Task 8: Sequence-based mutation analysis

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Lab journal task 8

Mutation selection

<figtable id="mutations">

Table 1: List of the 10 selected mutations from HGMD and dbSNP.

mutations
nucleotide change	amino acid change
	Val53Met
	His63Asp
	Arg67His
	Met97Ile
	Asn130Ser
	Glu168Gln
	Leu183Pro
	Thr217Ile
	Cys282Tyr
	Arg330Met

</figtable>

Mutation analysis results

<figtable id="summary">

Table 2: Summary of the results of analysis of all mutations.

reference	mutation	pyhsicochemical properties		strucural properties		conservation			prediction programms				consensus
		from	to	pymol visualisation	secondary structure	substitution matrix	PSSM	MSA	SIFT	Polyphen2	MutationTaster	SNAP
	Val53Met	brached chain, hydrophobic, nonpolar, neutral charge	sulfur containing, nonpolar, neutral charge	Val53Met mutation with valine in yellow and methionine in red.		E (beta sheet)
	His63Asp	aromatic ring, basic polar, mainly neutral charge	acidic polar, negative charge	His63Asp mutation with histidine in yellow and aspartic acid in red.
	Arg67His	aliphatic, basic polar, delocalised positive charge	aromatic ring, basic polar, mainly neutral charge	Arg67His mutation in the center with arginine in yellow and histidine in red.	C (loop)
	Met97Ile	sulfur containing, nonpolar, neutral charge	hydrophobic, nonpolar, neutral charge	Met97Ile mutation with methoinine in yellow and isoleucine in red.	C (loop)
	Asn130Ser	polar, neutral charge	polar, neutral charge	Asn130Ser mutation with asparagine in yellow and serine in red.	C (loop)
	Glu168Gln	acidic polar, negative charge	polar, neutral charge	Glu168Gln mutation with glutamic acid in yellow and glutamine in red.	H (helix)
	Leu183Pro	branched chain, hydrophobic, nonpolar, neutral charge	nonpolar, neutral charge	Leu183Pro mutation with leucine in yellow and proline in red.	H (helix)
	Thr217Ile	polar, neutral charge	hydrophobic, nonpolar, neutral charge	Thr217Ile mutation with threonine in yellow and isoleucine in red.	C (loop)
	Cys282Tyr	thiol side chain, nonpolar, neutral charge	polar, neutral charge	Cys282Tyr mutation with cysteine in yellow and threonine in red.	E (beta sheet)
	Arg330Met	aliphatic, basic polar, delocalised positive charge	sulfur containing, nonpolar, neutral charge	Could not be visualized because this resdiue is not contained in the the pdb structure.	-

</figtable>

All results from the mutation analysis are summarised in <xr id="summary"/>. Physicochemical properties are specified as characteristics of the aa, side chain polarity and charge.

Val53Met
The main effect of the change from valine to methonine is mainly due to the structure, because both aa are nonpolar and neutral. Methionine is linear and valine has a branched structure. As can be seen in the picture, this could lead to clashes with the alpha helix above the methionine. Also, valine is hydrophob whereas methionine is not.
His63Asp
This mutation is could be disease causing, because the basic aa histidine is replaced by the acidic aspartic acid. Besides, histidine is mainly neutral whereas the aspartic acid is negatively charged. But the pymol picture shows that residue 63 is located in a surface loop. Since both aa are not hydrophob, the implication of this aa exchange for the function of the protein is therefor not as severe as if this mutation would be located in the interior of the protein.
Arg67His
Arginine is able to form multiple H-bonds due to its delocalised positive charge and histidine is also able to form H-bonds. Both aa are basic polar. Thus, the main difference between arginine and histidine is the positive and neutral charge and that histidine contains an aromatic ring whereas arginine is aliphatic. The pymol visualisation of the mutation shows that the mutation is located in a surface loop and since both aa are not hydrophob, we think that the effect of the mutation should not be too severe.
Met97Ile
The main difference between methinine and isolecine is that methinine contains a sulfur atom and isoleucine not. Both aa are nonpolar and neutral charged, but isoleucine is hydropbob and methionine not due to its sulfur. The residue 97 is part of an alpha helix.
Asn130Ser
Asparagine can form H-bonds with the backbone and serine is also able to form H-bonds. The physicochemical proberties of both aa are very similar, therefore we think, that this mutations is probably not disease causing. This opinion is also supported by the 3D visualisation of the mutation which shows that the asparagine is located at the end of a beta sheet on the surface of the proteins. The isoleucine at this position does not disrupt the local structure.
Glu168Gln
Glutatimc acid as well as glutamie are both polar, but glutamic acid has a negative charge whereas glutamine is neutral. The only difference between the two aa is that glutamine has an NH group where glutamic acid contains an OH group. Consequently there is not big difference in 3D structure but only in physiochemical properties. This can also be seen in the pymol picture.
Leu183Pro
Proline is a unique aa in that it disrupts secondary structure. Since the leucine is located inside an alpha helix (see picture), the mutation to proline disrupts the helix and thus most probably leads to a disturbed function of the protein and causes the disease.
Thr217Ile
Threonine is polar whereas isoleucine is nonpolar and thus hydrophob. As the threonine is located on the surface of the protein, as can be seen in the pymol visualisation, a hydrophob residue at this position might lead to a structural change of the protein.
Cys282Tyr
Cystein can form an disulfide bonds with another cystein residue. Disulfide bonds are very important for the protein structure and stability. The picture shows that the cystein is part of a beta sheet and located in the interior of the protein. The polar threonine thus disrupts possible disulfide bonds and the protein structure. This mutation is therefore likely disease causing.
Arg330Met
Arginine and methionine have very different physicochemical properties. Arginine is basic polar and positive charged whereas methionine is nonpolar and uncharged. This mutation could therefore als have an effect on the protein function.