Difference between revisions of "Sequence-based mutation analysis BCKDHA"
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wether this mutation from Glutamine to Glutamic acid is neutral or not because the tools say completely different things. By looking at the protein itself it can be seen that there is a structural difference between the two proteins since it is not possible to superpose them perfectly. But the difference between the two structures to be minimal which can be the explanation that there are different predictions. It is also important to recognize that most of the tools are not completely sure with the prediction they made. For example the PSSM value is 0 which is exactly on the border between neutral or not. Also SNAP is not very sure at all because the reliability index is only 1. In contrast the preditino of SIFT is very save since the value for this mutation is 0.0 which is the lowest possible value. And also PolyPhen2 is unassured given that the two different datasets predict two different things. One says neutral and the other one non-neutral. Because of this many different results it can be assumed that if this mutation is non-neutral the influence is minimal. Since the experimental structure is different we would say that this mutation is non-neutral but as already said and as it can be seen the influence is minimal and so the difference is also not that grave.
Revision as of 00:33, 26 June 2011
We chose the following mutations for the sequence-based mutation analysis:
The mutation positions are relative to the Uniprot reference sequence.
A Protocol was created describing all steps for running the programs etc.
Amino Acid Properties
|Reference amino acid||Mutated amino acid||Structural Difference||Secondary Structure|
|29||G||tiny, small||E||charged, polar||C|
|125||Q||acidic, polar||E||charged, polar||C|
|166||Y||hydrophobic, aromatic, polar||N||acidic, polar, small||H|
|249||G||tiny, small||S||polar, small, tiny, hydroxylic||H|
|264||C||sulphur containing, hydrophobic, tiny, small, polar||W||hydrophobic, aromatic, polar||E|
|265||R||charged, positive (basic), polar||W||hydrophobic, aromatic, polar||[[File:||E|
|326||I||aliphatic, hydrophobic||T||hydroxylic, hydrophobic, small, polar||E|
|361||I||aliphatic, hydrophobic||V||aliphatic, hydrophobic, small||H|
|409||F||aromatic, hydrophobic||C||sulphur containing, hydrophobic, tiny, small, polar||C|
|438||Y||hydrophobic, aromatic, polar||N||acidic, polar, small||C|
Annotation: H = helix, E = beta-sheet, C = coil
To visualize the mutations in the three-dimensional protein structure, the PDB entry for BCKDHA, 1U5B, was used. As the PDB file only contains coordinate information about the protein itself, the signal peptide (45 first amino acids) are not annotated. Therefore the first mutation on position 29, which lies in the signal peptide, could not be visualized.
The Protocol describes in detail the way how we used pymol to visualize our mutations.
|29||G/E||-2||-4 (I, L)||7||0 (I, W, Y)||9||2 (W)||BLOSUM62 says that the mutation is not very likely, whereas PAM1 and PAM250 say that the mutation is not anomalous|
|125||Q/E||2||-3 (C, F, I)||27||0 (F, W, Y)||7||1 (C, F, W)||all three substitutionmatrizes show that this mutation occurs quite often|
|166||Y/N||-2||-3 (D, G, P)||3||0 (R, D, Q, G, K, M, P)||2||1 (A, R, D, Q, E, G, K, P)||Since the values of the three matrizes are low the mutation is not ofteh which shows that is not very probably|
