Difference between revisions of "Sequence-based mutation analysis BCKDHA"

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(Amino Acid Properties)
(PSSM)
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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.
 
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.
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== Secondary Structure ==
   
 
== Multiple Sequence Alignment ==
 
== Multiple Sequence Alignment ==

Revision as of 21:20, 25 June 2011

General

We chose the following mutations for the sequence-based mutation analysis:

  • G29E
  • Q125E
  • Y166N
  • G249S
  • C264W
  • R265W
  • I326T
  • I361V
  • F409C
  • Y438N

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
Position Residue Properties Structure Residue Properties Structure
29 G tiny, small E charged, polar C
125 Q acidic, polar
BCKDHAQ80E Q.png
E charged, polar
BCKDHAQ80E E.png
BCKDHAQ80E.png
C
166 Y hydrophobic, aromatic, polar
BCKDHA Y121N Y.png
N acidic, polar, small
BCKDHA Y121N N.png
BCKDHA Y121N.png
H
249 G tiny, small
BCKDHA G204S G.png
S polar, small, tiny, hydroxylic
BCKDHA G204S S.png
BCKDHA G204S.png
H
264 C sulphur containing, hydrophobic, tiny, small, polar
BCKDHA C219W C.png
W hydrophobic, aromatic, polar
BCKDHA C219W W.png
BCKDHA C219W.png
E
265 R charged, positive (basic), polar
BCKDHA R220W R.png
W hydrophobic, aromatic, polar
BCKDHA R220W W.png
[[File:
BCKDHA R220W.png
.png|thumb|100px]]
E
326 I aliphatic, hydrophobic
BCKDHA I281T I.png
T hydroxylic, hydrophobic, small, polar
BCKDHA I281T T.png
BCKDHA I281T.png
E
361 I aliphatic, hydrophobic
BCKDHA I316V I.png
V aliphatic, hydrophobic, small
BCKDHA I316V V.png
BCKDHA I316V.png
H
409 F aromatic, hydrophobic
BCKDHA F364C F.png
C sulphur containing, hydrophobic, tiny, small, polar
BCKDHA F364C C.png
BCKDHA F364C.png
C
438 Y hydrophobic, aromatic, polar
BCKDHA Y393N Y.png
N acidic, polar, small
BCKDHA Y393N N.png
BCKDHA Y393N.png
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.

Substitution matrices

Position AA1/AA2 BLOSUM62 PAM1 PAM250 result
score worst score worst score worst
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


BLOSUM62
PAM1
PAM250

PSSM


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.


Secondary Structure

Multiple Sequence Alignment

To find the homologue sequences to BCKDHA we used BLAST. It found 250 homologous sequences, 25 of them are mammals.

ID Accession Entry name
sp P11178 ODBA_BOVIN
sp P12694 ODBA_HUMAN
sp Q8HXY4 ODBA_MACFA
sp P50136 ODBA_MOUSE
sp A5A6H9 ODBA_PANTR
sp P11960 ODBA_RAT
tr Q6ZSA3 Q6ZSA3_HUMAN
tr E7ESE6 E7ESE6_HUMAN
tr B2R8A9 B2R8A9_HUMAN
tr Q658P7 Q658P7_HUMAN
tr E7EW46 E7EW46_HUMAN
tr B4DP47 B4DP47_HUMAN
tr Q59EI3 Q59EI3_HUMAN
tr F1N5F2 F1N5F2_BOVIN
tr B1PK12 B1PK12_PIG
tr E2RPW4 E2RPW4_CANFA
tr B2LSM3 B2LSM3_SHEEP
tr F1RHA0 F1RHA0_PIG
tr F1PI86 F1PI86_CANFA
tr D2HMT3 D2HMT3_AILME
tr Q2TBT9 Q2TBT9_BOVIN
tr Q3U3J1 Q3U3J1_MOUSE
tr Q99L69 Q99L69_MOUSE
tr Q5EB89 Q5EB89_RAT
tr B1WBN3 B1WBN3_RAT


Multiple Alignment of the homologous sequences of BCKDHA with CLUSTALW

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
29 0.72 0
125 0.96 0
166 1 0
249 1 0
264 1 0
265 1 0
326 1 0
361 0.92 0
409 0.92 0
438 0.92 0

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.

