Rs121907974

General Information

SNP-id	rs121907974
Codon	211
Mutation Codon	Phe -> Ser
Mutation Triplet	TTC -> TCC

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 created the possible effect that the mutation could have on the protein.

Phe	Ser	consequences
polar, tiny, hydrophilic, neutral	aliphatic, hydrophobic, neutral	Ile is much bigger than Ser and also is 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 mutated amino acid Serine looks very different to Phenylalanine. Phenylalanine has a huge aromatical ring. Contrary, Serine is very smaller and differs a little bit in the orientation. This shows that the amino acids have huge structural differences which will probably cause dramtical effects on protein structure and function.

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

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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 Phenylalanine to Serine has low values that are nearer to the values for the rarest subsitution for all three matrices. Therefore, an exchange at this position is very unlikely and a mutation there will almost certainly cause structural changes which can affect functional changes.

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	28 (Tyr)	0 (Asp, Cys, Glu, Lys, Pro, Val)	2	20 (Tyr)	1 (Arg, Asp, Cys, Gln, Glu, Gly, Lys, Pro)	-2	3 (Tyr)	-4 (Pro)

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PSSM Analysis

Besides, we looked additional at the position specific scoring matrix (PSSM) for ouer 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 Phenylalanine to Serine at this position is very low and near 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
-5	11	-7

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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 first colored column. Here we can see, that all the other mammalians havethe amino acid Phenylalanine on this position. Therefore, the mutation on this position is highly conserved and a mutation there will cause probably huge structural and functional changes for 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 at the position of the mutation a coil. This has as 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. Furthermore, HEXA_HUMAN does not posses any disordered regions and therefore, a mutation in a coiled region do not change a functional important region. 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 change.

JPred:
...EEEECCCCCEEEEEECCCCCCCHHHHHHHHHHHHHHCCCEEEEEEECCCCCCCCC...
PsiPred:
...EEECCCCCCCCCCCCCCCCCCCHHHHHHHHHHHHHCCCCEEEEEECCCCCCCEEC...

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 mutation position 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 extreme changes.

Mutation at position 211

Mutation at position 211 - 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 Serine which is the real mutation in this case. SNAP has a result that the exchange from Phenylalanine to Serine at this position is non-neutral with a high accuracy. This means that this certain mutation on this position cause very likely structural and functional changes of the protein.

Substitution	Prediction	Reliability Index	Expected Accuracy
S	Non-neutral	5	87%

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, the only substitution that is tolerated is the one to Phenylalanine itself. The substitution to Serine is not-tolerated at this position. This means that this mutation at this position is probably not neutral and will cause probably 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 y w v t s r q p n m l k i h g e d c a 211F 1.00 F

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PolyPhen2 Prediction

Finally, we also regarded the PolyPhen2 prediction for this muation. This prediction visualizes how 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 agreed in the most cases.

In this case both models predict that the mutation is probably damaging. This means that the mutation is not neutral and will probably destroy 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 and far away from the functional residues. Therefore, we do not know in which way this mutation affects the global structure of the protein.

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SCWRL Prediction

Because the mapping analysis does not give a good explanation why the mutation causes damages on the protein, we decided to analyse this mutation in more detail. Therefore, we looked for the structure of the original amino acid and the structure of the amino acid after the mutation event and compare them in size and orientation. For this purpose we used SCWRL.

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

As you can see on the picture, the original amino acid (Phenylalanine) has a ring structure and is therefore, an aromatic amino acid. The mutated amino acid is a Serine, which is smaller than Phenylalanine. Therefore there is no problem with the space for the amino acid. Serine is smaller than Phenylalanin and has enough space which means there should not be any clashes with other amino acid residues or with the backbone.

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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	-9594.637506

The total energy of the mutated structure is a little bit lower than the energy of the original protein structure. To have the possibility to compare these energy values with the values of the other analysis tools, we calculated a ratio between these energy values.

Ratio of the original protein	Ratio of the mutated protein	Difference
100	99.84	0.16

Therefore, we can see, that the mutated structure has only 0.16% less energy than the original structure.

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 Phenylalanine	Amino acid Serine	Picture which visualize the mutation

If we have a look at the pictures, we can see that the location of the residue of the two amino acids is very similar.

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 Serine residue.

H-bonds of the original amino acid	H-bonds of the mutated amino acid	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

On the pictures you can see, that neither the original amino acid nor the mutated amino acid has any H-Bonds with other residues or the backbone of the protein. Therefore, it is not possible to explain the damage of the protein by missing H-bonds. Furthermore, we can see, that there are no clashes between Serine and the rest of the protein, which means that the protein do not have to fold in another way because of clashing residues.

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Gromacs Energy Comparison

Comparing Energy:

To analyse the energy values calculated by Gromacs, we used the AMBER99SB-ILDN force field.

Here are the values of the original structure:

Energy	Average	Err.Est.	RMSD	Tot-Drift
Bond	1091.57	270	-nan	-1622.75
Angle	3326.81	62	-nan	404.076
Potential	-61304.1	960	-nan	-6402.44

Here you can see the values which gromacs calculated for the structure with the mutated amino acid:

Energy	Average	Err.Est.	RMSD	Tot-Drift
Bond	1166.24	390	3425.52	-2317.42
Angle	3275.2	50	185.491	324.728
Potential	-46177.4	3600	49139.5	-22414.8

One difference between gromacs and the other tools we used is, that gromacs also calculated the energy for the bonds and the angles. To compare the energies between the different tools we only consider the potential energy in our analysis, because the potential energy is the energy of the complete protein. Therefore, we calculated the ratio between the energies only for the potential energy.

Ratio original amino acid	Ratio mutated amino acid	Difference
100	75.32	24.68

The difference between the energys calculated by Gromacs is much higher than the difference of the energy values calculated by the other tools. But otherwise, Gromacs use a real phisical force field and therefore, it should be the most accurate method to analyse the energy of different structures. In this case the energy of the mutated structure is much lower than the energy of the original structure. Therefore it is possible, that the protein become instable with the mutation and therefore does not work any more.

Comparing Structure:

Gromacs also offers pictures of the mutated amino acids which can be seen in the following section.

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

This pictures is very similar to the pictures created by SCWRL. The mutated amino acid is much more smaller than the Phenylalanine and therefore does not need that much space. Otherwise it is possible, that because of the smaller amino acid, there are missing H-Bonds in the protein.

Visualization of H-bonds and Clashs:

To check if this is the case, we analysed the H-Bonds and clashes between the mutated amino acid and the rest of the protein.

H-Bonds of the original amino acid	H-bonds of the mutated amino acid	Clashes of the mutation
H-bonds of the original amino acid	H-bonds of the mutated amino acid	Possible clashes

Both amino acids do not have any H-Bonds with the rest of the protein. Therefore a missing H-Bond does not cause the damage on the protein.

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