PROTEIN CHEMISTRY STRUCTURAL BIOLOGY YEARS OF ‘DESIGNING’ BRAND AT THE UK’S LARGEST BIOCHEMICAL NEW PROTEINS PARTICLE ACCELERATOR 50 BREAKTHROUGHS : A BIOCHEMISTRY SPECIAL

THE MAGAZINE OF THE ROYAL SOCIETY OF BIOLOGY ⁄www.rsb.org.uk Vol 62 No 5 supplement • Oct/Nov 2015

FLUORO SCIENCE Nobel Prize winner Roger Tsien on the discovery and development of the green fluorescent protein Foreword About David Baulcombe Contents this issue Volume 62 No 5 Oct/Nov 2015 Produced in partnership with the Biochemical Society, this mini special issue aims to showcase the fascinating and important work that biochemists do. Biochemistry has created the tools, techniques and knowledge on which modern bioscience depends. It is at the very core of how all life on Earth works Welcome and its output underpins many other areas of the life sciences ROYAL SOCIETY OF BIOLOGY Charles Darwin House, including medicine, pharmaceuticals, agriscience and 12 Roger Street, London WC1N 2JU biotechnology. Our content here is focused towards the iochemistry is sometimes compared to cookery. Tel: 020 7685 2550 exciting chemistry of proteins, just one of many different types Chefs and biochemists both mix ingredients and Fax: 020 3514 3204 of macromolecule studied by biochemists. We can never do wait with excited expectation for the result – either a [email protected] justice to such a remarkable field in just 16 pages, but we delicious new dish or an experimental outcome. They www.rsb.org.uk hope this one-off biochemistry special whets your both follow recipes, although biochemists refer appetite to find out more. to theirs as ‘experimental protocols’. Continuing this Managing editor Tom Ireland MRSB metaphor, I am reminded of the famous recipe for @Tom_J_Ireland Bjugged hare that is said to [email protected] start with “first catch Tom Ireland, managing editor, your hare”. Royal Society of Biology Biochemists do not normally race around the countryside chasing furry BIOCHEMICAL SOCIETY animals, but until recently Charles Darwin House, there was a parallel 12 Roger Street, London WC1N 2JU preliminary step in our Tel: 020 7685 2400 protocols. We had to process Fax: 020 7685 2467 litres of culture or extract [email protected] www.biochemistry.org kilograms of tissue before we could start work with Science editor, The Biochemist milligrams of the molecule Frederica Theodoulou of interest. freddie.theodoulou@ We now operate on a micro rothamsted.ac.uk scale way beyond the tiniest We are ‘bio’ chemists Community and press editor, amuse bouche in a nouvelle Biochemical Society cuisine restaurant. We can and our ultimate goal Helen Albert start with tiny amounts of [email protected] The Biochemist tissue and get information is to understand how This is an exciting time to be a biochemist as new tools and technologies are about thousands of living systems are offering unprecedented insights into the molecular workings of life. It has been a molecules, rather than just one as in the past. more than the sum of The Biologist and its supplements pleasure to help to develop this special issue for The Biologist and share a small are produced on behalf of With genomics and their parts, for the the Royal Society of Biology by sample of what biochemistry has to offer with a wider audience. If you like what molecular biology, we can Think Publishing Ltd you see in this taster issue, please do take a look atThe Biochemist, to explore isolate genes affecting benefit of humankind Capital House processes that were the wonderful world of molecular biosciences. 25 Chapel Street Roger Tsien on the previously inaccessible to the London NW1 5DH Professor Frederica Theodoulou, science editor, The Biochemist development of the biochemist. From the genes www.thinkpublishing.co.uk 020 3771 7200 green fluorescent protein we see the proteins, and from the proteins we find other Art director Matthew Ball PAGE 6 Designer Dominic Scott components of the Production editor Sian Campbell Breakthroughs in biochemical circuitry in the Professor David Baulcombe, Sub editor Kirsty Fortune IN THIS SUPPLEMENT biochemistry at the cell or organism. president of the Biochemical Society Publisher John Innes Diamond Light Source Metaphors normally break [email protected] 02 50 YEARS OF INNOVATION chemistry will help us down under close inspection and this one is no exception. Few people Views expressed in this magazine are not PAGE 12 necessarily those of the Biochemical Society Seven biochemists make hardier crops would compare modern molecular genetics to mere cookery (and or the Royal Society of Biology. pick their favourite 12 DIAMOND LIGHT biochemists certainly do not ‘cook’ their results…). There is, however, © 2015 Royal Society of Biology breakthroughs Lessons in structural one element of the cookery metaphor that still applies: slow food. (Registered charity no. 277981) 06 INTERVIEW biology from Slow food enthusiasts would relish catching the hare, and they may The Society permits single copying of Roger Tsien on his most the UK’s largest embrace new technology, but they do not wish to lose sight of the whole individual articles for private study or research, irrespective of where the copying is famous work, the green particle accelerator food chain. Biochemists need to remember this slow food movement as done. Multiple copying of individual articles fluorescent protein 14 DESIGNER PROTEINS we bury ourselves in the enormous amounts of data pouring out of our for teaching purposes is also permitted without specific permission. For copying or 10 PLANT POWER How biochemists are mass spectrometers, imaging devices and next generation sequencers. reproduction for any other purpose, written Joseph Jez on why making new proteins for permission must be sought from the Society. We are ‘bio’ chemists and our ultimate goal is to understand how Exceptions to the above are those institutions understanding plant medicine and research living systems are more than the sum of their parts, for the benefit and non-publishing organisations that have an agreement or licence with the UK of humankind. We should not lose sight of the biological hare that Copyright Licensing Agency or the US Facebook “f” Logo RGB / .ai Facebook “f” Logo RGB / .ai is our raison d’être. Are we succeeding? I believe we are. Readers Copyright Clearance Center. Access to the TWITTER FACEBOOK WEBSITE magazine is available online; please see the twitter.com/ www.facebook.com/ www.biochemistry.org of The Biologist can judge for themselves by reading this special Society’s website for further details. BiochemSoc biochemicalsociety issue on the field.

