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?
How proteins evolve • Throughout evolution proteins change • Some change more than others, and at different rates in different regions of the protein.
Why study distant homologies? • 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.
Aminoacids were not born equally Both substitution matrices and PSI-BLAST are designed to model the process by which AAs mutate.
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
Computing probability of Mutation (Mi,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.
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
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.
BLOSUM • Scores for each position are derived from observations of the frequencies of substitutions in blocks of local alignments in related proteins. • BLOSUM62contains all blocks whose members shared at most 62% identity with any other member of that block.
BLOSUM-X BLOCKS DB 50% similarity 50% similarity 32% similarity Substitution Matrix B Substitution Matrix A
PAM vs. BLOSUM BLOSUM are the substitution matrices in use
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
Example • Query: an uncharacterized (hypothetical) protein • Data Base: nr • Blast Program: BLASTP • Matrices: PAM30 / PAM250 BLOSUM45 / BLOSUM90
Position Specific Iterative BLAST Aimed to find more distant proteins than BLAST allows PSI-BLAST
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
Example We will use a sequence of an uncharacterized (hypothetical) protein:
Threshold for initial BLAST Search (default: 10) Threshold for inclusion in PSI-BLAST iterations (default: 0.005)
Cool Story of the day Why should we care about cellular fusion in worms?
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
Cellular fusion in C.elegans • 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. BeniPodbilewicz
“...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…”