Canavan Task 6 - Sequence-based mutation analysis

From Bioinformatikpedia
Revision as of 16:38, 13 June 2012 by Gatzmannf (talk | contribs) (Physico-chemical effects)

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">


Hydrophobicity: Kyte J, Doolittle RF (May 1982).Journal of Molecular Biology 157 (1): 105–32.
Volume: A.A. Zamyatin, Prog. Biophys. Mol. Biol., 24(1972)107-123
Pi: JC Biro, Theor Biol Med Model. 2006; 3: 15
Grantham Score: Grantham R (1974) Amino acid difference formula to help explain protein evolution. Science 185: 862–864
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.