Biochemistry Supplement / The Biologist / 1 LOCAL WORLD/REX SHUTTERSTOCK 50 2 /TheBiologist /Biochemistry Supplement DNA andwas ratherhard work. The was basedonchemicalcleavage of which Gilbert methodfromHarvard, came hard on the heels of the Maxam– Sanger sequencing. chain terminationmethod,or clever techniqueknownasthe DNA canbesequencedbya Sanger forthediscovery that I have tochooseFred fragment. length differenteach present at is nucleotide altered which detecting readby be can size,DNAsequence the and rankedbythen arefragments either A,or C,T.G These bases– specific at fragments synthesisedDNAnewly terminate nucleotides alteredchemically DNAsequencing,of method this In DNA sequencing European breakthroughs of thepast five decades* Seven leading biochemists pickthemost important OF BIOCHEMISTRY YEARS Seminal discoveries History The development ofthismethod

*This article isanedited version of ‘50 years of European Biochemistry’, first publishedin TheBiochemist, August 2014 best way tosequenceDNA fornearly universally adoptedandundisputed led tothehumangenomeproject. in timeitbecameautomated,which robust andsosimpletouse.Ofcourse, method isthatitwas so elegant,so amazing thingabouttheSanger UniversityBirmingham,UK of ProfessorBusby,Stephen any problem. an essentialexperimentalapproach to the riseandofbioinformatics as importance ofDNA modifications, and understanding howDNA evolves, the Sanger’s method remained the Sanger’s methodremainedthe 25 years, and it’s only in the past 25 years,andit’sonlyinthepast of the third kingdom of life, of thethirdkingdomlife, decade that it has been decade thatithasbeen genetics, thediscovery supplanted by myriad supplanted bymyriad simplifying and unifying simplifying andunifying of our thinking, including of ourthinking,including new higherthroughput impact on so many areas impact onsomanyareas methodologies. Ithadan

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Science luminaries, from left: James important biochemical questions:howdowe biology. Anditdidforoneofthe most these dimensions,unitingchemistry and chemiosmotic theoryusedphysics tobridge did three-dimensionalbiology. The dimensional chemistry, andcellsorgans elegant aswell asfunctional. showed methatbiochemistrycouldbe have takenavery differentstep.Thetheory textbooks 30yearsago,mycareermight graced myundergraduatelecturesand If itwasn’t forthebeautyofthistheory that (ATP).triphosphateadenosine molecule carrying energycreate the to cells membranesenabled mitochondrial movementhydrogentheof through demonstratedthat ions it famously Most processes.catalysebiological provideto energyusefulmembranescould biological through movementions theof illustratedhow Peter Mitchell’stheory chemiosmotic The chemiosmotic theory In the1950sand1960s,enzymes didtwo- the chemiosmotic theorywas. Thefirst understand how trulyparadigmshifting video reconstructions,itisdifficult to by highresolutiongraphicsand conceptual century world viewofmitochondriashaped 21st our into theclarityof1980s.With converting themessinthisfield1960s strands of theory and confusing experiments, movement, development andreproduction? into ausefulformthatcanpower the foodwe eatandtheoxygenwe breathe efficiently convert thepotentialenergyfrom This theory beautifully explained disparate This theorybeautifullyexplaineddisparate years on, we now know many of the the yearsof manyknow nowon,we requiredcourse,division.Of cellfor 25 cyclins, was to which binding and proteinregulatedfromyeastwasby that a of identification the described he which fromPaulNurseDundee,in talk heard a I cyclins.called be later wouldproteins that cyclecell– eachsynchronously with down and levelswhoseup went urchinseaeggs in detected been had laboratories.proteins offamily A small ResearchCancer Fund’s ClareHall Imperial fromHunt’sthe Tim groupat me.on lost not waswork the ofsignificance the but liver– rat in metabolism regulatinglipid proteininvolveda on in working project– awayownmilesfrommy million division.a time,cellseemed the Atthis controllingkey rolein a had oocytesthat discoveredproteinfroma Colorado.in meeting Maller’s grouphad Denver,in laboratory a routeto en Maller’sto Jim visit brief a making 1980s the in back student PhD a rememberas I towardsdivision. cell’sinvolvedprogressionthe in chemicals other modifying important, most the(CDKs) kinasesamong are cyclecontrol, cyclin-dependent involved proteinscell many in the Of awry.goingpreventing division to cell processessentiala DNA– replication faithful and growthsufficient after divide only cells cyclethat ensurecell eukaryotic the in checkpoints Multiple The cell cycle Sometime later,Sometime postdocin a as afterwards,Shortly work heardabout I Xenopus

had acceptedthetheory. millennium, theworld, anditstextbooks, However, bythedawn ofthenew Prize toMitchell,therewere stilldissenters. paradigm shiftinginsociologybooks. famous thatitwas usedasanexampleof simultaneously. Thetheorybecameso bonds, andindeeditwas foundtheydidboth the transportofionsasformchemical entities emerged,equallyasabletocatalyse membrane enzymesasmultidimensional to beintegraltheirstructure. bilayer membranes,butwere notconsidered – theywere knowntoassociatewithlipid structure was stillmorethan20yearsaway understanding ofmembraneprotein UniversityEssex,of UK ProfessorCooper,E Chris Imperial CollegeLondon,Imperial UK ProfessorCarling,David elegancenatureitself.of the exampleofbeautiful a cyclecellis regulatingthe mechanisms fundamental Nonetheless,discovery the the of rule.exceptionthe the than ratherbe to results tend dried and cut moment,and the of heat the in clearcrystal things discoveriesscientific rarelyvery are– most naturereflectsof simplythe reality.fromthe farwas this time This together,slottedpieces the the at but all howsee easyto is it back looking and discovery.thing, wonderful a is Hindsight their for2001 in Medicine Physiologyor LeePrize Hartwell,Nobel in sharedthe Xenopusoocytes. in previouslyisolated had been that one very the MPF– complexas known kinase cyclinproteinB,together with a forms yeast,in gene cdc2 and bythe encoded (CDK1),proteinkinase dependent regulated:cyclecyclin-cellis the exquisitehow ofand intricatedetails Even aftertheaward ofthe1978Nobel After thetheory, anewconceptof Tim Hunt and PaulNurse,and Hunt togetherTim with Biochemistry Supplement /TheBiologist /3 Seminal discoveries

