Rs1054374

General Information

SNP-id	rs1054374
Codon	293
Mutation Codon	Ser -> Ile
Mutation Triplet	AGT -> ATT

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Sequence-based Mutation Analysis

Pysicochemical Properities

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

Ser	Ile	consequences
polar, tiny, hydrophilic, neutral	aliphatic, hydrophobic, neutra	Ile is much bigger than Ser and also branched, because it is an aliphatic amino acid. Therefore the structure of both amino acids is really different and Ile is too big for the position where Ser was. Therefore, there has to be a big change in the 3D structure of the protein and the protein probably will loose its function.

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Visualisation of the Mutation

In the next step, we created the visualization of the muation with PyMol. Therefore we created a picture for the original amino acid, for the new mutated amino acid and finally for both together in one picture whereas the mutation is white colored. The following pictures display that the original amino acid Serine looks different to Isoleucine. Serine is very small whereas Isoleucine is bigger and has two spreadin chains. The first part of the rest agrees in both amino acids. In this case the difference is not so heavy, but can also cause some structural changes which can have affects on the protein function. All in all, the mutation will probably have no structural or functional changes.

picture original aa	picture mutated aa	combined picture
Amino acid Serine	Amino acid Isoleucine	Picture which visualize the mutation

Subsitution 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 accoding amino acid substitution, the most frequent value for the substitution of the examined amino acid and the rarest substitution.

In this case, the substitution of Serine to Isoleucine acid has very low value that is nearer to the values for the rarest subsitution for PAM1. Contrary, for PAM250 the value for the amino acid subsitution Histidine to Aspartic acid is average. This means the most frequent subsitution value is almost as far as the rarerest subsitution from the the underlying value. The difference between the two PAMs can be ascribed to the different preparations of these two kind of substitutions matrices. For the PAM1-matrix the substitution rate is 1% which means the probability that one amino acid changes is 1% and that there is 99% similarity. Contrary, PAM250 means that 250 mutations have been fixed per 100 residues which has as result only similarity about 20%. A possible reason that PAM250 has a better value for the amino acid substitution is that the similarity is low and the amino acids are probably dissimilar. BLOSOUM62 has like PAM1 for this substitution a very low value that is nearer to the values for the rarest subsitution. Therefore, according to PAM1 and BLOSOUM62 a mutation at this position will almost certainly cause structural changes which can affect functional changes. The value from PAM250 is not realy significant and therefore we are not able to determine effects on the protein.

PAM 1			Pam 250			BLOSOUM 62
value aa	most frequent substitution	rarest substitution	value aa	most frequent substitution	rarest substitution	value aa	most frequent substitution	rarest substitution
2	38 (Thr)	1 (Leu)	5	9 (Ala, Gly, Pro, Thr)	3 (Phe)	-2	1 (Ala, Asn, Thr)	-3 (Trp)

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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 the substitution rate for Serine to Isoleucine at this position is very low and near to the value for the rarest substitution. This means this substitution at this position is likely very uncommon which indicates that this substitution has bad effects as a consequence. Therefore, we concluded that this mutation will probably cause protein structure changes as well as functional changes.

PSSM
value aa	most frequent substitution	rarest substitution
-3	4	-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. Here we can see, that many other mammalians have another amino acid at this position. Only four other mammalian agrees and have a Serine at this position. Therefore, the mutation at this position is not highly conserved and a mutation there will probably cause no structural and functional changes in the protein.

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 drastical the mutation can be. In this case both tools agree and predict a coil at the position of the mutation. This has a result, that the mutation at this position would not destroy or split a secondary structure element. It will probably only changes the coil between two secondary structure elements, but this can sometimes also cause a change of the the following secondary structure. Our protein do not posses any disordered regions and therefore, this mutation will not destroy any functional very important coil regions. We think that a drastical change of the protein structure and its function is unlikly because the mutation does not affect a secondary struture element. The change of the coil will probably only take places between two secondary structure elements which will probably not changes the protein.

JPred:
...HHHHHHHHCCCEEEECCCCCHHHHHHHHHCCCCCCCCCCCCCCCCCCCCCCCCCC...
PsiPred:
...HHHHHHHHCCCEEEECCCCCHHHHHHHHCCCCCCCCCCCCCCCCCCCCCCCCCCH...

Comparison with the real Structure:

Afterwards we also visualize the position of the muation (red) in the real 3D-structure of PDB and compare it with the predicted secondary structure. The visualisation 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 mutationposition almost agree with the position of the predicted secondary structure and is within a coil. Like explained above this means a mutation will probably not destroy a secondary structure element which affects no drastical structural change. Otherwise it can cause a change of the position of the two nearest secondary structure element which can has a functional loose as a consequence. We think that a structural change is unlikely, because it is not within a secondary structure element and will therefore not cause extrem changes of the protein.

Mutation at position 293

Mutation at position 293 - detailed view

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SNAP Prediction

Next, we looked at the result of the SNAP prediction. For this prediction we took the amino acid of the certain position and checked every possible amino acid mutation. Afterwards we extract the result for Isoleucine which is the real mutation in this case. SNAP has a result that the exchange from Serine to Isoleucine at this position is neutral with a relative high accuracy. This means that this certain mutation at this position cause very likely no structural and functional changes of the protein.

Substitution	Prediction	Reliability Index	Expected Accuracy
I	Neutral	2	69%

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

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SIFT Prediction

Next, we used SIFT Prediction which displays if a mutation is neutral or not. Therefore, it first shows a row which contains a score for the particular mutationposition to a certain amino acid. The amino acid which are not tolerated at this position are colored red. Besides, it also constructs a table which lists the amino acids that are predicted as tolerated and not-tolerated.

