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Addresses*University of Cambridge, Department of Chemistry, Lensfield Road,Cambridge CB2 1EW, UK; e-mail: jc162@cam.ac.uk †Oxford Centre for Molecular Sciences, University of Oxford, NewChemistry Laboratory, South Parks Road, Oxford OX1 3QR, UK; e-mail: chris.dobson@chem.ox.ac.uk

Current Opinion in Structural Biology 2001, 11:67–69

0959-440X/01/$ — see front matter© 2001 Elsevier Science Ltd. All rights reserved.

Protein folding remains one of the major intellectual chal-lenges for structural biologists. As well as being a topic oftremendous interest in its own right, it represents anopportunity to dissect the link between protein sequence,structure, stability and, ultimately, function. As we under-stand more about the folding process, however, thequestions remaining get more complex and the tech-niques required to examine them need to be moreingenious. The subject of protein folding, which we taketo include the assembly of protein complexes, has movedforward rapidly from the elegant ground-breaking studiesof model systems that dominated the field until the late1990s. A great deal of effort has recently been expendedin developing new techniques that are able to follow moresubtle and complex aspects of protein folding, allowingmore sensitive and rapid observations with more diverseprobes of a wider range of systems. We have also seen thedevelopment of strong links between experiment andtheory, an advance that has contributed much to our pre-sent understanding of the underlying mechanism of thefolding process.

The reviews in this section have as a firm foundation theresults of many fundamental studies using a wide range ofapproaches. The underlying theme of many of the reviewsis the search to find unifying principles from the gatheringtogether of data on a number of different systems, chosensystematically or simply through serendipity. As a numberof relatively simple empirical ‘rules’ describing the foldingof different proteins are to be established, the task is todefine and understand the physical principles behindthese rules. The field can then move towards other goals,such as structure prediction and protein design. As we shallsee in these reviews, studies of small proteins in vitro aregenerally much more advanced than those of complex,macromolecular systems or, indeed, of any systems in vivo.In particular, the importance of native state topology indetermining folding behaviour, established through stud-ies of related proteins, is stressed again and again. But thepicture is not always simple. Some closely related proteinsfold in a manner such that specific contacts between

residues are apparently more important than topologicalconstraints. Encouragingly, the use of the physical toolsand analytical methods developed for studying simple pro-teins is already revealing some details of the folding andassembly mechanisms of even the most complex systemsunder investigation at the present time.

The first review, by Grantcharova et al. (pp 70–82), is an‘overview’ of advances in our understanding of the mecha-nisms of protein folding and covers studies both of smallproteins in isolation and of larger proteins in the presenceof GroEL. The first half of the review describes in vitroexperiments that have concentrated on the folding of anumber of single-domain proteins that fold with two-statekinetics. The authors stress the attractively simple rela-tionship that has been observed between protein foldingkinetics and the topology of the native states of the pro-teins involved. In this review, the balance betweentopological constraints and variations of the local free ener-gies of interactions within the folding nucleus is discussedin the context of recent experimental data. Proteins withthe same topology do not always fold in identical ways. Forexample, in cases in which local structural interactions areimportant in the formation of the folding nucleus, differ-ences in the stabilities of these local interactions mayresult in the population of alternative folding routes. Manyof the points made in this opening review concerning thebehaviour of small proteins are taken up in more detail inthe next three review articles.

One of the principal areas of study that has, over the pastfew years, resulted in the elucidation of some simple prin-ciples underlying the folding of small molecules is thestudy of the behaviour of homologous protein families.The article by Gierasch and co-workers (pp 83–93) is acomprehensive and timely review of these studies. One ofthe points made is that there is still no consensus view ofwhether or not evolution has selected for fast folding.Proteins have to fold fast enough to avoid aggregation, butthere is no conclusive evidence to show that the rate ofprotein folding is an evolutionary pressure in a more gen-eral sense. The data described, however, show thattransition-state structures are frequently conservedthrough evolution. The transition states tend to be native-like and it is this characteristic that apparently leads to thecompelling correlation between contact order, a measure ofthe separation in the sequence between residues thatinteract with each other in the native structure, and foldingrates. As the authors of this review point out, however, it is,in fact, the contact orders of the transition states them-selves that are the important factors; these are not yet

Folding and bindingEmerging themes in protein folding and assemblyEditorial overviewJane Clarke* and Christopher M Dobson†

known in detail for any system. What is known, however,is that all-α or mixed α/β proteins have, on the whole, lessclosely related transition states than all-β proteins. Theauthors present an attractive analysis to explain a numberof apparently contradictory observations in the literature.They propose that, in some protein families, folding ratesare determined primarily by contact order, essentially anentropic consideration, whereas in other families, folding isdominated by specific interactions in the native-like tran-sition state and the folding rates are determined by thetotal free energy change involved in the folding process.