History

MICHEL DELARUE, ISM/SCIENCE PHOTO LIBRARY 4 /TheBiologist /Biochemistry Supplement of Auckland,NewZealand ProfessorPeterShepherd, University research would notexistaswe know it. in thecell.Withoutthem,modern biological analysis ofvirtuallyanybiological function comprehensive setoftoolstoassistinthe in thelast20yearsprovideuswith a mainstays ofmoderncellbiology. electron microscopy, have becomethe could linkantibodiestocolloidalgoldfor antibodies, togetherwithmethodsthat limited thebroaduseoftheseassays. molecules, buttheuseofradioactive isotopes revolutionised thequantitationofbiological Nicholas HalesandPhilipRandle,this precision. LaterperfectedbyCharles concentrations ofantigenswithextreme which allowed biochemiststomeasure by RosalynYalow andSolomonBerson, development oftheradioimmunoassay in biochemistrywas facilitated bythe biochemical research. both reagentsareessentialformodern of polyclonalandmonoclonalantibodies, Although we maydebatetherelative merits to identifyandstudyproteinseasily. of well characterisedantibodieswe canuse protocols. Now, therearetensofthousands large partreliantontediouspurification modifications altertheway theywork. localisation changesandpost-translational proteins riseandfall,theirsub-cellular are ever changingasthelevels ofindividual and theiractivitiescontributions power ofthehumbleantibody. reasons we candosuchexperimentsisthe computer screen.Ofcourse,oneofthemain or fromlookingatlittleblackbandsona performed inafewmicrolitresofclearliquid make anyconclusionsfromanexperiment they arealways amazedthatscientistscan When Ishowvisitorsaroundmylaboratory them? without be wewouldWhere technologies Antibody Seminal discoveries History Advances inantibodybasedtechnologies Techniques thatusefluorescentlylabelled The firstlargescaleuseofantibodies In the1960sstudyofproteinswas in Proteins domostofthework inanycell, Rosalyn Yalow

viral replication.viral successfulinhibit to means a as dsRNAs foreigndegrade and detect to mechanisms replication.virus haveCellsevolvedduring recognisingproduceddsRNA species by infection virus detect cells animal and plant infection:response virus to immune innate the of part important recognisedan been as (dsRNA)had RNA,categorywereof discovered. RNAs,entirelynew non-coding an small when 1990s the in dismantled be beganto expressiongene in molecule intermediary protein.translatedto is which mRNA, transcribedto DNAis (mRNA): messengerroleas RNA functional its is cell the understoodRNAbestin roleof synthesis.the perhaps and second The protein out carry ribosomesthat the scaffoldexample,building fora as acting for – structuralmolecule a be First,can it cells.therewererolesRNAin oftwo main yearsaround15 ago, Until thought was it againstpathogens. protecting and regulationgene in role keya play can (RNA) acid ribonucleic formation,protein in role its as wellAs ribonucleic acids small interfering The discovery of Foryears,many double-strandedRNA an mainly RNAas of view The UniversityGlasgow,of UK ProfessorGraham,Sheila interferingRNAs? small using expressiongenemanipulate to means the without bioscientistlaboratorydo biochemistry.today’swould what And of field entirelynew an seeded has RNAspecies tinythese at look tobiologists infections.viralcancers and targetingdiseases,genetictrials clinical 30 morethan recently,in used been has RNAi pathwaysbiochemicalcells. in More of dissection the forexperimentaltools providedinterferencehas essential (RNAi) cells. in haveRNAsessentialfunctions expression.words,other In short these regulatedevelopmentalalso gene can elements,geneticthey mobile but and defenceagainstin viruses important only arenot RNAs plant ultra-shortubiquitous bases21–24long. only is (siRNA)interferingRNAs average short so-calledthesesizeof the but 2,000length RNAbasesis in cells.plant averagein The messenger RNAsilencing this of phenomenon the mediated that RNAmolecules small discoveredpresenceextremelythe of Norwich, Laboratoryin Sainsbury the AndrewHamilton,at working The decision by a group of plant plant groupof bya decision The Crucially,discoverythe RNA of these that wassurprise second The Baulcombe,David togetherwith

NATIONAL LIBRARY OF MEDICINE/SCIENCE PHOTO LIBRARY; ILLUSTRATION BY DAVID S GOODSELL, THE SCRIPPS RESEARCH INSTITUTE

ALFRED PASIEKA/SCIENCE PHOTO LIBRARY/SCIENCE PICTURE CO/SCIENCE PHOTO LIBRARY contains 6.4×10 diameter, yet just 5–10μ nucleus measures human cell,the problem. Inatypical The nucleushasabig nucleus? the packedinto genomeentire the is How structure nucleosome Solving the H3 and H4. This forms an eight H3 andH4.This formsaneight each ofthehistones H2A,H2B, particle consistsoftwo copies known that the nucleosome core in Cambridge.Itwas already Laboratory of Molecular Biology Klug’s laboratoryattheMRC earnest inthe1980sAaron the atomiclevel beganin structure ofthenucleosomeat higher orderedstructures. are themselves arrangedinto proteins intonucleosomes,which is wrappedupbyasetofhistone is packaging(pictured).TheDNA small space.Thesolutiontothis DNA crammedintothis metres’ worth of That’s over two base pairsofDNA. Work tounderstand the m in m in 9

