1. August 26, 2009 Biochemistry 201 Thang Chiu, MEB 7E1, x2024 Sequence Alignments and Database Searching Adapted from DKW lecture

2. Why compare protein sequences? Significant sequence similarities allow associations based upon known functions. Protein A of interest to you. ornithine decarboxylase?

3. Extracted from ISMB2000 tutorial, WR Pearson, U. of Virginia Possible for proteins to possess high sequence identity/ similarity between segments and not be homologous 1)Homologous proteins (ie having similar structures) need not posess high sequence identity / similarity: S. griseus trypsin 36% S. griseus protease A 25% Homology vs. similarity 2)cytochrome c4, has reasonably high sequence identity/ similarity with trypsins, yet does not have common ancestor, nor common fold. 3)subtilisin has same spatial arrangement of active site residues, but is not related to trypsins

4. Homologous proteins always share a common three- dimensional fold, often with common active or binding site. Proteins that share a common ancestor are homologous. Proteins that possess >25% identity across entire length generally will be homologous. Proteins with <20% identity are not necessarily not homologous Homology vs. similarity

5. Homologous sequences are either: 1) orthologous, or 2) paralogous For orthologs - sequence divergence and evolutionary relationships will agree. For paralogs - no necessary linkage between sequence divergence and speciation. Extracted from ISMB2000 tutorial, WR Pearson, U. of Virginia Orthologous cyctochrome c isozymes Hemoglobins contain both orthologs and paralogs Orthologs - sequence differences arises from divergence in different species (i.e. cyctochrome c) Paralogs - sequence differences arise after gene duplication within a given species (i.e. GPCRs, hemoglobins) Homology vs. similarity

6. We’ve all seen and/or used sequence alignments, but how are they accomplished? Sequence searches and alignments using DNA/RNA are usually not as informative as searches and alignments using protein sequences. However. DNA/RNA searches are intuitively easier to understand: AGGCTTAGCAAA........TCAGGGCCTAATGCG |||||||| ||| ||||||||||| ||| AGGCTTAGGAAACTTCCTAGTCAGGGCCTAAAGCG The above alignment could be scored giving a “1” for each identical nucleotide, A zero for a mismatch, and a -4 for “opening a “gap” and a -1 for each extension of the gap. So score = 25 – 11= 14

7. Protein sequence alignments are much more complicated. How would this alignment be scored? ARDTGQEPSSFWNLILMY.........DSCVIVHKKMSLEIRVH | | | | | ||| | | || ||| AKKSAEQPTSYWDIVILYESTDKNDSGDSCTLVKKRMSIQLRVH Unlike nucleotide sequence alignments, which are either identical or not identical at a given position, protein sequence alignments include “shades of grey” where one might acknowledge that a T is sort of equivalent to an S etc. But how equivalent? What number would you assign to an S-T mismatch? And what about gaps? Since alanine is a common amino acid, couldn’t the A-A match be by chance? Since Trp and Cys are uncommon, should those matches be given higher scores? Do you see that accurately aligning sequences and accurately finding related sequences are  the same problem?

8. Needleman-Wunsch global sequence alignment (JMB (1970), 48, 443-453) A B C N J R AJCJNRAJCJNR A 1 0 0 0 0 0 J 0 1 1 1 2 1 C 0 1 1 0 0 0 J 0 1 0 0 1 0 N 0 1 0 1 0 0 R 0 1 0 0 0 1 SUM S(I,j) with max of S(I,j) of previous column/row A B C N J R A 1 0 0 0 0 0 J 0 1 1 1 2 1 C 0 1 2 1 1 2 J 0 1 1 2 3 2 N 0 1 1 3 2 3 R 0 1 1 2 2 4 A B C N J R A 1 0 0 0 0 0 J 0 0 0 0 1 1 C 0 0 1 0 0 0 J 0 0 0 0 1 0 N 0 0 0 1 0 0 R 0 0 0 0 0 1 A B C N J R A 1 0 0 0 0 0 J 0 0 0 0 1 0 C 0 0 1 0 0 0 J 0 0 0 0 1 0 N 0 0 0 1 0 0 R 0 0 0 0 0 1 Assign score to all cells A B C N J R A 1 0 0 0 0 0 J 0 1 1 1 2 1 C 0 1 2 1 1 2 J 0 1 1 2 3 2 N 0 1 1 3 2 3 R 0 1 1 2 2 4 Traceback A B C N J R A J C J N R A B C N J R A J C J N R OR