|249||G/S||0||-4 (I, L)||21||0 (I, W, Y)||11||2 (W)||All three values are high what means that this mutation is often and so probably not very damaging|
|264||C/W||-2||-4 (E)||0||0 (N, D, Q, E, G, L, K, M, F, W)||1||1 (R, N, D, Q, E, L, K, M, F, W)||The scores are all low. This reflects that the mutation is rare and because of this it is very likely that it influences the function of the protein|
|265||R/W||-3||-3 (W, V, F, I, C)||8||0 (D, E, G, Y)||7||1 (F)||PAM1 and PAM250 have high values whereas BLOSUM62 has a low value. So BLOSUM62 says that this mutation is rare and probably damaging and PAM1 and PAM250 say that the mutation is quite often and so not very damaging|
|326||I/T||-1||-4 (G)||7||0 (G, A, P, W)||4||1 (W)||Again the three matrizes have a different result. Whereas BLOSUM62 says that the mutation is rare, PAM1 and PAM250 says that the mutation have no bad influence in the protein and thats why it is probably.|
|361||I/V||3||-4 (G)||33||0 (G, H, P, W)||9||1 (W)||All three scores are high so that the mutation is often and because of that it is very possibly not damaging|
|409||F/C||-2||-4 (P)||0||0 (D, C, Q, E, K, P, V)||1||1 (R, D, C, Q, E, G, K, P)||The three values are all very low which means that this mutation is very rare. This hypothesize that the mutation damages the function and the structure of the protein|
|438||Y/N||-2||-3 (D, G, P)||3||0 (R, D, Q, G, K, M, P)||2||1 (A, R, D, Q, E, G, K, P)||Again the scores are all low which indicates the damaging effect of the mutation|
Last position-specific scoring matrix computed, weighted observed percentages rounded down, information per position, and relative weight of gapless real matches to pseudocounts
A R N D C Q E G H I L K M F P S T W Y V A R N D C Q E G H I L K M F P S T W Y V 29 G 1 0 0 -2 1 0 -2 4 1 -1 -3 -1 -3 -2 -3 1 -1 -4 -1 -1 10 6 5 1 3 4 1 37 3 5 2 3 0 2 0 9 2 0 2 6 0.37 0.77 125 Q -1 -1 -3 -3 -5 8 0 -4 0 -3 -4 -1 -1 -6 -4 -1 1 -5 -4 -4 4 2 0 0 0 74 2 0 1 1 1 2 1 0 0 2 9 0 0 0 1.46 1.28 166 Y 3 -3 -4 -4 3 0 -3 -4 1 -2 -2 -3 0 -2 -4 -1 1 7 3 1 24 1 0 0 7 5 1 1 3 1 3 1 2 1 0 3 8 15 12 11 0.62 1.29 249 G 5 -4 -3 -4 -4 -3 -2 4 -4 -5 -5 -3 -4 -5 -4 1 -2 -5 -5 -4 54 0 0 0 0 0 3 35 0 0 0 0 0 0 0 8 1 0 0 0 1.12 1.21 264 C -2 -5 -3 -5 9 -5 -5 -5 -5 3 -2 -5 -3 -4 -1 -3 -3 -5 -4 4 1 0 2 0 45 0 0 0 0 15 2 0 0 0 4 1 1 0 0 29 1.43 1.18 265 R -3 4 2 -3 -5 5 2 -4 0 -2 -4 -1 -2 -5 -4 -2 -2 -5 -2 0 0 25 12 0 0 34 15 0 1 2 0 1 0 0 0 1 1 0 1 7 0.88 1.21 326 I -3 -5 -6 -6 -4 -5 -6 -6 -6 7 0 -5 0 -2 -5 -5 -3 -5 -4 4 0 0 0 0 0 0 0 0 0 66 6 0 1 1 0 0 0 0 0 26 1.40 1.17 361 I -3 -5 -6 -6 -4 -5 -6 -6 -6 6 3 -5 1 -1 -5 -5 -3 -5 -4 2 0 0 0 0 0 0 0 0 0 55 27 0 3 1 0 0 0 0 0 14 1.22 1.21 409 F -4 -3 -6 -6 -3 -5 -5 -6 -3 0 1 -5 1 8 -6 -5 -3 0 1 -1 1 1 0 0 1 0 0 0 0 5 11 0 3 69 0 0 1 1 3 4 1.56 1.31 438 Y 0 -2 -2 -4 -2 -1 -1 -3 3 -3 -3 -3 -3 1 -5 -3 -3 3 8 -2 9 2 1 0 1 3 3 1 6 0 1 0 0 1 0 0 0 3 66 2 1.34 0.89
The values in the pssm reflect the grade of conservation in an multiple alignment. The higher the values, the better the conservation and therefore a substitution of the corresponing amino acids is usually tolerated, as both alleles have been passed on successfully. The pssm values for our mutations have been colored orange. For most of the mutations the pssm score is negative and therefore this substitution is not conserved and not likely to be tolerated.
The Q125E mutation has a score of 0.