UniversityManchester,of UK ProfessorRichardReece, J nucleosome–DNA complexat solved thestructureof Richmond andhiscolleagues tour deforce,Timothy are wrapped. roughly 150basepairsofDNA histone structurearoundwhich to betheway theyare. we appreciatewhythingshave more we understand,themore is, however, farfrom it. The nucleus islikeaballofstring.It the two metresofDNA ineach regulation ofgeneexpression. biological significanceinthe modifications have enormous arrangements. These create higher-orderfolding with nearbynucleosomesto forming tails that can interact forming tailsthatcaninteract In an x-ray crystallography In anx-raycrystallography It might be easy to think that It mightbeeasytothinkthat near atomic resolution some near atomicresolutionsome tRNA, andthelarge purple structures are ribosomes. RNA hasmany functions histones protrude out, histones protrudeout, 13 yearslater. At this maroon molecules are Goodsell, mRNA inan ends of some of the ends ofsomethe resolution, the path resolution, thepath E.coli illustration by David white. The L-shaped revealed thatthe of theDNA helixas The structure also The structurealso it encircled the it encircledthe within cells. Inthis could be traced. could betraced. histone octamer cell isshown in bacteria andviruses defend against foreign objectslike proteins used by theimmunesystem to Below: Humanantibodies, the Y-shaped but the antibodies wereantibodiesweak the but culture,in produceantibody to continued and immortal questions.such study them help would that sourceproduction antibody of reproduciblestable a for Milstein’s looking groupwas diverseof antibodies? extraordinaryan rangesuch systemimmune produce the doeshow was:question antibodies.monoclonal modified havedrugsbeen recentlyapproved therapeutic approximatelyall thirdof a extraordinary that statistic the resultedin and industry multibillion-pound a spawned cancer.as such has It diseasestreatment major for biology,direct therapeutic to cell ofcountless aspects and purification,isolation gene pathology,protein everythingfrom diagnostic on impact enormous an had colleagues,and Winter has exploitationbyGreg medical for adaptation bytheir Georgesand Köhler, followed Milstein byCésarantibodies inventionmonoclonal ofThe substance.that purify targetdetect,or to used and produced be now substancecan anyalmost to specifically biologist’sbind molecular toolkit.that Antibodies any of essentialpart an are antibodies Monoclonal their exploitation Monoclonal antibodies and Myeloma cells wereMyelomacells mid-1970s,the In crucial a Biochemistry Supplement /TheBiologist /5 Cambridge,UK University of ProfessorRonLaskey, approaches.and tools immunodiagnostic extraordinaryan range of immunofluorescence, and components,cellular other proteinsand of purification to isolationfromgene haveexpected. rangeThese not could Köhler and Milstein extraordinary waysthat exploitedin havebeen antibodies monoclonal 1984. in Medicine Prize Nobel Physiologyforor wereawardedKöhler and the understatement, Milstein as use”,industrial and classic a medical forvaluable be could that culturesinsight “such profoundthe with ended 1975 indefinitely.antibody single produceda which of cells,producinghybrids, each myeloma immortal to cells antibody-producingspleen problemsbythesefusing overcameKöhler and Milstein research.for unsuitable and Seminal discoveries Since that time,that Since classic the resultedin This Naturepaper, which

History Interview Roger Y Tsien The light fantastic The remarkable American biochemist Roger Y Tsien tells us how he made his most famous discovery

n 2008 Roger Y Tsien shared the alone full sized proteins, we would have to Nobel Prize in Chemistry for the adopt the techniques of molecular biology discovery and development of the rather than synthetic chemistry. green fluorescent protein (GFP). I started in around 1988 by discussing a This glowing molecular tool has collaboration with Alexander Glazer revolutionised many areas of on phycobiliproteins, a family of biochemistry research, allowing fluorescent proteins from blue-green algae. Iresearchers to visualise the expression However, these needed a separate partner of certain genes or certain protein to insert the chromophore, molecules within cells. the part of the molecule Molecular biologists have responsible for its colour. since found countless uses for GFP and Why was the similar molecules, fluorescent protein of and fluorescent the jellyfish Aequorea proteins are now an victoria so useful? essential part of In 1992, Douglas biochemists’ Prasher at the Woods molecular toolkit. Bacteria expressing Hole Oceanographic fluorescent proteins used Institution cloned and Before you discovered as ‘paint’ on a petri dish sequenced the gene for GFP GFP, your work involved from Aequorea victoria. looking for dyes that could Although he was unable to help image neuronal activity. What work on GFP any further himself, inspired you to work in this field? he was willing to give samples of its DNA The visual system is the only sensory to requestors, of which there were two: system with the ability to display lots of Martin Chalfie and me. Marty’s lab events in spatiotemporal detail, so one discovered that GFP didn’t need help has to use one’s own visual system to from any other protein in the jellyfish, investigate another creature’s nervous so GFP had both availability and system. From very early on in graduate autonomy. It has taken us almost 30 school, I was attracted to developing more years to engineer an easily techniques for visualising neuronal expressible phycobiliprotein. activity as the best way to resolve many neurons firing simultaneously.

What led you to look at fluorescent I was attracted to proteins and their related genes? developing techniques My colleagues and I had painstakingly built dyes such as Fura-2 and Indo-1 – with for visualising molecular weights near 840 – for recognising neuronal activity as and visualising small calcium ions, whose molecular weight was only 40. So it seemed the best way to resolve that for the more general problem of many neurons firing recognising biochemical messengers such

SIPA PRESS/REX SHUTTERSTOCK PRESS/REX SIPA as cyclic AMP (molecular weight 329), let simultaneously

6 / The Biologist / Biochemistry Supplement Biochemistry Supplement / The Biologist / 7 Interview Interview Roger Y Tsien Roger Y Tsien

We want to use AFTERBEFORE biochemical differences between the tumour and normal tissue to © WENN LTD/ALAMY WENN © Left to right: Nobel Prize winners Paul make the tumour Krugman, Martin Chalfie and Roger Tsien with the then US president George W Bush fluorescent

Did you ever imagine that GFP and its BEFORE derivatives would be used by so many LIGHTING THE WAY researchers in so many different ways? A matched pair of photographs I knew that an autonomously fluorescent showing a tumour about to protein module would be of immense be excised, viewed without value, but I didn’t anticipate it would have and with the aid of tumour quite so many uses. imaging peptides

