1. Russell Group, Protein Evolution _________ ____

2. Russell Group, Protein Evolution _________ ____ Rob Russell Cell Networks University of Heidelberg Putting it all together to answer a “real” question

3. Russell Group, Protein Evolution _________ ____ Domains assemble to form higher-order structures Pawson & Nash, Science, 2003

4. Russell Group, Protein Evolution _________ ____ Case study 1: GabaB R1/R2 Family 3 GPCRs Subunit R1 binds ligands, R2 signals, but not vice versa Why?

5. Russell Group, Protein Evolution _________ ____ Analysis of intrinsic features Low complexity Signal peptide Coiled coil region Transmembrane helices >gi|3776094|emb|CAA09940.1| GABAB receptor, subunit 1b [Homo sapiens] MGPGAPFARVGWPLPLLVVMAAGVAPVWASHSPHLPRPHSRVPPHPSSERRAVYIGALFPMSGGWPGGQACQPAVEMALEDVNSRRDILPDYELKLIHHDSK CDPGQATKYLYELLYNDPIKIILMPGCSSVSTLVAEARMWNLIVLSYGSSSPALSNRQRFPTFFRTHPSATLHNPTRVKLFEKWGWKKIATIQQTTEVFTSTLDDL EERVKEAGIEITFRQSFFSDPAVPVKNLKRQDARIIVGLFYETEARKVFCEVYKERLFGKKYVWFLIGWYADNWFKIYDPSINCTVDEMTEAVEGHITTEIVMLNPA NTRSISNMTSQEFVEKLTKRLKRHPEETGGFQEAPLAYDAIWALALALNKTSGGGGRSGVRLEDFNYNNQTITDQIYRAMNSSSFEGVSGHVVFDASGSRMAW TLIEQLQGGSYKKIGYYDSTKDDLSWSKTDKWIGGSPPADQTLVIKTFRFLSQKLFISVSVLSSLGIVLAVVCLSFNIYNSHVRYIQNSQPNLNNLTAVGCSLALAA VFPLGLDGYHIGRNQFPFVCQARLWLLGLGFSLGYGSMFTKIWWVHTVFTKKEEKKEWRKTLEPWKLYATVGLLVGMDVLTLAIWQIVDPLHRTIETFAKEEPK EDIDVSILPQLEHCSSRKMNTWLGIFYGYKGLLLLLGIFLAYETKSVSTEKINDHRAVGMAIYNVAVLCLITAPVTMILSSQQDAAFAFASLAIVFSSYITLVVLFVPK MRRLITRGEWQSEAQDTMKTGSSTNNNEEEKSRLLEKENRELEKIIAEKEERVSELRHQLQSRQQLRSRRHPPTPPEPSGGLPRGPPEPPDRLSCDGSRVHLL YK PFAM analysis Homology to known structure can be used to create model

6. Russell Group, Protein Evolution _________ ____ Family III GPCRs 1234567 R1 Ligand binding domain IC1 IC2 IC3 Cterm EC1 EC2 EC3 - - - G-protein dimerisation cut

7. Russell Group, Protein Evolution _________ ____ Robbins et al, J. Neurosci, 21, 8043, 2001 R1 binds ligand R2 signals

8. Russell Group, Protein Evolution _________ ____ GabaB R1/R2 12345671234567 R1 R2 Ligand binding domain IL1 IL2 IL3Cterm EL1 EL2 EL3 IL1 IL2 IL3Cterm EL1 EL2 EL3 - - - - - - - G-protein (none) blocked

9. Russell Group, Protein Evolution _________ ____ Case study 2: Human RYK an inactive tyrosine kinase

10. Russell Group, Protein Evolution _________ ____ Human RYK model Insulin receptor YK (template) Human RYK (model) Katso, Russell, Ganesan, Mol Cell Biol, 19, 6427, 1999

11. Russell Group, Protein Evolution _________ ____ Van Noort et al, Mol Sys Biol, 2012 Case study 3: What are phosphorylation sites doing?

12. Russell Group, Protein Evolution _________ ____ Katso, Russell, Ganesan, Mol Cell Biol, 19, 6427, 1999 MPN134 is phosphorylated at Serine 392

13. Russell Group, Protein Evolution _________ ____ What do modifications do to interfaces? Van Noort et al, Mol Sys Biol, 2012 From positively charged to polar From polar to negatively charged Modelled MPN134 homodimer: From a polar-polar interaction to a pair of negative charges in proximity

14. Russell Group, Protein Evolution _________ ____ Homology modelling + algorithm

15. Russell Group, Protein Evolution _________ ____ Homology modelling steps Identify the homologue of known structure Get the best alignment of your sequence to the structure Model building –Side-chain replacement –Loop building –Optimisation/relaxation/minimisation

16. Russell Group, Protein Evolution _________ ____

17. Russell Group, Protein Evolution _________ ____ Problem with loops Two subtilisin-like serine proteases