1. Tutorial 4 Substitution matrices and PSI-BLAST 1

2. Agenda Why study distant homologies? Substitution Matrices – PAM - Point Accepted Mutations – BLOSUM - Blocks Substitution Matrix PSI-BLAST Cool story of the day: Why should we care about cellular fusion in worms? 2

3. How proteins evolve Throughout evolution proteins change Some change more than others, and at different rates in different regions of the protein. 3

4. When we study a new organism we may find a lot of unknown sequences that we would like to characterize. We might not be able to find any close homologies. Substitution matrices model different evolutional distances. PSI-BLAST enable to find more distant relations between proteins. 4 Why study distant homologies?

5. Amino acids were not born equally 5 Both substitution matrices and PSI-BLAST are designed to model the process by which AAs mutate.

6. Substitution Matrix Scoring matrix S of size 20x20 S i,j represents the gain/penalty due to substituting AA j by AA i (i – line, j – column) – Based on likelihood this substitution is found in nature – Computed differently in PAM and BLOSUM Each matrix is tailored to a particular evolutionary distance 6

7. Computing probability of Mutation (M i,j ) PAM - Point Accepted Mutations – Based on a small set of proteins that are closely related – Other than PAM1 the matrices are theoretical. BLOSUM - Blocks Substitution Matrix – Based on a wider database of proteins that includes families of proteins with conserved regions. – The matrices are empirical. 7

8. PAM Based on a small set of proteins that are closely related PAM1 Captures mutation rates between close proteins – protein with 1% divergence Problematic when comparing distant proteins. The 1% divergence does not capture more sporadic mutations 8

9. PAM-X In order to apply for more distant proteins PAM-1 was self-multiplied. This models the evolutionary process of accumulation of mutations. The higher the number of the matrix – the more suitable it is to find distant homologies. Other than PAM1 the matrices are theoretical. 9

10. Scores for each position are derived from observations of the frequencies of substitutions in blocks of local alignments in related proteins. BLOSUM62 contains all blocks whose members shared at most 62% identity with any other member of that block. 10 BLOSUM

11. 11 50% similarity 32% similarity Substitution Matrix B BLOSUM-X Substitution Matrix A BLOCKS DB

12. PAM vs. BLOSUM PAMBLOSUM Based 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 no more than 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. 12 BLOSUM are the substitution matrices in use

13. PAM100 ~ BLOSUM90 Closely Related PAM120 ~ BLOSUM80 PAM160 ~ BLOSUM60 PAM200 ~ BLOSUM52 PAM250 ~ BLOSUM45 Highly Divergent Query lengthMatrixGap costs <35PAM309,1 35-50PAM7010,1 50-85BLOSUM8010,1 >85BLOSUM6211,1 Use Recommendations 13 http://www.ncbi.nlm.nih.gov/blast/html/sub_matrix.html

14. Example Query: an uncharacterized (hypothetical) protein Data Base: nr Blast Program: BLASTP Matrices: PAM30 / PAM250 BLOSUM45 / BLOSUM90 14

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17. PSI-BLAST Position Specific Iterative BLAST Aimed to find more distant proteins than BLAST allows 17

18. PSI-BLAST Steps 18 1.Search a query against a protein database 2.Constructs a specialized multiple sequence alignment based on the top results. 3.Creates a position-specific scoring matrix (PSSM). 4.The PSSM is used as a query against the database 5.PSI-BLAST estimates statistical significance (E values) Repeat steps 3-5 iteratively. Protein DB Search Query PSSM Results Iterations

19. Example 19 We will use a sequence of an uncharacterized (hypothetical) protein:

20. 20 Threshold for initial BLAST Search (default: 10) Threshold for inclusion in PSI-BLAST iterations (default: 0.005)

21. 21 The results are all hypothetical proteins

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23. Cool Story of the day Why should we care about cellular fusion in worms?

24. Cellular fusion In cellular fusion two cells unite and form one cell Fertilization Muscle cells are composed of rows of fused cells Placenta is made up of powerful multinucleated cells that are actually numerous individual cells that have fused The eyes' lenses are formed of rows of fused cells In bones too cellular fusion occurs. The fusion processes are also involved in cancer, viral infections and stem cells. 24 http://www1.technion.ac.il/_local/includes/blocks/sci- news-items/100513-elegans/news-item-en.htm

25. 25 Beni Podbilewicz The exact way fusion takes place is still not completely clear and is the focus of work in Prof. Podbilewicz's lab. The worm suits cell fusion research because in its skin intensive cell-cell fusion processes take place and can be easily followed. They identified the protein responsible for the worm's fusion activity - the EFF-1 protein. The researchers showed that in mutant worms skin cells do not fuse and the cells begin to migrate through the body. Cellular fusion in C.elegans

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27. 27 “...we identified fusion family (FF) proteins within and beyond nematodes, and divergent members from the human parasitic nematode Trichinella spiralis and the chordate Branchiostoma floridae could also fuse mammalian cells…”