Do you have a favourite way in which GFP has been used? In this ‘brainbow’ image of a mouse’s It was satisfying when we got a phenomenon brain, different neurons glow with called fluorescence resonance energy different fluorescent proteins, allowing transfer (FRET) working between mutants researchers to visualise brain circuits of GFP. FRET senses the proximity of two fluorophores of different colours and had been a major goal when we set out. fluorescent substrates that are triggered synapses – and thus serve as molecular washed away by the next big wave or high But that’s now long in the past. Aequorea victoria by these enzymes to enter cells and substrates for memory. tide. Perhaps that’s a metaphor for much become trapped, and also to change of my career. How else do you think fluorescence might colour by modulating FRET (the same You hold around 100 or so patents for be used in the future? phenomenon mentioned above). A small various other biotechnology tools. Which You have a long tradition of engineering I can’t foresee a limit to future applications biotech company partly founded by me are you most proud of? in the family. Do you consider yourself a of fluorescence. After all, fluorescence is has just started a clinical trial with such In 1994, we started a biotech company chemist, biologist, bioengineer or what? an unusual and very useful property of molecules, together with the called Aurora Biosciences to use new I’m a muddled mix. When I was applying a small proportion of molecules. Under instrumentation for surgeons to see the fluorescence assays to speed up drug for my first faculty position, several biology the right circumstances it can be observed fluorescence as they operate. screening in the pharmaceutical industry. departments rejected me on the grounds in anything from single molecules to One of the projects Aurora took on was that I was a chemist, and at least one oceans, over nanoseconds to many days, What else is your lab working on to find drugs to help cystic fibrosis. Most chemistry department turned me down using the naked eye to the most at the moment? experts thought Aurora’s chances were as too much of a biologist. Almost all my sophisticated instruments. We are trying to gather evidence for a negligible, as the market for such rare work has been involved with tool building, ‘chromophore’ in the centre – it is new hypothesis for how and where the disease remedies was thought to be too but I have never had a formal engineering Can you tell us a little about fluorescence What is GFP? thought just three amino acids in the brain might store permanent memories small, and gene therapy was considered course or appointment. Fortunately, assisted cancer surgery? GFP stands for green fluorescent protein. protein chain create the fluorescent at the molecular level1. a much more promising approach. However, most forward looking departments In cancer surgery, fluorescence guidance It is a protein that glows green in the ‘chromophore’. It is stable, non-toxic to Previous hypotheses have assigned the Cystic Fibrosis Foundation backed have now adopted a more flexible and would be helpful because tumour tissue presence of UV or blue light, originally most organisms when expressed in cells, the site of memory storage to be various Aurora’s efforts, and fluorescence screening interdisciplinary viewpoint. Personally, doesn’t look any different from normal found in the bioluminescent and and requires only UV/blue light and proteins within synapses, the places at found the drug that was recently lauded by I don’t care much for labels. tissue under ordinary white light fluorescent jellyfish Aequorea victoria. oxygen to emit its eerie glow, making it which neurons communicate with each President Obama as an example of illumination. We want to use biochemical In 1992, the gene for GFP was perfect for in vivo applications. other. The difficulty with these hypotheses ‘precision medicine’. Such a long time is differences between the tumour and normal sequenced by American biologist Douglas Green fluorescent protein has since is that proteins inside synapses undergo required before one knows whether one References tissue to make the tumour fluorescent, so Prasher. The first to express the gene in been used in thousands of different continuous rapid turnover and replacement, has success or not. 1) Tsien, R. Y. Very long-term memories may be stored in the pattern of holes in the perineuronal net. Proc. Natl. that the surgeon can decide where to cut another organism was Martin Chalfie, an ways. Replacing a gene with the gene so that memories would require recopying Acad. Sci. USA 110(30), 12456–12461 (2013). with realtime guidance. American biochemist who shared the for GFP can result in GFP being expressed very many times over an animal or Were you interested in science as a child? Unfortunately, one cannot use GFP or its Nobel Prize with Tsien. He inserted the in the organism only in the places where person’s lifetime. I was always obsessed by pretty colours homologues, because they can be linked to gene for GFP into the bacteria E. coli and the original gene would have been Instead, we are looking at the and by technologies that seem useful. One Roger Y Tsien is professor of pharmacology malignancy only by sophisticated gene nematode worm C. elegans. The resulting expressed, creating a bright visual glycoproteins (proteins plus carbohydrates) of my earliest memories is of a beach that and professor of chemistry and biochemistry at the University of California, therapy that is not practical yet or ethical organisms then glowed green in the pattern of expression. By selectively known to form a coating just outside had a zone of coarse pebbles surrounded by San Diego. After graduating from Harvard, in human patients. Instead, we are presence of UV or blue light. labelling specific proteins, we can create synapses. We are accumulating two zones of sand. I tried to lay down a Tsien also held posts at Cambridge exploiting extracellular enzymes that The protein itself is a barrel shaped images to see exactly where those evidence that this coating, once formed, bridge of sand across the pebbles to make and Berkeley. He is also a noted biochemical inventor who holds more than 100 patents. He are turned on in practically all solid molecule with a colour producing proteins are present. is basically stable but can be locally crossing more comfortable for my tiny bare shared the Nobel Prize in Chemistry in 2008

malignant tumours. We have engineered © LISA WERNER/ALAMY STOCK PHOTO LIVET/WWW.OLYMPUSBIOSCAPES.COM JEAN DR remodelled to strengthen individual feet. Of course, the bridge would have been for his development of GFP.

8 / The Biologist / Biochemistry Supplement Biochemistry Supplement / The Biologist / 9 10 10 / TheBiologist /Biochemistry Supplement P and usetheirmetabolic arsenalstothrive andsucceed. their seedlands, plantsaretheconsummateDIYers adapting toachangingenvironment. Growingwhere plants; stockingseedsforthenext generation;and pollinators; communicatingwith soilmicrobesorother create hardierandmoreusefulcrops, writesJoseph Jez Understanding thechemistryofplantswillenable usto sometimes eachother;attracting pathogens, insects,herbivores and cellular buildingblocks;fighting off harvesting light;makingnutrientsand molecules forallsortsofpurposes: They createadizzyingarrayofsmall lants aretheoriginalmasterchemists. green chemists Little Little other useful materials. materials. other useful and molecules feedstock of production chemical fuels, such as or drought, stresses for the environmental under food production to maintain plant metabolism could help systems these us engineer Understanding challenges. by to plants grow and meet environmental molecular machinery that generates the molecules used and ecologists. geneticists biochemists, and for lessons chemists, opportunities offers new metabolism their understanding Unravelling plants’ molecular bounty and bounty molecular plants’ Unravelling My research group focuses on understanding the My on group focuses research understanding

