Difference between revisions of "Sequence-based mutation analysis BCKDHA"
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== Comparison ==
Revision as of 21:14, 23 June 2011
A Protocol was created where all steps for running the programs etc are described.
Subset of mutations
|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)|
|125||Q/E||2||-3 (C, F, I)||27||0 (F, W, Y)||7||1 (C, F, W)|
|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)|
|249||G/S||0||-4 (I, L)||21||0 (I, W, Y)||11||2 (W)|
|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)|
|265||R/W||-3||-3 (W, V, F, I, C)||8||0 (D, E, G, Y)||7||1 (F)|
|326||I/T||-1||-4 (G)||7||0 (G, A, P, W)||4||1 (W)|
|361||I/V||3||-4 (G)||33||0 (G, H, P, W)||9||1 (W)|
|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)|
|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)|
In order to get a human-readable PSSM-File PsiBlast was run using the following command: blastpgp -i sequence.fasta -j 5 -d /data/blast/nr/nr -C profile.ckp -u 1 -J T
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 one. So we removed those sequences and realigned the other sequences.
With this new multiple Alignment we could analyze the 10 positions 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|
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|
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