Tutorial 4Comparing Protein Sequences

Intro to Bioinformatics

1

Amino acids were not born equally

2

Comparing Protein Sequences

Substitution MatricesPAM - Point Accepted MutationsBLOSUM - Blocks Substitution Matrix

Advance comparison toolsPsi-BLASTPhi-BLAST

3

Substitution Matrix

Scoring matrix S20x20 for protein alignment (Amino-acid)

Si,j represents the gain/penalty due to substituting AAj by AAi (i – line , j – colomn)Based on likelihood this substitution is found in

natureComputed differently in PAM and BLOSUM

4

Computing probability of Mutation (Mi,j)

PAM - Point Accepted MutationsBased on closely related proteins (X% divergence)

Matrices for comparison of divergent proteins computed

BLOSUM - Blocks Substitution MatrixBased on conserved blocks bounded in similarity (at least X% identical)

Matrices for divergent proteins are derived using appropriate X%

5

PAM-1

Captures mutation rates between close proteins1% divergenceMi,j = AB / #A

Problematic when comparing far proteinsThe 1% divergence does not capture more sporadic mutations

PAM250 is theoretical (extrapolation based)

6

PAM-1

7

Captures mutation rates between divergent proteins

Why is BLOSUM62 called BLOSUM62? Basically, this is because all blocks whose members shared at least 62% identity with ANY other member of that block were averaged and represented as 1 sequence.

BLOSUM62

8

BLOSUM62

The idea of BLOSUM matrices is to get a better measure of differences between two proteins specifically for more distantly related proteins.

Similar AA have high score

9

PAM & BLOSUM

PAM BLOSUMBased on global alignments of closely related proteins.

Based on local alignments.

The PAM1 is calculated from comparisons of sequences with no more than 1% divergence.

BLOSUM 62 is calculated from comparisons of sequences with at least 62% identity in the blocks.

Other PAM matrices are extrapolated from PAM1.

All BLOSUM matrices are based on observed alignments. They are not extrapolated from comparisons of closely related proteins.

10

PAM100 ~ BLOSUM90 Closely RelatedPAM120 ~ BLOSUM80PAM160 ~ BLOSUM60 PAM200 ~ BLOSUM52PAM250 ~ BLOSUM45 Highly Divergent

Query length Matrix Gap costs

<35 PAM30 9,1

35-50 PAM70 10,1

50-85 BLOSUM80 10,1

>85 BLOSUM62 11,1

Use Recommendations

11

ExampleQuery: >ADRM1_HUMAN

(Proteasomal ubiquitin receptor)Data Base: nr on Human genome.Blast Program: BLASTPMatrices: PAM30,BLOSUM45

12

PAM 30 BLOSUM45

•With BLOSUM45 we found related and divergent sequences.

•With PAM30 we found only related sequences.

What difference do we observe?

13

PAM 30

BLOSUM45

With BLOSUM45 we can discover interesting relations between proteins

...

Mucin-13:a glycosylated membrane protein that protects the cell by binding to pathogens

14

With PAM 30

With BLOSUM45

Using different scoring matrices can produce slightlyDifferent alignments:

15

A same alignment can be solved in many ways, specially when using a matrix for highly divergent sequences (BLOSUM45):

16

PSI-BLAST

Position Specific Iterative BLAST

We will analyze the following Archeal uncharacterized protein: >gi|2501594|sp|Q57997|Y577_METJA PROTEIN MJ0577MSVMYKKILYPTDFSETAEIALKHVKAFKTLKAEEVILLHVIDEREIKKRDIFSLLLGVAGLNKSVEEFENELKNKLTEEAKNKMENIKKELEDVGFKVKDIIVVGIPHEEIVKIAEDEGVDIIIMGSHGKTNLKEILLGSVTENVIKKSNKPVLVVKRKNS

17

18

Threshold for initial BLAST

Search (default:10)

Threshold for inclusion in PSI-BLAST iterations

(default:0.005)

19

The query itself

Orthologous sequences in two other archaeal species

Other homologous sequences

20

21

...

...

...

Is MJ0577 a filament protein?

Is MJ0577 a cationic amino

transporter?

Is MJ0577 a universal stress

protein?22