JON WILSON/SCIENCE PHOTO LIBRARY reactive oxygen molecules. molecules. oxygen reactive make to used be that help other molecules the the protect plant against substrate its lets enzyme the down for consequences the Slowing metabolic plant. larger stress. environmental is that enzyme the for by produced by toggled oxygen molecules the reactive switch on/off an becomes effect altering performs, it change howin much product it The makes. structural chemistry the slow to changed is is what to 5’-phosphosulfate happens kinase. adenosine on acids the amino a This modify They protein. also can and development. to plant growth affect metabolism and physiology cellular perturb can molecules changes, generate reactive oxygen molecules. These drought and temperature pollution, to metals, heavy of synthesis the in soil molecules. various the from sulphur use to plants which allows 5’-phosphosulfate kinase, adenosine the the bark of water a of infusing tree because headache, for tasting paint, berries the particular and mashing looking gatherer hunter for and leaves the the nuts, right to fruits – eat, mixing of species a as pre-dawn the in history began our diversity chemical their move can from freely atoms to ecosystems. worldwide. However, who scientists it requires also both basic and applied research motivates drought, such as stresses under environmental production food maintaining while and materials, molecules bio-based production of fuels, chemical feedstock cracks the between the from disciplines. comes science new because biology, and systems but is that blending important biology, and computer cellular genetics science, the lines between protein chemistry, molecular New technologies and changing perspectives blur FASCINATIONTIMELESS avenues for how work. deciphering plants new are opening technologies of genetic manipulation reagents and and technology, imaging a variety growing (see page 14),determination new structure protein for sources radiation synchrotron spectrometry, mass of the sensitivity and ever RNA sequencing, improving the and ease relative data, low ofcosts whole genome widening. is palette And the experimental plant metabolism. manipulating and to both understanding are fundamental substrates of synthesis biology techniques and other and molecular products, assays, enzyme purification, of combination protein traditional The powerful and find’, but has brought us the new 21st century tools. that are ideal. less conditions than change, or climate under growing during production crop to plants such as meet maintaining challenges for insights provide can modifying to response stresses how change such in proteins understanding Ultimately, MORE NO ‘GRIND TOFIND’ These seemingly subtle atomic level differences have have differences level atomic subtle seemingly These the protein’s structure Following modification, exposure including stresses, environmental Many A good of example is this as the known protein Our fascination with plants and explorations of and explorations plants with Our fascination for metabolism plant engineering of possibility The tools and genomic of bioinformatics The availability was Once, ‘grind of the mantra plant biochemists

practical purpose? a forchange that use we Can function? its alter that does protein structureand the changewe Can DNA sequence? a proteinor another metabolite,particular proteinrecognise a the doeshappen? Why chemistry doeshow generator:hypothesis our proteinis a in everyis atom wherenearly Knowing understandingmetabolism keyto The Protein structure machine crystallography An x-ray offers new opportunities andlessons and understanding theirmetabolism Unravelling plants’ molecular bounty Joseph Jez isprofessor of biologyat Washington University inStLouis sustainable future. sustainable change the way we of in ask pursuit questions a of the workings inner and plants are revealing also applications and biomedical of diseases understanding Today, our that advance the same technologies of and fuels. fossil the harnessing pharmaceuticals, of and the many houses, discovery transportation the of creation advent paper,of agriculture, a tool the or through continued a That tradition fire. leaves, the or best special wood with finding for a spear, the crystal,x-raysthe the in atomsthose hits x-raythe beam When focusedx-ray beam. a intocrystal a place interest.of Next, we protein our ofcrystals growing with begins experimentaltool proteins.of This structure dimensional three-unveilthe crystallography to x-rayof use our effortsthoseis At the heart of of heart the At Biochemistry Supplement /TheBiologist /11 Plant biochemistry Metabolic pathways structurelike.looks protein the what revealareand atoms wherethe of map crystal, generatewe a the to back spots the relates that maths the solve computershelp to dimensions.Using three proteinin crystal the everyin atom of location the about information contains detector. pattern That x-rayan on spots of patternproduce a is,arediffracted.they that – off bounce then The diffractedx-raysThe

