Rs121907967

General Information

SNP-id	rs121907967
Codon	329
Mutation Codon	Trp -> TER
Mutation Triplet	TGG -> TAG

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Sequence-based Mutation Analysis

Pysicochemical Properties

First of all, we explored the amino acid properties and compared them for the original and the mutated amino acid. Therefore we thought about the possible effects that the mutation could have on the protein.

Trp	TER	consequences
aromatic, polar, hydrophobic	TER	By this change, the protein is not complete, therefore it is not possible for the protein to fold and to function.

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Visualization of the Mutation

In the next step, we created the visualization of the mutation with PyMol. Therefore we created two pictures: one which displays the original amino acid (Figure 1) and one that displays the consequence of the resulting termination (Figure 2). The grey and the red parts of 3D-structure are the original protein whereas the red part shows the remaining protein if there is an exchange of Tryptophan to a stop codon. Here we can see that the remaining red part has only the half size of the protein. Furthermore, the missing part can have an effect on the folding of the remaining part, which this one can fold in a completely other way. Therefore, this mutation will have engraving effects on the protein. The protein will probably loss its whole function and is not usable anymore.

picture original amino acid	consequence for the whole protein
Figure 1: Amino acid Tryptophan	Figure 2: Visualization of the mutated protein

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Substitution 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 according amino acid substitution, the most frequent value for the substitution of the examined amino acid and the rarest substitution.

In this case, we get no informations because there is no entry for the substitution of Tryptophan to a stop codon. Therefore we can not say what the possible consequences for the protein are only by looking at the substitution matrices. However, a stop codon will if course always have a drastic effect on the protein and its function.

PAM 1			Pam 250			BLOSOUM 62
value amino acid	most frequent substitution	rarest substitution	value amino acid	most frequent substitution	rarest substitution	value amino acid	most frequent substitution	rarest substitution
X	2 (Arg)	0 (all, except Arg, Phe, Ser, Tyr)	X	2 (Arg)	0 (all, except Arg, His, Leu, Phe, Ser, Tyr)	X	2 (Tyr)	-4 (Asn, Asp, Pro)

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PSSM Analysis

Besides, we looked additional at the position specific scoring matrix (PSSM) for our sequence. In contrast to PAM and BLOSOUM, the PSSM contains a specific substitution rate for each position in the sequence. Therefore, the PSSM is more position specific than PAM or BLOSOUM. We extracted the substitution value for the underlying mutation, the value for the most frequent substitution and the rarest substitution.

In this case, we got no information because there is no entry for the substitution of Tryptophan to a stop codon. Therefore we can not say what the possible consequences for the protein are by only looking at the substitution matrices. However, a stop codon will if course always have a drastic effect on the protein and its function.

PSSM
value amino acid	most frequent substitution	rarest substitution
X	12	-5

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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 colored column on Figure 3. Here we can see, that all other mammalians have at this position the same amino acid. Therefore, the mutation at this position is highly conserved and in a normal case this would be a indication for structural and functional changes. In this special case it will cause anyway structural and functional changes, because it is a substitution with a stop codon and therefore only a part of the protein will be translated.

Figure 3: Mutation in the multiple alignment

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Secondary Structure Mutation Analysis

As a next step we compared the different results of the secondary structure prediction tools JPred and PsiPred. Afterwards we can examine in which secondary structure element and where therein the mutation takes place. This can give an overview of how drastic the mutation can be. In this case both tools agree and predict at the position of the mutation a coil. This normally has as a result, that the mutation at this position would not destroy or split a secondary structure element which would have no drastic changes for the protein. In this special case with a substitution to a stop codon there will be anyway structural changes followed by functional loss.

JPred:
...CCCCCCCCCCCCHHHHHHHHHCCCCCCHHHHHHHHHHHHHHHHHHCCCEEEEECC...
PsiPred:
...CCCCCCCCCCCCHHHHHHHHHCCCCCCHHHHHHHHHHHHHHHHHHCCCCEEEECC...

Comparison with the real Structure:

Afterwards we also visualize the position of the mutation (red) in the real 3D-structure of [PDB] and compare it with the predicted secondary structure. The visualization can therefore like above the predicted secondary structure display if the mutation is in a secondary structure element or in some other regions.

Here in this case the mutation position does not agree with the position of the predicted secondary structure and is within a alpha helix (Figure 4 and Figure 5). Normally, this means that the mutation will probably destroy or split the helix which has structural and functional changes of the protein as result. In this special case with a substitution to a stop codon there will be anyway structural changes followed by functional loose.

Figure 4: Mutation at position 329

Figure 5: Mutation at position 329 - detailed view

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SNAP Prediction

No prediction available, because the protein ends here. However, in this special case with a substitution to a stop codon there will be anyway a drastic protein structure change which is followed by the the functional loose of the protein.

A detailed list of all possible substitutions can be found [here]

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SIFT Prediction

SIFT Matrix:
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 (Figure 7).

Figure 6: Legend

Figure 7: Shift Matrix

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PolyPhen2 Prediction

In this case the substitution is from Tryptophan to a stop codon. Therefore, we made no PolyPhen2 prediction, because it is clear that it will cause a damage of the 3D-structure of the protein. Furthermore, it will of course affect a function of the protein hardly and probably the protein is useless afterwards.

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Structure-based Mutation Analysis

Mapping onto Crystal Structure

In Figure 8 you can see the mapping of the functional residues onto the crystal structure of the protein.

Figure 8: Visualization of the mutation and important functional sites
Color declaration:
* red: position of mutation
* magenta: maintained protein
* green: position of active side
* yellow: position of glycolysation
* cyan: position of Cysteine

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SCWRL Prediction

In this case the mutation is from Tryptophan to a stop codon. Therefore, we made no SCREWL prediction, because it is clear that it will cause a damage of the 3D-structure of the protein. Furthermore, it will of course affect a function of the protein hardly and probably the protein is useless afterwards.

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FoldX Energy Comparison

It was not possible to calculate the energy of the mutated protein, because the mutation leads to a stop codon in the sequence. Therefore, we know surly, that the protein is damaged, but we can not calculate any energy values for this structure.

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Minimise Energy Comparison

It was not possible to calculate the energy of the mutated protein, because the mutation leads to a stop codon in the sequence. Therefore, we know surly, that the protein is damaged, but we can not calculate any energy values for this structure.

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Gromacs Energy Comparison

It was not possible to calculate the energy of the mutated protein, because the mutation leads to a stop codon in the sequence. Therefore, we know surly, that the protein is damaged, but we can not calculate any energy values for this structure.

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