The G249S substitution has a score of +1, which is quite good. This indicates a small rate of conservation and therefore this mutation might be tolerated in nature.
The highest score for our substitutions is +2 for the I361V mutations. This mutation is conserved and likely to be tolerated. This can be argumented by the fact that isoleucin and valin show a structural and physiochemical similarities.
To find out if the mutation has an influence on the secondary structure of the protein we compared the secondary structure of the sequence without mutations and the sequence including the mutations. To get the secondary structure of the two sequences we used psipred
We compared the structure for each position:
seq: SQAALLLLRQPGARGLARSHPPRQQQQFSSLDDK non-mut: HHHHHHHHCCCCCCCCCCCCCCCCCCCCCCCCCC mut: HHHHHHHHCCCCCCCCCCCCCCCCCCCCCCCCCC
seq: KEKVLKLYKSMTLLNTMDRILYESQRQGRISFYMTNYG non-mut: HHHHHHHHHHHHHHHHHHHHHHHHHHCCCCCCCCCCCC mut: HHHHHHHHHHHHHHHHHHHHHHHHHHCCCCCCCCCCCC
seq: EAGVLMYRDYPLELFMAQCYG non-mut: HHHHHHHCCCCHHHHHHHHCC mut: CHHHHHHCCCCHHHHHHHHCC
seq: VVICYFGEGAASEGDAHAGFNFAATLECP non-mut: EEEEEECCCCCCHHHHHHHHHHHHHHCCC mut: EEEEEECCCCCCCHHHHHHHHHHHHCCCC
seq: IIFFCRNNGYAISTPTSEQYRGD non-mut: EEEEEECCCCCCCCCCCHHCCCC mut: EEEEEECCCEEECCCCCHHCCCH
seq: IIFFCRNNGYAISTPTSEQYRGD non-mut: EEEEEECCCCCCCCCCCHHCCCC mut: EEEEEECCCEEECCCCCHHCCCH
seq: RAVAENQPFLIEAMTYRIGHHSTSDDSSAYRS non-mut: HHHCCCCCEEEEEECCCCCCCCCCCCCCCCCC mut: HHHCCCCCEEEEEECCCCCCCCCCCCCCCCCC
seq: VDEVNYWDKQDHPISRLRHYLLSQGWWD non-mut: HHHHHHHHHCCCCHHHHHHHHHHCCCCC mut: HHHHHHHHHCCCCHHHHHHHHHHCCCCC
seq: KPKPNPNLLFSDVYQEMPAQL non-mut: CCCCCHHHHHHHHHCCCCHHH mut: CCCCCHHHHHHHHHCCCCHHH
seq: QEMPAQLRKQQESLARHLQTYGEHYPLDHFDK non-mut: CCCCHHHHHHHHHHHHHHHHHCCCCCCCCCCC mut: CCCCHHHHHHHHHHHHHHHHHCCCCCCCCCCC
As we can see by comparing the secondary structure of all the positions, most of the mutations have no influence on the secondary structure since there are no changes on the position of the mutation or in the neighbourhood. On the positions 166, 249, 264 and 265 the mutations have an influence on the structure. The mutation on position 166 has an influence on the secondary structure 6 residues earlier because the helix which starts normally at position 160 now starts at position 161. Because of the mutation on position 249 the surrounding helix is shorter because it starts one residue later and ends one residue earlier than without mutation. Because the mutations 264 and 265 are next to each other it is not clear which of them is responsibel for the change in the secondary structure or if it is the combination of the two mutations. Nevertheless there is a change in the neighbourhood of these mutations because four or five residues after the mutation occurs a beta sheet which is not in the wildtype structure. Additionally the helix which should start 19 or 20 residues after the mutation starts one position earlier.
Multiple Sequence Alignment
To find the homologue sequences to BCKDHA we used BLAST. It found 250 homologous sequences, 25 of them are mammals.
With this 25 results we made a multiple alignment by using CLUSTALW. The alignment with all mammalian homologous was quite bad because of the sequences "Q6ZSA3" and "E2RPW4". These two sequences are much longer than the other ones. So we removed those sequences and realigned the other sequences.
