©CMBI 2008 Aligning Sequences The most powerful weapon in the bioinformaticist’s armory is sequence alignment. Why? Lets’ think about an alignment. It is a representation of a whole series of evolutionary events, which left traces in the sequences. Things that are more likely to happen during evolution should be most prominently observed in your alignment.

©CMBI 2008 Why align sequences? Lots of sequences with unknown structure and function. A few sequences with known structure and function If they align, they are likely to be similar If they are similar, then they very likely have same structure and/or function If one of them has known structure/function, then alignment to the other yields insight about how the structure or function works

©CMBI 2008 Sequence Alignment The purpose of a sequence alignment is to line up all residues in the sequence that were derived from the same residue position in the ancestral gene or protein gap = insertion or deletion A B B A

©CMBI 2008 Alignment To carry over information from a well studied protein sequence and its structure to a newly discovered protein sequence, we need an sequence alignment that represents the protein structures today, a structural alignment.

©CMBI 2008 Alignment The implicit meaning of placing amino acid residues below each other in the same column of a protein (multiple) sequence alignment is that they are at the equivalent position in the 3D structures of the corresponding proteins!! Two very simple examples: 1) the 3 active site residues H, D, S, of the serine protease we saw earlier 2) Cys-bridges: STCTKGALKLPVCRK TSCTEG--RLPGCKR

©CMBI 2008 Things one can do with a good alignment Carry information from a well studied to a less well studied protein. Such information can be: Phosphorylation sites Glycosylation sites Stabilizing mutations Membrane anchors Ion binding sites Ligand binding residues Cellular localization Typically what one finds in the FT records of Swissprot!

©CMBI 2008 Significance of alignment One can only transfer information if the similarity is significantly high between the two sequences. Schneider (group of Sander) determined the “threshold curve” for transferring structural information from one known protein structure to another protein sequence: If the sequences are > 80 aa long, then >25% sequence identity is enough to reliably transfer structural information. If the sequences are smaller in length, a higher percentage of identity is needed. Structure is much more conserved than sequence!

©CMBI 2008 Significance of alignment (2)

©CMBI 2008 Aligning sequences by hand Most information that enters the alignment procedure comes from the physico-chemical properties of the amino acids. Examples: which is the better alignment (left or right)? 1) CPISRTWASIFRCW CPISRTWASIFRCW CPISRT---LFRCW CPISRTL---FRCW 2)CPISRTRASEFRCW CPISRTRASEFRCW CPISRTK---FRCW CPISRT---KFRCW

©CMBI 2008 Aligning sequences by hand (2) Procedure of aligning depends on information available: 1)Use “only” identity of amino acid and its physico-chemical properties. This is more or less what alignment programs do. 2)Also use explicitly the secondary structure preference of the amino acids. 3)Use 3D information if one or more of the structures in the alignment are known. In most cases you will start with a alignment program (e.g. CLUSTAL) and then use your knowledge of the amino acids to improve the alignment, for instance by correcting the position of gaps.

©CMBI 2008 Helix

©CMBI total H H H H H ALA CYS ASP GLU PHE GLY HIS ILE LYS LEU MET ASN PRO GLN ARG SER THR VAL TRP TYR Helix preferences

©CMBI 2008 Helix preferences and alignment 1) S G V S P D Q L A A L K L I L E L A L K 2) G T S L E T A L L M Q I A Q K L I A G S G V S P D Q L A A L K L I L E L A L K G T S L E T A L L M Q I A Q K L I A G

©CMBI 2008 Helix preferences and alignment 1)S G V S P D Q L A A L K L I L E L A L K 2)G T S L E T A L L M Q I A Q K L I A G S G V S P D Q L A A L K L I L E L A L K G T S L E T A L L M Q I A Q K L I A G

©CMBI 2008 Helix preferences and alignment S G V S P D Q L A A L K L I L E L A L K G T S L E T A L L M Q I A Q K L I A G Final alignment: S G V S P D Q L A A L K L I L E L A L K - G T S L E T A L L M Q I A Q K L I A G

©CMBI 2008 A ‘real’ example of threading If you know that in structure 1 the Ala is pointing outside and the Ser is pointing inside: Where does the Arg in structure 2 go? (and what will CLUSTAL choose?) 1 2

©CMBI 2008 An even more real example AILE CYS ARG LEU PRO GLY SER ALA GLU ALA VAL B1VAL CYS ARG THR PRO GLU ALA ILE B2VAL CYS ARG THR PRO GLU ALA ILE

©CMBI 2008 An even more real example AILE CYS ARG LEU PRO GLY SER ALA GLU ALA VAL B1VAL CYS ARG THR PRO GLU ALA ILE B2VAL CYS ARG THR PRO GLU ALA ILE IVVIVV CCCCCC RRRRRR LT-LT- PP-PP- G- -G- - S-TS-T A-PA-P EEEEEE AAAAAA V I I

©CMBI 2008 Multiple sequence alignments can confirm or improve pair-wise sequence alignments: CWPVAASYGR CWPT---YGR CWPTA-SYGR CWPTLGLFGR Multiple sequence alignment

©CMBI 2008 Multiple sequence alignment Multiple sequence alignments can reveal structural information: ASCTRGCIKLPTCKKMGRCTGY STCTKGALKLPVCRKMGKSSAY ATSTHGCMKLPCSRRFGKCSSY TSCTEGCLRLPGCKRFGRCTSY TTCTKGLLKLPGCKRFGKSSAY ASSTKGCMKLPVSRRFGRCTAY

©CMBI 2008 Multiple sequence alignment Multiple sequence alignments can validate PROSITE search results. In N-{P}-[ST]-{P} the N is the glycosylation site. The chance of finding N-{P}-[ST]-{P} is rather high. So how can you be sure? Look at the multiple sequence alignment: ASLRNASTVVTIGDTITGNLTLASYHW GSIKNGSSVITLPGTMEGNLSTTTYHY ATLRNASTVMEINGTITGDLTLASFHW

©CMBI 2008 Summary Bioinformatics is all about obtaining information. Everything you can find in a database saves you doing experiments. Sequence alignment is important for carrying over information between ‘similar proteins’. To align sequences, you need to understand the amino acids.