SNAP

To run SNAP we used the command:

snapfun -i BCKDHA.fasta -m mutations.txt -o SNAP.out

nsSNP Prediction Reliability Index Expected Accuracy
G29E Neutral 0 53%
Q125E Non-neutral 1 63%
Y166N Non-neutral 2 70%
G249S Neutral 1 60%
C264W Non-neutral 4 82%
R265W Non-neutral 4 82%
I326T Non-neutral 3 78%
I361V Neutral 4 85%
F409C Non-neutral 4 82%
Y438N Non-neutral 4 82%

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.

SIFT

The following table displays the SIFT results. The threshold for intolerance is 0.05.

The amino acids are colored in the following way:

  • nonpolar
  • uncharged polar
  • basic
  • acidic

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

Position Reference AA Mutated AA SIFT prediction SIFT Matrix Prediction
Predict Not Tolerated Seq Rep Predict Tolerated BCKDHA aa.PNG
29 G E 0.37 wcmPdIGnqRhVTkeSFLAy BCKDHA 29.PNG tolerated
125 Q E ywvtsrpnmlkihgfedca 0.98 Q BCKDHA 125.PNG not tolerated
166 Y N cpdmeqkngrtisval 1.00 FHYW BCKDHA 166.PNG not tolerated
249 G S whyfimrqnlckdvtps 1.00 EGA BCKDHA 249.PNG not tolerated
264 C W ywvtsrqpnmlkihgfeda 0.98 C BCKDHA 264.PNG not tolerated
265 R W ywvtsqpnmlkihgfedca 1.00 R BCKDHA 265.PNG not tolerated
326 I T hdwpneqcrsgkytaM 1.00 FLVI BCKDHA 326.PNG not tolerated
361 I V hwdgnqryekspcfmtlA 1.00 VI BCKDHA 361.PNG tolerated
409 F C hndkrqgecpstamvwiy 1.00 LF BCKDHA 409.PNG not tolerated
438 Y N wvtsrqpnmlkihgfedca 1.00 Y BCKDHA 438.PNG not tolerated

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.

Polyphen2

Position AA1/AA2 HumDiv HumVar
prediction Score Sensitivity Specificity prediction Score Sensitivity Specificity
29 G/E benign 0.025 0.96 0.80 benign 0.018 0.96 0.52
125 Q/E possibly damaging 0.759 0.85 0.93 benign 0.285 0.87 0.75
166 Y/N probably damaging 0.997 0.40 0.98 probably damaging 0.964 0.59 0.93
249 G/S benign 0.145 0.93 0.86 benign 0.292 0.86 0.75
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
361 I/V benign 0.039 0.95 0.82 benign 0.178 0.89 0.70
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.

Comparison

Position AA1/AA2 BLOSUM62 PAM1 PAM250 PSSM Multiple Alignment SNAP SIFT Polyphen2
Prediction Conservation wildtype Conservation mutant Prediction Prediction HumDiv HumVar
29 G/E non-neutral neutral neutral non-neutral neutral 0 neutral neutral neutral neutral
125 Q/E neutral neutral neutral non-neutral non-neutral 0 non-neutral non-neutral non-neutral neutral
166 Y/N non-neutral non-neutral non-neutral non-neutral non-neutral 0 non-neutral non-neutral non-neutral non-neutral
249 G/S non-neutral neutral neutral neutral non-neutral 0 neutral non-neutral neutral neutral
264 C/W non-neutral non-neutral non-neutral non-neutral non-neutral 0 non-neutral non-neutral non-neutral non-neutral
265 R/W non-neutral neutral neutral non-neutral non-neutral 0 non-neutral non-neutral non-neutral non-neutral
326 I/T non-neutral neutral neutral non-neutral non-neutral 0 non-neutral non-neutral non-neutral non-neutral
361 I/V neutral neutral neutral neutral neutral 0 non-neutral neutral neutral neutral
409 F/C non-neutral non-neutral non-neutral non-neutral non-neutral 0 non-neutral non-neutral non-neutral non-neutral
438 Y/N non-neutral non-neutral non-neutral non-neutral non-neutral 0 non-neutral non-neutral non-neutral non-neutral

position 29:

position 125:

position 166:

position 249:

position 264:

position 265:

position 326:

position 361:

position 409:

position 438:

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