9. Databases Nucleotide: GenBank (NCBI), EMBL, DDBJ Protein: SwissProt, TrEMBL, GenPept(GenBank) Huge databases – share much information. Many entries linked to other databases (e.g. PDB). SwissProt small but well “curated”. NCBI non-redundant (nr) protein sequence database is very large but sometimes confusing. These databases can be searched in a number of ways. Can search only human or metazoan sequences. Can eliminate entries made before a given Date. Etc.

10. Protein Sequence Records from PRF A series of digits (often six or seven) followed by a letter, e.g.: 1901178A RefSeq Nucleotide Sequence Records Two letters, an underscore bar, and six digits, e.g.: mRNA records (NM_*): NM_000492 genomic DNA contigs (NT_*): NT_000347 complete genome or chromosome (NC_*): NT_000907 genomic region (NG_*): NG000019 Type of RecordSample accession format GenBank/EMBL/DDBJ Nucleotide SequenceRecords One letter followed by five digits, e.g.: U12345 Two letters followed by six digits, e.g.: AY123456, AF123456 GenPept Sequence Records (which contain the amino acid translations from GenBank/EMBL/DDBJ records that have a coding region feature annotated on them) Three letters and five digits, e.g.: AAA12345 Protein Sequence Records from SWISS-PROT and PIR All are six characters: Character/Format 1 [O,P,Q] 2 [0-9] 3 [A-Z,0-9] 4 [A-Z,0-9] 5 [A-Z,0-9] 6 [0-9] e.g.: P12345 and Q9JJS7 What do all those numbers mean? NCBI

11. RefSeq Protein Sequence RecordsTwo letters (NP), an underscore bar, and six digits, e.g.: NP_000483 RefSeq Model (predicted) Sequence Records from the Human Genome annotation process Two letters (XM, XP, or XT), an underscore bar, and six digits, e.g.: XM_000583 Protein Structure RecordsPDB accessions generally contain one digit followed by three letters, e.g.: 1TUP MMDB ID numbers generally contain four digits, e.g.: 3973 The record for the Tumor Suppressor P53 Complexed With DNA can be retrieved by either number above Continued…. GI numbers: a series of digits that are assigned consecutively by NCBI to each sequence it processes. Version numbers: consist of the accession number followed by a dot and a version number. Nucleotide sequence: GI: 6995995 VERSION: NM_000492.2 Protein translation: GI: 6995996 VERSION: NP_000483.2 >gi|897557|gb|AAA98443.1| TIAM1 protein NCBI http://www.ornl.gov/sci/techresources/Human_Genome/posters/chromosome/geneguide.shtml

12. We’ve got the data, now how do we score/search? First, we need a way to assign numbers to “shades of grey” matches. Genetic code scoring system – This assumes that changes in protein sequence arise from mutations. If only one point mutation is needed to change a given AA to another (at a specific position in alignment), the two amino-acids are more closely related than if two point mutations were required. Physicochemical scoring system – a Thr is like a Ser, a Trp is not like an Ala…… These systems are seldom used because they have problems. Why try to second guess Nature? Since there are many related sequences out there, we can look at some (trusted) alignments to SEE which sub- stitutions have occurred and the frequency with which they occur.