With this new multiple alignment we could analyze the 10 positions of our mutations to find out how good they are conserved.
|position||conservation wildtype||conservation mutant|
The results show that all amino acids on the observed positions are really good conserved since the value is always nearly 1. Only on position 29 the conservation of Glycin is only about 72%. This is not that high as the other results but it is still good conserved. Regions in the proteins which are good conserved are propably very important for the structure and the function of the protein. Because of the fact that all amino acids are very good conserved, the mutations on these positions can be very damaging and can have a huge impact on the protein and its function.
To run SNAP we used the command:
snapfun -i BCKDHA.fasta -m mutations.txt -o SNAP.out
|nsSNP||Prediction||Reliability Index||Expected Accuracy|
The output of SNAP shows us that most of the mutations would have a damaging effect on the structure and function of the protein. Only the mutations on position 29, 249 and 361 would not have an influence on the protein.
A second SNAP run was performed where all ten chosen mutation positions were mutated by all possible substitutions.
The following table displays the SIFT results. The threshold for intolerance is 0.05.
The amino acids are colored in the following way:
- uncharged polar
Capital letters: amino acids appear in the alignment
Lower case letters: amino acids result from prediction
Seq Rep:fraction of sequences that contain one of the basic amino acids
The only substritutions SIFT predicts not to affect protein function are G29E and I361V. The first substitution may be tolerated, as this position is not within the actual protein sequence. The second tolerated amino acid exchange is from isoleucin to valin, which are both 'branched-chain' amino acids. These two amino acids quite similar concerning their structure and physiochemical properties, so an exchange can be tolerated.
|166||Y/N||probably damaging||0.997||0.40||0.98||probably damaging||0.964||0.59||0.93|
|264||C/W||probably damaging||1.000||0.00||1.00||probably damaging||1.000||0.00||1.00|
|265||R/W||probably damaging||1.000||0.00||1.00||probably damaging||1.000||0.00||1.00|
|326||I/T||probably damaging||0.997||0.40||0.98||probably damaging||0.998||0.16||0.99|
|409||F/C||probably damaging||0.998||0.27||0.99||probably damaging||0.939||0.64||0.92|
|438||Y/N||probably damaging||1.000||0.00||1.00||probably damaging||0.987||0.49||0.96|
Polyphen2 uses two different datasets to do the prediction. As the results show the two predictions are not always the same. The predictions with the HumDiv dataset says that there are three mutations that possibly have no grave effect on the function or structure of the protein whereas the result of HumVar is that there are four mutations that perhaps would have no damaging influence. The three mutations which are in both datasets marked as "benign" are on the positions 29, 249 and 361. The mutation which is only in the HumVar dataset predicted as benign is on position 125.
|Position||AA1/AA2||BLOSUM62||PAM1||PAM250||PSSM||Secondary Structure||Multiple Alignment||SNAP||SIFT||Polyphen2|
|Prediction||Conservation wildtype||Conservation mutant||Prediction||Prediction||HumDiv||HumVar|
On position 29 is the mutation from Glycine to Glutamic acid. Except of BLOSUM62 and PSSM all of the other tools and sources point out that this mutation is neutral. The fact that this position is not very conserved with only 72% shows that this amino acid changes quite often. Because of this actuality it is possible that the mutation is neutral.
Basing on the predictions of the different tools it is not possible to decide wether this mutation from Glutamine to Glutamic acid is neutral or not because the tools say completely different things. By looking at the protein itself it can be seen that there is a structural difference between the two proteins since it is not possible to superpose them perfectly. But the difference between the two structures seems to be minimal which can be the explanation that there are different predictions. It is also important to recognize that most of the tools are not completely sure with the prediction they made. For example the PSSM value is 0 which is exactly on the border between neutral or not. Also SNAP is not very sure at all because the reliability index is only 1. In contrast the preditino of SIFT is very save since the value for this mutation is 0.0 which is the lowest possible value. And also PolyPhen2 is unassured given that the two different datasets predict two different things. One says neutral and the other one non-neutral. Because of this many different results it can be assumed that if this mutation is non-neutral the influence is minimal. Since the experimental structure is different we would say that this mutation is non-neutral but as already said and as it can be seen the influence is minimal and so the difference is also not that grave.
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