Evolutionary Rate Heterogeneity in Proteins with Long Disordered RegionsOutlineProtein Evolution–Patterns of Evolution–Rates of EvolutionDisordered ProteinDNAAGCTRNAAGCU20 amino acidsProteinPhenotypeSerine-Threonine PhosphataseMutations vs SubstitutionsNot all mutations persist over time• Selection• Random lossDetecting SubstitutionshADH-G CCTCCTAAGGCTCATGAAGTTCGCATTAAGhADH-A CCTCCTAAGGCTCATGAAGTTCGCATTAAGhADH-B CCTCCTAAGGCTTATGAAGTTCGCATTAAGbADH-B CCTCCTAAGGCTTATGAAGTTCGCATTAAGChange in Protein SequencehADH-G PPKAHEVRIK hADH-A PPKAHEVRIKhADH-B PPKAYEVRIK bADH-B PPKAYEVRIKPatterns of EvolutionModeling Protein EvolutionhADH-A PPKAHEVRIKhADH-B PPKAYEVRIKSubstitution MatricesSerine-Threonine PhosphataseUses of Substitution MatricesAligning sequencesFinding sequence homologuesCalculating genetic distancesGenetic Distance0.1hADH-A PPKAHEVRIKhADH-B PPKAYEVRIKSubstitution MatricesREMARDPV 26 2 -2 1D E=R D P V R E M AREMARDPVRates of EvolutionSubstitution rate heterogeneity among proteinsSubstitution rate heterogeneity within proteinsVariation among ProteinsDickerson, 1971, J. Mol. Evol. 1:26millions of years since divergence of species being comparedVariation within ProteinsLi, 1997, Molecular EvolutionEstrogen ReceptorDisordered ProteinProtein with no fixed secondary and/or tertiary structure that occurs as an ensemble of structuresLac Repressor DNA binding domainW C F I Y V L H M A T R G Q S N P D E K-1-0.500.51(Disorder – Order) / Orderdis XRAY (2844 aa)dis NMR (4019 aa)dis CD (10554 aa) Differences in Amino Acid CompositionDisorder and FunctionLinkers, spacers, bristles, clocks, springs, detergents, self-transportAcetylation, fatty acylation, glycosylation, methylation, phosphorylation, ADP-ribosylation, ubiquitination, proteolytic digestionGlobular oligomers, linear polymers, hetero complexes, phages, viruses Inter- and Intra-protein, ssDNA, dsDNA, tRNA, rRNA, mRNA, nRNA, bilayers, ligands, co-factors, metalsDescriptionEntropic ChainsProtein ModificationMolecular Assembly / DisassemblyMolecular RecognitionCategoryUnknown17 36>13114Number8020406080>=30 >=40 >=50 >=60 >=70 >=80 >=90 >=100% of proteinsconsecutive disorder predictions Cancer-associated proteins Signaling proteins SwissProt O_PDB Select25 PONDR Disorder EstimatesProteins with Disorder and Rate HeterogeneityCalcineurinTopoisomerase IIRibosomal Protein S4Voltage-gated potassium channelsFlagellinIdentify disordered proteinIdentify homologous proteinsPDB, PubMedBLAST Align homologuesCLUSTALW DOCreate aligned Order and Disorder setsCreating Disordered Protein FamiliesCalculating Pair-wise Genetic DistanceD340.080.25 0.290.35 0.38 0.42D =Calculate average difference between genetic distances0.080.25 0.290.35 0.38 0.42D =0.050.2 0.250.25 0.27 0.35O =Δ = {(0.05 - 0.08) + (0.2- 0.25) + (0.25 - 0.29) + (0.25 - 0.35) + (0.27 - 0.38) + (0.35 - 0.42)} / 6 = -0.07Calculating Average Differences, ΔHypothesis TestH0: Any given position in the alignment is as likely to be in a region of disorder as in a region of orderRandom AssignmenthADH-G PPKAHEVRIK hADH-A PPKAHEVRIKhADH-B PPKAYEVRIK bADH-B PPKAYEVRIK P(D) = #Disordered residues/totalP(O) = 