Protein evolution - Department of Chemical Engineering - Caltech

Protein evolutionNICOLAS PUTMAN / UC BERKELEYteam traced in detail how evolutiontweaked the ancestral structure overtime to yield new functions.They found that in many receptorlineages, a few mutations subtlychanged the size and shape ofthe cavity where the signallingcompound binds, causing receptorsto evolve partnerships withhormones or other new signals.Other members of the receptorfamily became independent ofchemical signals; these proteins, likeswitches stuck in the ‘on’ position,evolved when simple mutationsincreased the intrinsic stability ofthe protein’s active conformation,removing the need for a chemicalsignal to activate gene expression.Ancestral complexity‘If you just compare the receptors inmodern humans, the evolutionaryevents by which they could haveevolved are not obvious. It may lookas if the complex functions of eachprotein evolved independently,’ saysThornton. ‘But when we traced theseproteins from their ancestor throughtime, we saw how evolution tinkeredwith the ancestral form, producingan incredible diversity of proteinfunctions and the ability to interactwith many different chemical signals.’Nuclear receptors are a ‘greatcase study in protein evolution’,says Thornton, adding that it islikely that other protein families,when studied in similar detail,will turn out to have diversifiedby a similar kind of tinkering. ‘Wepredict that, when sufficient dataare gathered to allow detailedevolutionary reconstructions, itwill become apparent that mostprotein superfamilies diversifiedby subtle modification and partialdegradation of ancient, deeplyhomologous functions. Invoking theevolution of wholesale ‘‘novelty’’will seldom be necessary.’ 1That’s all very interesting, ofcourse, but is it really useful? Verymuch so, Thornton explains:‘Structure-function relationships arethe fundamental object of knowledgein protein chemistry; they allow usto rationally design drugs, engineerproteins with new functions, andunderstand why mutations causedisease.’ Unfortunately, revealing howthe sequence of a protein determinesits function using traditionallaboratory manipulations is difficult,because the number of possiblesequence variations in any proteinfamily is too great. ‘The solution liesin studying evolution, which has beena massively parallel experiment indiversifying and optimising proteins,conducted over vast periods of time’says Thornton.One person who has found proteinevolution both interesting anduseful is Frances Arnold, a chemicalengineer at California Institute ofTechnology (Caltech) in Pasadena,US. Arnold, together with Willem‘Pim’ Stemmer, chief executive of theCalifornian biopharmaceutical firmAmunix, has just been awardedthe Charles Stark Draper prize –widely considered the equivalentof the Nobel prize for engineeringin the US – for developingdirected evolution: ‘a methodused worldwide for engineeringNuclear receptorevolution has beentraced in the seaanemone N. vectensisand other species fromthe relatively distantevolutionary pastProteins with flexibleconformations, like thissugar-binding lectin, aremore likely to evolve awide variety of functionsnovel enzymes and biocatalyticprocesses for pharmaceutical andchemical products.’Inspired by the real-life evolutionof proteins, Arnold developed asystem that literally ‘directs’ theevolution of proteins by repeatedrounds of mutation and selection.‘Evolution happens by theaccumulation of beneficial mutationsone at a time through functionalproteins,’ says Arnold. ‘And that’sa very simple algorithm; it doesn’trequire that you screen large numbersof things if you can go through singlemutational stops.’Mimicking evolutionArnold began to evolve proteinsby randomly mutating the geneof a known protein, screening itsmutant offspring a few-hundred or afew-thousand at a time, finding onebeneficial mutation, and repeatingthe process starting from that mutantgene. ‘That’s just mimicking howadaptive evolution happens,’ she says.‘It doesn’t fully mimic it, becausewe miss the entire neutral mutationpathways,’ she says, ‘but peopleare starting to try to insert neutralmutations into directed evolutionas well.’ Neutral mutations will bepassed on from one generation tothe next if they pose no threat to anorganism’s survival. They shouldn’tbe ignored because, further on downthe line, they might help give rise to abeneficial mutation.Another important lesson fromadaptive, real-life, evolution has comefrom recognising which proteins andwhat functions are evolvable. ‘We’velearnt that the proteins that evolveDAN TAWFIK44 | Chemistry World | March 2011www.chemistryworld.org

