Tutorial 4 Substitution matrices and PSI-BLAST

1. Tutorial 4Substitution matrices and PSI-BLAST

2. Agenda • 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?

3. Studying distant homologies • When we study a new organism/ protein we may find a lot of unknown sequences that we would like to characterize. We might not be able to find any close homologies. • In the evolution, three-dimensional structures of proteins may be conserved even after considerable erosion of their sequence similarity. https://www.ncbi.nlm.nih.gov/books/NBK2590/

4. Multiple alignment of the new protein families of the HSP70-actin fold O-sialoglycoproteases (OSGP) and related proteins Newly identified proteins with the HSP70-actin fold UDPases and extracellular ATPases Classic HSP70 and sugar kinases Aravind and Koonin, Journal of Molecular Biology, 1999

5. Comparison of the HSP70 structure and a structural model of the O-sialoglycoprotease Aravind and Koonin, Journal of Molecular Biology, 1999

6. Substitution matrices model different evolutionary distances. • PSI-BLAST enable to find more distant relations between proteins.

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

8. Substitution Matrix • Scoring matrix S of size 20x20 • Si,jrepresents the gain/penalty due to substituting AAj by AAi(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.

9. PAM vs. BLOSUM BLOSUM are the substitution matrices in use

10. Use Recommendations PAM100 ~ BLOSUM90 Closely Related PAM120 ~ BLOSUM80 PAM160 ~ BLOSUM60 PAM200 ~ BLOSUM52 PAM250 ~ BLOSUM45 Highly Divergent http://www.ncbi.nlm.nih.gov/blast/html/sub_matrix.html

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

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

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

16. PSSM The PSSM captures the conservation pattern in alignment and stores it as a matrix of scores for each position in the alignment. This profile is used in place of the original substitution matrix for a further search of the database to detect sequences that match the conservation pattern specified by the PSSM. http://www.ebi.ac.uk/training/online/course/introduction-protein-classification-ebi/what-are-protein-signatures/signature-types/what-are https://www.ncbi.nlm.nih.gov/books/NBK2590/-

17. PSI-BLAST Example Cellular DNA polymerase enzymes tend to dissociate from DNA after adding a few nucleotides and require an accessory factor to tether them to DNA while elongating the growing DNA chain. In eukaryotes: PCNA In prokayotes: β-subunit of DNA polymerase encoded by the dnaN gene https://www.ncbi.nlm.nih.gov/books/NBK2590/

18. E.Coli (dimer) Human (trimer)

19. Querying the human protein Changed to 1000

22. Summarize results by organism

26. Marked in yellow are sequences scoring below threshold on previous iteration

27. Iteration 2

28. Iteration 3

29. Iteration X Alignment of human proliferating cell nuclear antigen (PCNA) and Escherichia coli DNA polymerase III β-subunit. Hydrophobic AAs: V, L, F, A Polar AAs: E , D, K,R, N, Y

30. Example 2 We will use a sequence of an uncharacterized (hypothetical) protein:

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

32. The results are all hypothetical proteins

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

35. 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. http://www1.technion.ac.il/_local/includes/blocks/sci-news-items/100513-elegans/news-item-en.htm

36. Cellular fusion in C.elegans • How exactly two cells fuse is not is the focus of work in Prof. Podbilewicz's lab. • The worm suits cell fusion research because intensive cell-cell fusion processes take place in its skin 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. BeniPodbilewicz

38. “...we identified fusion family (FF) proteins within and beyond nematodes, and divergent members from the human parasitic nematode Trichinellaspiralis and the chordate Branchiostomafloridae could also fuse mammalian cells…”