1- P(D)OrderPPAHEKPPAHEKPPAYEKPPAYEKDisorderKVRIKVRIKVRIKVRI0150300450600750900Δ0FrequencyRPATBSV-1-0.8-0.6-0.4-0.200.20.4Sampling DistributionsDisordered Protein Families26 Families with DR > 30 amino acids–17 by X-ray crystallography–6 by NMR–1 by both X-ray and NMR–2 by circular dichroism and limited proteolysisFamily size from 4 to 80 sequencesDifferences in Rates of EvolutionDisorder > Order 19Disorder = Order 5Disorder < Order 2A Rapidly Evolving Disordered RegionFlexibility and ConservationRNL AGEVGVKIGNPVPYNEGHAQQQAVSAPASAATPPASKPQPQNGSLGVGSTVAKAYGASKPFGKPAGTGLLQPTSGTHSL AEAVGVKIGNPVPYNEGLGQPQVAPPAPAASPAASSRPQPQNGSSGMGSTVSKAYGASKTFGKAAGPSLSHTSGGTNCL LGCPEKMGDPQPLGPRSAEPQQNPNLGSTGFYGVKSEPTQDTKPQFPRQMPSRNASGGQGSSTATL ETIGNPTIFGETDTEAQKTFSGTGNIPPPNRVVFNEPMVQHSVNRAPPRGVNIQNQANNTPSFRPSVQPSYQPPASYRNHGPIMKNEAOSL LEVVFKALDSEIKCEAEKQEEKPAILLSPKEESVVLSKPTNAPPLPPVVLKPKQEVKSASQIVNEQRGNAAPAARL02468101 10 20 30 40 50 60 70 80 90HSL J(0) vnRNL J(0) vnOSL J(0) vnATL J(0) vnJ(0) (ns)RNL AGEVGVKIGNPVPYNEGHAQQQAVSAPASAATPPASKPQPQNGSLGVGSTVAKAYGASKPFGKPAGTGLLQPTSGT HSL AEAVGVKIGNPVPYNEGLGQPQVAPPAPAASPAASSRPQPQNGSSGMGSTVSKAYGASKTFGKAAGPSLSHTSGGT NCL LGCPEKMGDPQPLGPRSAEPQQNPNLGSTGFYGVKSEPTQDTKPQFPRQMPSRNASGGQGSST ATL ETIGNPTIFGETDTEAQKTFSGTGNIPPPNRVVFNEPMVQHSVNRAPPRGVNIQNQANNTPSFRPSVQPSYQPPASYRNHGPIMKNEA OSL LEVVFKALDSEIKCEAEKQEEKPAILLSPKEESVVLSKPTNAPPLPPVVLKPKQEVKSASQIVNEQRGNAAPAARL Compact Globular Protein00.20.40.60.81AAAAAAAAAEGEEEEEEDDEAAVVAAEVVVVVVVVVGGGGGGGGGLVVVVVSLGKKKKKKKKKIIIIIIIIIGGGGGGGGGNNNNNNNNNPPPPPPPPPVVVVVVVVVPPPPPPPPPYYYYYYYYYNNNNNNNNNEEEEEEEEEGGGGGGGGGYHLLLHHHQGAGGGGGGGQQQQQQQQQQQPPPPQQQAAVVVAVAASAPPPPPSAPPPPPPPPPAAAAAVVVSSSPPPSSSPAAAAAAAAAAAAAAGTATTTSSSSSNNPPPPPPPPPNNNNNHNNNGGGGGGGGGSSSSTSSSSLLSSSSPSSGGGGGGGAGMVMVAAMMLGGGGGGAGGSSSSSSSFSTTTTTTTTTAVVVVVAAAAASSSSSSTKKKKKKKKKAAAAAAATSYYYYYYFYFGGGGGGGSGAAAAAAAAGSSSSSSSSSKKKKKKKKKPPTTTTTTTFFFFFFFFFGGGGGGGGGKKKKKKKKKPPAAAAAPVAAAAAGGGGGGGGGGGGNTTPPPPTTAGGSSSSSSSQQHHHNNSSPPTTNTSSTSTSSSSSSPGSGGGGGGGGGGGGGGGGTTTTTTTTSNeutral vs Purifying SelectionFunctions of Rapidly Evolving Disordered RegionsReplication protein A, Topo II 2Entropic ChainsTopo II, Gonadotropin, Bcl-xL 3Protein ModificationNF-KB, RGS4, Topo II, Calcineurin, cFos, Thyroid TF, F-tRNA synthetase, TBSV & SV coat proteins, Histone H5, Telomere BP 12Molecular RecognitionProteinsNumberCategoryG-tRNA synthetase, Sulfotransferase, Cytochrome BC1, DNA-lyase 4UnknownFunctions where Disorder = OrderEpidermal growth factor1UnknownGlycine N-methyltransferase 1AutoregulatorySmall HSP, Prion, SBMV coat protein3Molecular RecognitionProteinsNumberCategoryFunctions of Slowly Evolving Disordered Regions ssDNA BP1Entropic ChainsFlagellin 1Molecular AssemblyssDNA BP, Flagellin2Molecular RecognitionProteinsNumberCategoryWhy was Order Faster?The ordered region of Flagellin is antigenicMultiple functions or specificity of binding of ssDNA BP?Why was Disorder Faster?Differences in amino acid compositionLow complexity sequences evolve rapidly, and often disordered proteins are low complexityDisorder has no function and therefore evolves at a neutral rateWhy was Disorder Faster?Fewer constraints because there are many ways to be