13. PAM (Percent Acceptable Mutation) matrices Are derived from studying global alignments of well-characterized protein families. PAM1 = only 1% of residues has changed (ie short evolutionary distance) Raise this to 250 power to get 250% change of two sequences (greater evolutionary distance), or about 20% sequence identity. Therefore, a PAM 30 would be used to analyze more closely related proteins, a PAM 400 is used for finding and analyzing distantly related proteins. PAMx = PAM1 x (Dayhoff, Atlas of Protein Sequence and Structure, vol. 5, suppl 3, p 345-352)

14. Are derived from studying local alignments (blocks) of sequences from related proteins that differ by no more than X%. (Henikoff & Henikoff, PNAS ‘92, 89, p10915-10919) 1)In other words, one might use the portions of aligned sequences from related proteins that have no more than 62% identity (in the portions or blocks) to derive the BLOSUM 62 scoring matrix. 2)One might use only the blocks that have <80% identity to derive the BLOSUM 80 matrix. Block substitution matrices (BLOSUM) 3) BLOSUM and PAM substitution matrices have the opposite effects: a)The higher the number of the BLOSUM matrix (BLOSUM X), the more closely related proteins you are looking for. a)The higher the number of the PAM matrix (PAM X), the more distantly related proteins you are looking for.

15. Extracted from ISMB2000 tutorial, WR Pearson, U. of Virginia PAM250 matrix Note that for identical matches, scores vary depending upon observed frequencies. That is, rare amino acid (i.e. Trp) that are not substituted have high scores; frequently occuring amino acids (i.e. Ala) are down-weighted because of the high probability of aligning by chance. Amino acid substitution matrices Negative scores - unlikely substitutions

16. Gap penalties – Intuitively one recognizes that there should be a penalty for introducing (requiring) a gap during identification/alignment of a given sequence. But if two sequences are related, the gaps may well be located In loop regions which are more tolerant of mutational events and probably have little impact on structure. Therefore, a new gap should be penalized, but extending an existing gap should be penalized very little. Filtering – many proteins and nucleotides contain simple repeats or regions of low sequence complexity. These must be excluded from searches and alignments. Why? Significance of a “hit” during a search - More important than an arbitrary score is an estimation of the likelihood of finding a hit through pure chance. Ergo the “Expectation value” or E-value. E-values can be as low as 10 -70.

17. E-value So, for sufficiently large databases (so can apply statistics): E = Kmne - S m- query length n - database length E - expectation value K - scale factor for search space (database) - scale factor for scoring system S - score, dependent on substitution matrix, gap- penalties, etc. Doubling either sequence string doubles number of sequences with a given expectation value; similarly, double the score and expectation value decreases exponentially Expectation value - probability that given score will occur by chance given the query AND database strings

18. Extracted from ISMB2000 tutorial, WR Pearson, U. of Virginia Must account for increases in similarity score due to increase in sequence length searched. Scaling with against the sequence length allows the detection of distantly- related sequences. solids = individual sequence opens = average score Removing length bias from scoring statistics

19. Extracted from ISMB2000 tutorial, WR Pearson, U. of Virginia Global scores require alignment of entire sequence length. Cannot be used to detect relationships between domains in mosaic proteins. Global versus local alignments Local alignments are necessary to detect domains within mosaic proteins, internal duplications.

20. 1)Break query up into “words” e.g. ASTGHKDLLV AST WORDS STG TGH 2) Generate expanded list of words that would match with (i.e. PAM250) a score of at least T – You’re acknowledging that you may not have any exact matches with original list of words. 3) Use expanded list of words to search database for exact matches. 4) Extend alignments from where word(s) found exact match. Basic local alignment search tool (BLAST) Heuristic algorithm – Uses guesses. Increases speed without a great loss of accuracy (BLASTP, FASTA (local Hueristic), S-W local rigorous, Needleman-Wunsch global, rigorous)

21. Pictorial representation of BLAST algorithm (Basic Local Alignment Search Tool). Query sequence Words (they overlap) Expand list of words Search database, find exact hits, extend alignments Report sorted list of hits