Structural biology Structural biology Diamond Light Source Diamond Light Source

of foot and mouth disease, creating a Magnets (left) drive ‘lookalike’ empty shell, devoid of the viral the electron beam component (RNA). The shell vaccine then around the ring of induces an immune response without any the Diamond Light Source synchrotron threat of infection. Currently in clinical particle accelerator. trials, this method has the potential to yield The Oxfordshire safer, cheaper and more effective vaccines, facility (below) and scientists are also looking to use it to continues to shed develop a new vaccine for polio. light on how biological THE IMMUNE RESPONSE IN REAL TIME molecules function, as well as A team from Cardiff University is currently the structure using the machine to study T cells, a variety of viruses. of white blood cell responsible for finding and destroying unwelcome cells. The scientists have been studying receptors on the outside of T cells that help identify cells containing foreign proteins. Scientists have been able to observe this process in astonishing detail and are now looking to develop ways of enhancing patients’ own T cells so that they can recognise and bind to cancerous proteins, destroying tumours. In separate research, a group from Queen’s University Belfast is exploring the potential of gold nanoparticles to enhance radiotherapy. The scientists are using Diamond to study chemical reactions between nanoparticles and surrounding tissue when exposed to radiation. Their research shows that by injecting nanoparticles to the site of a tumour, it may be possible to increase the therapeutic impact of radiotherapy, enabling doctors Diamond currently has five beamlines dedicated to use less radiation. to protein crystallography and a further 19 Shedding lightlight on molecules FILLING PROTEINS’ POCKETS Approximately 30% of drugs target providing other techniques useful to bioscience Structural biology owes much to the intense biochemical breakthroughs being made at G-protein-coupled receptors (GPCRs), the end of the synchrotron beamline. making them the largest and most beam of the UK’s largest particle accelerator important family of drug targets in the in forms that cannot be processed by ago, this profoundly complex biological WATCHING VIRUSES UP CLOSE human body. Using the synchrotron, enzymes in the human gut. Scientists are component would have been impossible to echnology is the use it to investigate the atomic and Viruses are one of the oldest and most researchers from Heptares Therapeutics using Diamond’s spectroscopy capabilities – study in any detail. However, advances in cornerstone of scientific molecular nature of matter. pervasive elements of the natural world, have identified the structure of one of the including elemental mapping, fluorescence technology have opened up more progress. Throughout From viruses and drugs to engineering and have been evolving constantly over receptors in the brain responsible for the and x-ray tomography – to explore methods sophisticated techniques, enabled scientists history, developments in components and nanotechnology, the UK millions of years. Scientists from Diamond stress response. of changing the way the grains store their to collect data more quickly, and made it equipment, techniques and synchrotron is used for research in virtually and the University of Oxford are using the By visualising this stress protein receptor nutrients so that they become easier for our possible to explore more deeply into cells expertise have all been all areas and its high-tech capabilities are synchrotron’s high containment virus at the atomic level, they have identified a guts to access. than ever before. crucial to supporting pushing the boundaries of what is possible facilities to chart developments in viruses ‘pocket’ in the structure. These findings will Commenting in a Royal Society paper Tdiscovery and advancing our understanding for scientists everywhere. However, about and their structure over time, building up enable scientists to design a drug to fit RING OF POWER celebrating Diamond’s scientific of the world. Resembling a giant silver 40% of the work that takes place at Diamond our knowledge of individual virus families precisely into the pocket, inhibiting the Work that once took decades to complete achievements, Ramakrishnan said progress ring the size of Wembley Stadium, one is biology related, making it the most at the atomic level. response of the receptor and offering new, can now be done at the synchrotron within in structural biology in the last two decades of the most iconic examples of technology popular area of research, and the This research is helping us to understand more targeted treatment options for anxiety an hour. However, it would be wrong to had been “truly remarkable. In large part, supporting research is the UK’s synchrotron supports a vast range of more about viruses and the way they evolve – and depression. assume that the field has become routine. this has been due to x-ray crystallography synchrotron science facility, research in this field, including biomedical work that will enable us to predict viruses Indeed, advanced technology is allowing using synchrotron radiation”. Diamond Light Source. studies, bioengineering, environmental that may emerge in the future. This work MOLECULES AND MALNUTRITION bioscientists to explore the structures and Bioscience is a rapidly evolving field and Diamond functions like a collection science and much more. could prove particularly important in the Away from biomedical research, scientists compositions of bigger complexes and more this is also true for the technology that of giant microscopes using x-rays, Diamond currently has five beamlines event of an epidemic, when knowing a virus’s at Rothamsted Research are using Diamond difficult proteins. supports it. Facilities such as Diamond are infrared and UV light. dedicated to protein crystallography and a next step could enable us to respond quickly. to look at wheat and its nutritional content. Venki Ramakrishnan, president-elect of constantly upgrading to allow scientists to A third generation synchrotron, further 19 providing other techniques Understanding the atomic structure of Wheat is one of the most popular foods in the Royal Society, was awarded the Nobel do more. Things certainly move incredibly or particle accelerator, the machine useful to bioscience, including spectroscopy, viruses is also helping in the development the world: combined with rice and maize, it Prize in Chemistry in 2009 for solving the quickly at the synchrotron, but staying still produces a beam of light at an energy of small-angle scattering, tomography and of next-generation vaccines. Some comprises 60% of all food consumed on structure of the ribosome, the large is not an option, and as Diamond grows, so 3 gigaelectronvolts, which is 10 billion powder diffraction, to name a few. researchers working with Diamond have Earth. The grains are packed with essential molecular machine that strings amino acids does our understanding of the biological times brighter than the sun. Scientists Here are just a few examples of the successfully replicated the atomic structure nutrients, but much of these are locked away together to create proteins. Not too long chemicals around us.

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Figure 1 (below): Consensus proteins are designed by aligning the sequences of homologous proteins from different organisms. Each row represents one protein, with its amino acid sequence represented by letters A-V. Conservation is indicated by colour, where red indicates absolute conservation across proteins and purple indicates no conservation. The most conserved (ie common) amino acid at each position is used to create a stable protein with the same structure as the natural homologue, to which new functions can be added. In positions where there is no conservation (ie position 2), the amino acid in the consensus protein is arbitrarily chosen.

1 2 3 4 5 6 7 8

ALLIGATOR E K S L K E K G

FRUIT FLY I S S V I A L G

BLUE JAY P D L Q T G L G

YEAST S Q E L A K L G

HUMAN A L E R K N E G

RABBIT A E R L K A E G A design for life CONSENSUS A D E L K A L G protein structure and function, and to apply safer for administration as drugs because of history (Figure 1). For a group of proteins that Proteins are the most diverse group of biological this understanding towards the creation of the greatly reduced risk of complications have similar structures but diverse functions, Proteins are molecules, able to perform a dizzying number of proteins with high value in research and from an immune response. the conserved amino acids can be interpreted particularly suitable biotechnological and clinical settings. Given the highly complex relationship as playing key roles in the protein structure. functions within living systems. Lynne Regan There are two main ways to design a between protein sequence and structure, The amino acids that play functional roles for development of protein. The first uses our knowledge of successful execution of both strategies should show poor conservation. advanced drug and colleagues from Yale University explain how existing protein sequences and structures to typically requires the testing of multiple By taking the most frequent amino acid at design a completely new molecule that has sequences to produce a protein that satisfies each position in the alignment, we can create delivery systems biochemists can design new ones to perform little or no relationship to natural proteins. the design goals. The knowledge gained an entire consensus protein that will nearly This is best illustrated by Top7 (see below), from careful analysis of successful and failed always be more stable and will have the same specific functions for medicine and research. a protein composed of typical protein designs can be applied iteratively to future structure as the aligned proteins, but will lack elements, but folded in a way that has never designs. In other words, we try a bunch of function. These consensus proteins make for protein design because it allows ver billions of years of scientific research and improve patient care been observed in natural proteins1. designs, see which ones work and figure out excellent frameworks on which a wide range researchers to specify the non-standard evolution, proteins have in our hospitals. And yet the current range The second strategy, sometimes referred what distinguishes successes from failures. of functions can be accommodated later. A amino acid and its position in the protein acquired an enormous of natural protein functions represents only to as protein redesign, uses natural proteins Of course, the key to protein design is nice example is the use of tetratricopeptide based on the placement of the hijacked stop variety of functions – from a fraction of the potential ‘protein as templates and makes judicious exploring the existing ‘protein universe’ to proteins in cancer treatment6 (see codon in the protein coding gene. tiny chemical messengers universe’. Designing proteins in modifications to alter the understand what makes natural proteins Outcompeting cancer, page 16). One example of protein design that uses such as hormones to the the lab allows us to explore protein’s structure and/or work. Of specific interest is identifying how The boundaries of the protein universe the above approach is the design of small tough molecules that the almost infinite number function2,3. A prime amino acids in a protein sequence contribute have traditionally been defined by the same proteins called TRAPs to interact with Oform hair, skin and muscle. Proteins can of combinations of amino example of protein to protein stability and function. 20 amino acid building blocks that make up specific phosphorylated peptides3. also act as catalysts for chemical reactions, acids that it is possible redesign is the This can help inform a process called all natural proteins. However, advances in our Phosphorylation is a very common protein or form mini-machines that perform to create. ‘humanisation’ consensus design. At the heart of this is the understanding of how proteins are made and modification and plays a key role in relaying specific tasks within cells. Many enable To explore and of therapeutic mouse premise that patterns of amino acid genome engineering have allowed scientists information about the environment to a organisms to survive in diverse expand the protein antibodies by grafting the conservation in similar proteins – across to break through these boundaries7. cell’s interior. Because phosphorylation can environments, from scalding deep sea universe, we use a functional components of many species – can be used to identify which For designing proteins with non- drastically alter a cell’s behaviour, many vents to frozen polar caps. technique called protein The artificial mouse antibodies onto amino acids are important in the proteins’ standard amino acids, we introduce a diseases are associated with improper 4,5 Outside of their cellular context, many design. The aim is to satisfy Top7 protein human antibody scaffolds. structure and function . The first step is the special codon in the protein-coding mRNA, regulation of phosphorylation signalling. of these molecules still carry out their two goals simultaneously: to Because the bulk of each alignment of the homologous protein and reprogram the cell to insert a non- Thus, the ultimate goal of the TRAP designs remarkable functions and form the further our understanding of ‘humanised’ antibody has a sequences to identify which amino acids have standard amino acid in response to that is to produce a set of proteins, each of which