NATIONAL LIBRARY OF MEDICINE / SCIENCE PHOTO LIBRARYreadily in nature also evolve readily inthe laboratory,’ says Arnold.‘For example, secondary metabolicpathway enzymes, like cytochromeP450s and other enzymes whosespecificities and functions havevaried all over the map tend to readilychange in the laboratory,’ she says.‘And the converse is true, proteinswhose functions have not showngreat diversity in nature tend to bemore difficult to engineer’Arnold comes from what she callsan engineering and problem solvingbackground. ‘I was looking for themost effective means to engineerproteins, the sequence basis of whosefunctions we just don’t understand.So a rational design pathway toachieve very specific functions justwasn’t realistic, and evolution seemedto be a much more promising route.’Just as Arnold keeps up with theworld of natural protein evolution,particularly work from Thornton’slab, protein evolutionists are nowreading the directed evolution work.‘We have one huge advantage,’ saysArnold, ‘and that’s data.’Although directed evolutionstarted from an engineeringperspective, researchers fromdifferent backgrounds and interestshave come to it specifically to answerquestions about how proteinsevolve. ‘If you look at people likeJoe Thornton or a number ofpeople whose primary interest is inevolution, they are now using directedevolution to try to understand howproteins can adapt,’ she says.The traditional view that proteinspossess absolute functional specificityand a single, fixed structure conflictswith their marked ability to adapt andevolve new functions and structures,says Dan Tawfik of the WeizmannInstitute of Science in Israel.‘Natural selection is yieldingmolecular machines withbreathtaking performance,’ saysTawfik. ‘New functions can evolvewithin years or even months, ashappens naturally with things likedrug resistance.’Tawfik’s team argues that proteinsexhibit ‘functional promiscuity’,which is an essential component of aprotein’s evolvability. He correlatespromiscuity with conformationaldiversity. In antibodies, for example,increased affinity for a ligand –clearly an essential characteristicof an antibody – leads to decreasedbinding-site flexibility. Converselythe P450s, as mentioned by Arnold,demonstrate increased promiscuity– relatively broad substrate scope –with increasing flexibility.But is it really chemistry?The study of protein evolution liesat the interface of chemistry andbiology, and relies to a large extenton engineering. Each discipline canclaim it as their own, but it is oftenChristian Anfinsenone of a host of proteinscientists who haveclaimed the Nobel prizefor chemistry‘Naturalselectionis yieldingmolecularmachines withbreathtakingperformance’dismissed as ‘not really biology’ or‘not really chemistry’.It’s definitely chemistry,says Arnold the engineer, whonevertheless files directed evolutionfirmly under ‘synthetic biology’.Apart from anything else, scientistshave a habit for winning Nobel prizesin chemistry for work on proteins.Back in 1946, the Nobel prize inchemistry went to James Sumnerof Cornell University, US, who wonhalf the prize ‘for his discovery thatenzymes can be crystallised’; andJohn Northrop together with WendellStanley, both of the RockefellerInstitute for Medical Research inPrinceton, US, shared the other half‘for their preparation of enzymes andvirus proteins in a pure form’.In 1972, half the chemistry Nobelprize went to Christian Anfinsenof the US National Institutes ofHealth with the other half shared byStanford Moore and William Stein,both from Rockefeller Universityin New York, US, for fundamentalwork in protein chemistry. Anfinsenhad shown that the information fora protein assuming a specific threedimensionalstructure is inherentin its amino acid sequence, whichwas the starting point for studiesof protein folding. Moore and Steinreceived the prize for discoveringanomalous properties of functionalgroups in an enzyme’s active site, as aresult of the protein fold.In 2009, the prize went toVenkatraman Ramakrishnan, ThomasSteitz and Ada Yonath ‘for studiesof the structure and function of theribosome’. In 2008 it was awardedjointly to Osamu Shimomura, MartinChalfie and Roger Tsien ‘for thediscovery and development of thegreen fluorescent protein, GFP’.On Frances Arnold’s website, 3 teammembers have contributed to a ‘Howwould you explain synthetic biologyto Darwin?’ section.In Arnold’s words: ‘You had itjust right! And now I can build newcomponents of life – for example,the proteins of which we are made– by artificial selection. When Idecide who gets to reproduce,these components evolve just likethe organisms that make them do,because they are encoded by the samestuff and follow the same principlesof evolution that you showed usfor all forms of life.’References1 J T Bridgham et al, PLoS Biol., 2010, 8,e1000497, (DOI: 10.1371/journal.pbio.1000497)2 N Tokuriki and D S Tawfik, 2009, Science,324, 2033 www.che.caltech.edu/groups/fhawww.chemistryworld.org Chemistry World | March 2011 | 45