22. Nucleotide BLAST looks for exact matches Protein BLAST requires two hits GTQITVEDLFYNI SEI YYN ATCGCCATGCTTAATTGGGCTT CATGCTTAATT neighborhood words exact word match one hit two hits NCBI BLAST

23. FASTA Instead of breaking up query into words (and then generating a list of similar words), find all sequences in the database that contain short sequences that are exact or nearly exact matches for sequences within the query. Score these and sort. Sort of reverse methodology to BLAST Query sequence Database sequence

25. Protein database

27. mouse over

28. link to entrez sorted by e values 5 X 10 -98 LocusLink S’ = (λS –lnK)/ln2 E=mn2 -S’

29. Identifying distant homologies (use several different query sequences) Examine output carefully. A lack of statistical significance doesn’t necessarily mean a lack of homology! Extracted from ISMB2000 tutorial, WR Pearson, U. of Virginia Also remember - If A is homologous to B, and B to C, then A should be homologous to C

30. PSI-BLAST Very sensitive, but must not include a non-member sequence! 1)Regular BLAST search 2)Sequences above a certain threshold (< specified E-value) are included. Assumed to be related proteins. This group of sequences is used to define a “profile” that contains the essence of the “family”. 3)Now with the important sequence positions highlighted, can look for more distantly related sequences that should still have the essence of the protein family. 4)Inclusion of more distantly related sequences modifies the profile further (further defines the essence) and allows for identification of even more distantly related sequences. Etc. Note: PSI-BLAST may find and then subsequently lose a homologous sequence during the iteration process! “Drifting” of the program, would be the gradual loss of close homologs during the iteration process.

31. >gi|113340|sp|P03958|ADA_MOUSE ADENOSINE DEAMINASE (ADENOSINE AMINOH MAQTPAFNKPKVELHVHLDGAIKPETILYFGKKRGIALPADTVEELRNIIGMDKPLSLPGFLAKFDYY VIAGCREAIKRIAYEFVEMKAKEGVVYVEVRYSPHLLANSKVDPMPWNQTEGDVTPDDVVDLVNQGLQ EQAFGIKVRSILCCMRHQPSWSLEVLELCKKYNQKTVVAMDLAGDETIEGSSLFPGHVEAYEGAVKNG RTVHAGEVGSPEVVREAVDILKTERVGHGYHTIEDEALYNRLLKENMHFEVCPWSSYLTGAWDPKTTH VRFKNDKANYSLNTDDPLIFKSTLDTDYQMTKKDMGFTEEEFKRLNINAAKSSFLPEEEKKELLERLY e value cutoff for PSSM PSI-BLAST: initial run NCBI

32. PSI-BLAST: initial run NCBI

33. Other purine nucleotide metabolizing enzymes not found by ordinary BLAST PSI-BLAST: first PSSM search NCBI

34. iteration 1 iteration 2 PSI-Blast of human Tiam1 PSI-BLAST: importance of original query (remember, if A is like B….)

35. iteration 2 iteration 1 iteration 3 Ras-binding domains PSI-Blast of mouse Tiam2 (~90% identity with human Tiam1) PSI-BLAST: importance of original query