The basis behind much of this work, for small simple sys-tems, has been detailed analysis of transition states usingφ-values determined from protein engineering experi-ments. The details of the structures of folding transitionstates are considered further in the review by Oliveberg(pp 94–100). What does a folding transition state repre-sent? For many small proteins, the transition-stateensemble is apparently a collection of molecules withbroadly similar structures, each of which is an expandedform of the native state. Oliveberg describes how the energylandscape for folding may be changed by mutation or bythe presence of a denaturant, and his work has led to adescription of transition-state barriers as broad regions ofthe energy surface that may be either ‘rough’ or ‘smooth’.He suggests that shifts in the transition state as a result ofmutation can be used to map the formation of structure asthe protein moves along the reaction coordinate for fold-ing. Analysis of the energetics of related and circularlypermuted proteins may explain at least some of the exper-imentally observed changes in the folding transitionstates. In his conclusion, Oliveberg raises an interestingquestion — why does folding seem to be so strongly dom-inated by local interactions? He suggests that evolutionmay have selected against sequences that are likely tomisfold. This could be mediated by the action of ‘gate-keepers’, strategically placed residues that preventproteins following folding trajectories that do not leadefficiently to the native structure.

Although much has been learnt about protein folding,there are still many features that we do not understand,notably the nature of the sequence-specific signals thatguide and modulate folding behaviour. This question isone of the topics addressed in the review by Guerois andSerrano (pp 101–106). These authors, like those earlier inthis section, draw attention to the results of experimentssupporting the important roles that topology and chainconnectivity play in directing the folding process. A fea-ture of modern folding studies is an infectious enthusiasmfor using experimental data to guide the design of com-puter-based folding algorithms. Several groups havedeveloped simple potentials to model folding landscapesand to predict the broad features of folding reactions. Newideas described in this review involve refining these mod-els to take account of the experimental observation thatspecific sequence details can modulate the effects of

topology, for example by increasing the likelihood of onefolding route relative to another. Guerois and Serrano sug-gest that the new algorithms will do more than be able tomake predictions about the folding of native proteins.This might permit the design or optimisation of particulartypes of folding behaviour; for example, to encourage pro-ductive folding as a means of preventing aggregation.These suggestions throw down a challenge to protein fold-ers. Can we take the principles we are learning from ourexperiments or calculations and use them in the rationaldesign of new sequences with predictable properties?

In the second part of the introductory review,Grantcharova et al. discuss whether the simple conceptsderived from the folding of small proteins apply to chaper-one-assisted protein folding. Why, in particular, do manylarger proteins require chaperones in order to fold effi-ciently? The authors suggest that, since a majorcontribution to the barriers limiting the folding rates ofsmall proteins is apparently the formation of nonlocalstructure, these barriers are likely to be much greater forlarger proteins. Chaperones, therefore, may be required toprevent the misfolding and aggregation events that com-pete with productive folding. Structural, kinetic andthermodynamic studies of the GroEL/GroES system inparticular are being used to dissect the mechanisms ofchaperonin action. The authors conclude that some basicprinciples are now understood, but there is some way to gobefore the folding of larger proteins in vivo is amenable todescription by a set of well-defined rules.

GroEL is one of the macromolecules whose structuralanalysis over the past few years has been of particularinterest to protein folders. In the past year, spectacularstructural descriptions of another key complex in the fold-ing field, the ribosome, have begun to emerge. Thesestructures will undoubtedly provide the basis for under-standing the starting point of protein folding in vivo.Bamford et al. (pp 107–113) describe the main features ofthese studies, which have revealed the remarkable role ofRNA, not simply as a scaffold but also as a key part of thecatalytic machinery. The authors also describe some of theadvances in our understanding of the assembly of macro-molecular complexes, focusing particularly on therelatively well understood world of viruses. Studies ofthese systems have demonstrated the power of combiningexquisite structural descriptions of very large complexeswith the results of biophysical techniques designed toanalyse the assembly and function of these large complexes.The assembly of viruses has proved to be particularlyamenable to in vitro studies. The use of time-resolvedstructural techniques has permitted the assembly processto be monitored in some detail. The authors draw parallelsbetween the assembly of a virus and the folding of a pro-tein, with the process in each case dominated by ‘transitionstates’ as the system moves from one energy minimum toanother. It seems probable, therefore, that the assembly ofmacromolecular complexes will be dissected in much the

68 Folding and binding

same way as protein folding, that is, by a combination ofhigh-resolution structural analysis of intermediates andkinetic studies of their formation and disappearance.