In this case, there are seven substitutions that are tolerated: Methionine, Isoleucine, Alanine, Serine, Valine, Threonine and Leucine. The substitution to Isoleucine is tolerated at this position. This means that this mutation at this position is probably neutral and will not cause any structural and function changes of the protein.

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.

SIFT Table
Threshold for intolerance is 0.05.
Amino acid color code: nonpolar, uncharged polar, basic, acidic.
Capital letters indicate amino acids appearing in the alignment, lower case letters result from prediction.

Predict Not Tolerated																				Position	Seq Rep	Predict Tolerated
							w	g	h	y	d	r	n	f	q	e	k	c	p	293S	1.00	M	l	A	S	V	T	I

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PolyPhen2 Prediction

Finally, we also regarded the PolyPhen2 prediction for this muation. This prediction visualizes have strongly demaging the mutation probably will be. Therefore it gives the result for two possible cases: HumDiv and HumVar. HumDiv is a prefered model for evaluation rare allels, dense mapping of regions identified by genome-wide assiociation studies and analysis of neutral selection. In contrast, HumVar is a prefered model for diagnostic of Mendelian diseases which require distinguishing mutations with drastic effects from all remaining human variations including abundant mildly deleterious allels. We decided to look at both possible models, which are agrees in the most cases.

In this case both models predict that the mutation is benign. This means that the mutation is neutral and will probably not damage the structure and the function of the protein.

HumDiv prediction

HumVar prediction

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Structure-based Mutation Analysis

Mapping onto Crystal Structure

Visualization of the mutation and important functional sites

Color declaration:

red: position of mutation
green: position of active side
yellow: position of glycolysation
cyan: position of Cystein

First of all, we colored the important residues and also the mutated residue in the crystal structure, to see if the mutation is near of far away from the functional residues. As you can see on the picture, the mutation is located within a loop near to a cystein residue. Therefore, if the mutation causes changes in the structure it is possible, that the cystein can not build a disulfid bond any longer. This could causes dramatical changes in the structure of the protein which leads to an inactive protein structure.

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SCWRL Prediction

Next, we analysed the mutation in more detail. Therefore we looked directly to the structure and orientation of the residue.

picture original aa	picture mutated aa	combined picture
Amino acid Serine	Amino acid Isoleucine	Picture which visualize the mutation

On the picture you can see that the amino acids are very different. The Isoleucine (mutated amino acid) is bigger than the original amino acid Serine. This could lead to clashes and additional H-Bonds within the protein. If there are clashes, the protein has to fold in another way, which destroy the disulfid bond between the two cystein residues which could destroy the complete structure and function of the protein.

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FoldX Energy Comparison

One important point in the analysis of mutations is to look at the energy of the protein with the original and with the mutated amino acid. Often the energy increases dramatically with the mutated amino acid. This means, that the protein becomes very instable and therefore, it is often possible that the protein can not bind its ligands any longer. Otherwise, it is also possible, that the protein with the mutated amino acid has a lower energy than the original protein. This means, that the protein is too rigid and loses its flexbility. Than it is also possible, that the protein can not bind the ligands any longer.

Therefore, we compared the energy of our protein with different methods. Here we want to present the result of FoldX.

Original total energy	Total energy for the mutated protein	Strongest energy changes within the mutated protein
-154.17	-152.15	Energy_vdwclash

The energy of the mutated structure is a little bit lower than the energy of the original structur, but the values do not differ that much. The strongest energy changes within the mutated protein are vdwclashes, which means that there is a clash in the van-der-Waals binding. So it seems that there is not a problem with the H-Bonds but instead there is a problem with electrostatic interactions within the protein. This is a strong hint, that this mutation destroy the protein. To have the possibility to compare the energy values with values from other programs, we calculated a ratio between these two values.

Ratio of the original structure	Ratio of the mutated structure	Differences
100	98.79	1.21

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Minimise Energy Comparison

Next we use the minimise energy tool to compare the energy values of the two different structures.

Comparing Energy:

Original total energy	Total energy for the mutated protein
-9610.467157	-6189.246312

As before the energy of the structure is lower. But in this case the difference between the two energy values is much stronger than by our analysis with FoldX. To compare the values in a stricter way we calculated again a ratio between the energy values.

Ratio of the original structure	Ratio of the mutated structure	Difference
100	64.40	35.60

In this case the ratio between the energy is about 35%, which is a very high value.

Comparing Structure:

This tool also gives as output a pdb file with the position of the original and the mutated amino acid.

picture original aa	picture mutated aa	combined picture
Amino acid Serine	Amino acid Isoleucine	Picture which visualize the mutation

The pictures show, that the structure of the amino acids differ extremly. The orientation of the amino acids is similar to that shown with SCWRL.

Visualization of H-bonds and Clashs:

To get more insight in the effects of the mutated amino acid on the structure, we also analysed the H-bonds and clashes of the Asparagine residue.

H-bonds of the original amino acid	H-bonds near the mutation	Clashes of the mutation
H-bonds of the original amino acid (colored in magenta)	H-bonds of the mutated amino acid (colored in red)	Possible clashes

We can see, that the original amino acid has two H-Bonds to another residue in the protein. These two bonds are lost, if we look at the picture with the mutated amino acid. Therefore, there is also a problem with missing H-Bonds in the protein. This means, that the protein with the mutated amino acid is not that stable as the original structure.

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