36. Active site serine Weakly conserved serine Position specific scoring matrix (PSSM) (learning from your “hits”) NCBI

37. A R N D C Q E G H I L K M F P S T W Y V 206 D 0 -2 0 2 -4 2 4 -4 -3 -5 -4 0 -2 -6 1 0 -1 -6 -4 -1 207 G -2 -1 0 -2 -4 -3 -3 6 -4 -5 -5 0 -2 -3 -2 -2 -1 0 -6 -5 208 V -1 1 -3 -3 -5 -1 -2 6 -1 -4 -5 1 -5 -6 -4 0 -2 -6 -4 -2 209 I -3 3 -3 -4 -6 0 -1 -4 -1 2 -4 6 -2 -5 -5 -3 0 -1 -4 0 210 S -2 -5 0 8 -5 -3 -2 -1 -4 -7 -6 -4 -6 -7 -5 1 -3 -7 -5 -6 211 S 4 -4 -4 -4 -4 -1 -4 -2 -3 -3 -5 -4 -4 -5 -1 4 3 -6 -5 -3 212 C -4 -7 -6 -7 12 -7 -7 -5 -6 -5 -5 -7 -5 0 -7 -4 -4 -5 0 -4 213 N -2 0 2 -1 -6 7 0 -2 0 -6 -4 2 0 -2 -5 -1 -3 -3 -4 -3 214 G -2 -3 -3 -4 -4 -4 -5 7 -4 -7 -7 -5 -4 -4 -6 -3 -5 -6 -6 -6 215 D -5 -5 -2 9 -7 -4 -1 -5 -5 -7 -7 -4 -7 -7 -5 -4 -4 -8 -7 -7 216 S -2 -4 -2 -4 -4 -3 -3 -3 -4 -6 -6 -3 -5 -6 -4 7 -2 -6 -5 -5 217 G -3 -6 -4 -5 -6 -5 -6 8 -6 -8 -7 -5 -6 -7 -6 -4 -5 -6 -7 -7 218 G -3 -6 -4 -5 -6 -5 -6 8 -6 -7 -7 -5 -6 -7 -6 -2 -4 -6 -7 -7 219 P -2 -6 -6 -5 -6 -5 -5 -6 -6 -6 -7 -4 -6 -7 9 -4 -4 -7 -7 -6 220 L -4 -6 -7 -7 -5 -5 -6 -7 0 -1 6 -6 1 0 -6 -6 -5 -5 -4 0 221 N -1 -6 0 -6 -4 -4 -6 -6 -1 3 0 -5 4 -3 -6 -2 -1 -6 -1 6 222 C 0 -4 -5 -5 10 -2 -5 -5 1 -1 -1 -5 0 -1 -4 -1 0 -5 0 0 223 Q 0 1 4 2 -5 2 0 0 0 -4 -2 1 0 0 0 -1 -1 -3 -3 -4 224 A -1 -1 1 3 -4 -1 1 4 -3 -4 -3 -1 -2 -2 -3 0 -2 -2 -2 -3 Serine scored differently in these two positions Active site nucleophile Position specific scoring matrix (PSSM) NCBI

38. Multiple sequence alignments (MSAs) In this example, an MSA is used to identify regions of high sequence conservation presumably reflecting structural and functional constraints. Useful for delimiting known domains and potential new functional regions (e.g. the Ras-binding domain in yellow and the blue box of currently unknown function).

39. Fun with MSA... MSA used to locate functional residues and domain boundaries in homologs of Dbl-proteins with known structure (Dbs and Tiam1). Red amino acids directly interact with GTPases. Blue residues directly interact with phosphoinositides.

40. Tutorial on Jalview for MSA 1)Determining domain boundary for construct to express 2)Secondary, possible 3D structural information to help narrow down 5’ and 3’ regions for PCR primers

41. HomologyIf two proteins are homologous, they have a common fold and a common ancestor If two proteins have >25% identity across their entire length, they are likely to be Homologs. However, sometimes true homologs have quite low sequence identity! OrthologsHomologous (and equivalent) proteins from different species. Arise from speciation. ParalogsHomologous (and equivalent) proteins found in same species. Divergence of sequences NOT from speciation. AlignmentsHow to score? Minimum # of mutations?, Physicochemical properties (as perceived by us)?, Or learn from nature? Scoring schemesPAM, BLOSUM What you should know

42. E valuesWhat it means in words E = Kmne -λS Alignment algorithmsBLAST (Basic Local Alignment Search Tool) FASTA (Fast Alignment) Needleman-Wunsch (Global alignment) Why use local alignment algorithm?