backbone for reagents that advance how amino acid sequences affect human origin, these antibodies are © SCIENCE PHOTO LIBRARY/ALAMY been conserved throughout evolutionary codon. This approach is particularly useful interacts with a specific phosphorylated

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Outcompeting cancer Designing tetratricopeptide repeat (TPR) proteins to fight cancer

Statistical analysis was used to identify regions of TPR proteins that are involved in binding to a major anticancer target known as Hsp90. Hsp90 is a ‘chaperone protein’ that assists other proteins to fold properly, and is associated with the folding of many cancer associated proteins. We then grafted the putative binding regions onto a very stable consensus TPR framework. The new protein binds to Hsp90 with high affinity. This designed TPR protein outcompetes natural Hsp90 co-factors in binding to Hsp90. This competition prevents Hsp90 from carrying out its cellular function. As a result, levels of HER2, a cancer associated binding partner of Hsp90, fell in HER2-positive breast cancer cells6. Similar use of consensus proteins as design templates has been applied to a variety of proteins, simultaneously enhancing our understanding of protein structure and creating novel proteins that Dividing breast can fulfil current therapeutic needs. cancer cell

peptide, and so provides the means for in Hydrogel layer = Small molecule drugs Lynne Regan is professor of molecular biophysics, vitro and in vivo detection of aberrant biochemistry and chemistry at Yale University. The Regan Lab is interested in the relationships between the phosphorylation signalling. structure, function and stability of macromolecules. How else can we take full advantage of the expanding protein universe? Proteins are Danielle Williams is a graduate student in the department of molecular biophysics and biochemistry at particularly suitable for development of Yale University. Her current research involves the design advanced drug delivery systems and tissue and creation of new protein-based nanomaterials. engineering. Specifically, proteins’ diverse Nicholas Sawyer is a graduate student in the department functionality and biocompatibility give them of Molecular Biophysics and Biochemistry at Yale an advantage over many synthetic materials University. His current research focuses on designing novel in that they can be reabsorbed by the body proteins to detect protein post-translational modifications. after use and are less likely to cause complex immune responses. Normal cells Tumour cells Normal cells Our lab has created a new drug delivery References system based on ‘smart’ hydrogels8,9. Like Figure 2: The targeted delivery of small 1) Kuhlman, B. et al. to guide repeat protein jelly, the hydrogels can encapsulate small molecule drugs (depicted as yellow stars) to Design of a novel globular design. J. Mol. Biol. 425, tumour cells (pink) using ‘smart’ hydrogels. protein fold with atomic- 1826–1838 (2013). molecules and respond to mechanical stress The acidity of the tumour cells causes the level accuracy. Science 6) Cortajarena, A. L. et al. 302, 1364–1368 (2003). Designed TPR modules as like natural tissues. Unlike jelly, however, hydrogel layer (green) to break down at the 2) Jackrel, M. E. et al. novel anticancer agents. the bonds that hold our smart hydrogels tumour interface while staying intact at the Redesign of a protein- ACS Chem. Biol. 3, together are genetically encoded to respond normal cell layer (blue), allowing for the peptide interaction: 161–166 (2008). to physiological stimuli, such as a change in controlled release of molecular cargo. characterization and 7) Rovner, A. J. et al. applications. Protein Sci. Recoded organisms pH or ionic strength. These hydrogels 18, 762–774 (2009). engineered to depend on dissolve in low pH conditions, such as those existing protein universe. Ongoing efforts 3) Sawyer, N. et al. synthetic amino acids. observed in cancer microenvironments, and have been greatly enhanced by the creation of Designed phosphoprotein Nature 518, 89–93 (2015). recognition in Escherichia 8) Grove, T. Z. et al. therefore can deliver their encapsulated highly stable consensus proteins and an ever coli. ACS Chem. Biol. 9, Stimuli-responsive smart cargo specifically to cancer cells (Figure 2). expanding repertoire of amino acid units. 2502–2507 (2014). gels realized via modular All of the advances above illustrate how Clever assembly of these new proteins into 4) Main, E. R. G. et al. protein design. J. Am. Design of stable α-helical Chem. Soc. 132, 14024– protein design is being used to create complex hydrogel networks allows the direct Arrays from an idealized 14026 (2010). amazing new molecules that enable us to translation of relatively simple designed TPR motif. Structure 11, 9) Grove, T. Z. et al. A develop innovative tools for biomedical proteins into valuable biomedical materials 497–508 (2003). modular approach to the 5) Sawyer, N. et al. All design of protein-based therapies and also better understand the for tissue engineering, targeted drug delivery repeats are not equal: a smart gels. Biopolymers

WWW.ROYALTYSTOCKPHOTO.COM evolutionary forces that have shaped our and other research and clinical applications. module-based approach 97, 508–517 (2012).

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