disease related |
synon. SNPs - not disease related |
structural location | helix - binding pocket | loop C-terminus | beta-sheet | active site | active site | loop region | loop region | outer helix | outer loop | outer loop |
Sources | dbSNP SNPdbe OMIM HGMD |
dbSNP SNPdbe OMIM HGMD |
SNPdbe HGMD |
dbSNP SNPdbe HGMD |
SNPdbe | HGMD | dbSNP SNPdbe |
dbSNP | dbSNP | dbSNP |
</figtable>
Physico-chemical effects
<xr id="mutations_summary"/> is meant to give a first idea of the nature of the mutations. For each of the ten mutations, we included following properties for the amino acids:
- Hydrophobicity scores range from -7.5 (Arg) to 3.1 (Ile), where Arginine is most hydrophilic and Isoleucine most hydrophobic
- the volume, measured in Å3
- the isoelectric point, which is the pH at which the amino acid's overall charge is zero
- the Grantham score, which classifies amino acid substitutions into the classes conservative (0-50, coloured green), moderately conservative (51-100, yellow), moderately radical (101-150, orange), or radical (≥151, red)
TODO: Valine - Glutamic Acid - small-bulky-issue
<figtable id="mutations_summary">
Mutation | Amino acid | Hydrophobicity (and diff.) | Volume [Å3] (and diff.) | Isoelectric point pI(Charge) | Grantham Score | |||
E285A | Glutamic Acid | -3.5 (polar) | 5.3 | 138.4 (bulky) | 49.8 | 3.2 (negative) | 2.8 | 107 |
Alanine | 1.8 (nonpolar) | 88.6 (tiny) | 6.0 (neutral) | |||||
A305E | Alanine | 1.8 (nonpolar) | 5.3 | 88.6 (tiny) | 49.8 | 6.0 (neutral) | 2.8 | 107 |
Glutamic Acid | -3.5 (polar) | 138.4 (bulky) | 3.2 (negative) | |||||
G123E | Glycine | -0.4 (nonpolar) | 3.1 | 60.1 (tiny) | 78.3 | 6.0 (neutral) | 2.8 | 98 |
Glutamic Acid | -3.5 (polar) | 138.4 (bulky) | 3.2 (negative) | |||||
R71H | Arginine | -4.5 (polar) | 1.3 | 173.4 (bulky) | 20.2 | 10.8 (positive) | 3.2 | 29 |
Histidine | -3.2 (polar) | 153.2 (bulky) | 7.6 (neutral) | |||||
R71K | Arginine | -4.5 (polar) | 0.6 | 173.4 (bulky) | 4.8 | 10.8 (positive) | 1.1 | 26 |
Lysine | -3.9 (polar) | 168.6 (bulky) | 9.7 (positive) | |||||
K213E | Lysine | -3.9 (polar) | 0.4 | 168.6 (bulky) | 30.2 | 9.7 (positive) | 6.5 | 26 |
Glutamic Acid | -3.5 (polar) | 138.4 (bulky) | 3.2 (negative) | |||||
V278M | Valine | 4.2 (nonpolar) | 2.3 | 140.0 (small) | 22.9 | 6.0 (neutral) | 0.3 | 21 |
Methionine | 1.9 (nonpolar) | 162.9 (bulky) | 5.7 (neutral) |
</figtable>
Substitution matrices
Blossum62
This substitution matrix can be found in the protocol. The lowest score in Blosum62, that represents a really unlikely mutation event is -4 and the highest score, that represents a likely mutation is 11. Blossum substitution matrices are derived from non gapped local alignments of the BLOCKS database. Blossum62 is derived from local alignments of sequences with at most 62% sequence similarity. The scores in the matrix represent the log-odds values of a substitution. Negative scores meaning the substituion is less than randomly expected and positive values meaning the substitution is observed more than randomly expected.
PAM1
This substitution matrix can also be found in the protocol. The worst score in PAM1 is 0 for 0% mutation probability and the highest score is 56 for 0.56% mutation probability. A score in PAM1 expresses how probable a mutation from A to B will be, assuming the two proteins are 99% similar (1% AA mutations in both sequences). As we are just considering variations of the same protein within a family, mutation generally are very unlikely with the most probable mutation being Asp to Glu with 0.56%.
E285A
Location
This amino acid is located at the end of helix in the binding pocket. Yet is it not involved in substrate binding. DSSP assigned does not assign a state to E285.
physico-chemical properties
Glutamic acid and Alanine have different physico-chemical properties, which is represented in a moderate Grantham Score of 107. Whereas glutamic acid is negatively charged, alanine has no charge or polarity. Furthermore Alanine is much smaller than glutamic acid.
substitution matrices
In Blosum62 the substitution E --> A is scored -1. This means, that a substitution from E to A is very unlikely, since the lowest score in Blosum62 is -4 and negative scores refer to less than randomly expected events.
In PAM1 the substitution E --> A is scored 17 which equals a mutation probability of 0.17%.
Therefore both substitution matrices expect this mutation to be rather unlikely.
PSSM
SIFT
SIFT predicts an effect of this mutation with a score of 0.00. Amino acids with probabilities < .05 are predicted to be deleterious. The confidence for this prediction is 0.94 and therefore very high, so that the prediction seems reasonable.
Substitution at pos 285 from E to A is predicted to AFFECT PROTEIN FUNCTION with a score of 0.00. Median sequence conservation: 3.01 Sequences represented at this position:17
Furthermore, SIFT predicts ANY substitution of E at this position to be deleterious:
pos A C D E F G H I K L M N P Q R S T V W Y 285E 0.94 0.00 0.00 0.00 1.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
Polyphen2
This mutation is predicted to be probably damaging with a score of 1.000 (sensitivity: 0.00; specificity: 1.00)
Polyphen used 64 sequences of a multiple sequence alignment at position 285.
Evaluation
ashkenazi
A305E
As for E285A, the physico-chemical properties for Alanine and Glutamic acid are different, which is represented in a moderate Grantham Score of 107. Whereas glutamic acid is negatively charged, alanine has no charge or polarity. Furthermore Alanine is much smaller than glutamic acid.
location
A305 is located at the end of a beta sheet at the C-terminus of the protein (protein length: 313 AA). DSSP also assignes state E to this residue as the last residue in the respective sheet.
physico-chemical properties
As for E285A, the physico-chemical properties for Alanine and Glutamic acid are different, which is represented in a moderate Grantham Score of 107. Whereas glutamic acid is negatively charged, alanine has no charge or polarity. Furthermore Alanine is much smaller than glutamic acid.
substitution matrices
As Blossum is a symmetrical substitution matrix, the score for A305E is the same as for E285A: -1. The lowest score in Blosum64, that represents a really unlikely mutation event is -4 and the highest score, that represents a likely mutation is 11. This means, that a substitution from E to A is very unlikely.
In PAM1 the substitution A --> E is scored 10 which equals a mutation probability of 0.1%.
Therefore both substitution matrices expect this mutation to be rather unlikely.
Evaluation
non-ashkenazi
G123E
R71H
R71C
K213E
V278M
A93A
T277T
P280P
References
Henikoff, S. & Henikoff, J.G. (1992) "Amino acid substitution matrices from
protein blocks." Proc. Natl. Acad. Sci. USA 89:10915-10919.