The next paper, by Ellis (pp 114–119), is a provocativereminder to those who study protein folding in vitro thatthe intracellular environment may be more complex thanwe might like to believe. The phenomenon of ‘crowding’in the cell results from the very high density of macromol-ecular components, but is, at present, poorly understoodand frequently ignored. The review describes importantprinciples and some experimental evidence for the effectsof crowding on folding and assembly. The chief effects ofmacromolecular crowding are to increase the effective con-centration of solutes in the cell and the viscosity of themedium in which they are located. These changes canresult in substantial perturbation of thermodynamic andkinetic properties. In the first place, equilibria betweenmonomer and multimer may be effected by several ordersof magnitude, so that dissociation constants measured indilute solution may not reflect the values relevant to con-ditions in the cell. Thus, for example, assembly ofcomplexes may be substantially enhanced inside the cell.In addition, the native states of proteins may be stabilisedeither as a direct consequence of crowding or through asso-ciation with ligands. Furthermore, aggregation may beenhanced by macromolecular crowding, increasing theneed for chaperone action to prevent misfolding events.The kinetics of folding or assembly on the other hand maybe slowed through the effects of decreased rates of diffu-sion. Thus, although one can contend that studies ofproteins in dilute solution in vitro are revealing the funda-mental relationships between topology, sequence andfolding, this review provides food for thought when relatingthese principles to specific molecular characteristics in vivo.It also provides an interesting background to the nextreview — on folding in the endoplasmic reticulum (ER).

In prokaryotic cells, proteins usually fold in the cytosol,where the various chaperones are located. In more com-plex cells, there are specialised compartments in whichthe folding of particular proteins may take place. The ERis the complex organelle responsible for ensuring thatonly correctly folded proteins access the secretory path-ways. To achieve this end, folding in the ER is under tightcontrol. In their review, Bergeron and co-authors(pp 120–124) describe the ER as consisting of four ‘mol-ecular machines’ that interact to ensure the correctprocessing of folded proteins. This review provides aclear description of the current understanding of the func-tion of each of these machines. The basic process isprotein folding, often accompanied by a series of glycosy-lation reactions. The folding process is carefullymonitored and, where things go wrong, a quality controlsystem allows misfolded or misprocessed enzymes achance to recover. When the ER is under stress it activates the signalling machinery, which allows commu-nication with the nucleus so as to modulate protein

expression. Finally, the ER has a degradation mechanismto rid itself of incorrectly folded proteins that cannot berescued. The integration of these machines results in analmost inconceivably complex system that acts with stun-ning efficiency. It is particularly exciting that many of thefundamental questions are just becoming amenable toinvestigation using structural techniques.

The final review in this collection places protein folding inthe context of undoubtedly the most exciting future chal-lenge to structural biologists, that posed by the informationpouring out of genome projects. Sunyaev, Lathe and Bork(pp 125–130) identify three areas that link genomics toprotein folding and binding. The first concerns the analy-sis of the structures of the proteins encoded by completegenomes. The second concerns the use of genome infor-mation to predict interactions between specific proteins.The third is the structural analyses of disease-causingmutations and the significance of single nucleotide poly-morphisms (SNPs). The first two areas have been thesubject of intense discussion and considerable activity forsome time. In this review, the authors focus on the last ofthese issues, which is only now becoming a really ‘hottopic’ in genome analysis, with information being gatheredmore and more rapidly (see Figure 1 of the review). Locus-specific databases have already revealed the existence inbiological populations of thousands of amino acid variantsof well-characterised proteins. The simple rules derivedfrom folding studies should enable us to interpret or evenpredict the effects of these variations on folding, structure,stability or binding. This should provide a direct linkbetween the ‘molecular phenotype’ and the biologicalphenotype, and give insights into the relationship betweendisease and the functions of large numbers of proteins.The database of SNPs is growing rapidly and the pilot stud-ies described in this review present an exciting challengeto the protein folding community.

We have come a long way in the past few years in dissect-ing the manner in which proteins fold and a number ofattractive and, in some cases, simple concepts are emerg-ing. But the devil lies in the detail. The central issue ofhow a particular sequence encodes a specific fold and howsequence modulates folding behaviour is still not wellunderstood. Undoubtedly, much future effort will be con-centrated in this area of research and in using the ideas thatemerge to model and predict folding behaviour and theeffect of sequence changes. In the concluding remarks ofhis review, Oliveberg suggests that we might go a steptowards sharing information by compiling a ‘web bank’ ofaccumulated kinetic and thermodynamic data on the fold-ing of small proteins and their mutational variants. Thisresource would facilitate the systematic analysis of theincreasingly large amount of information that is becomingavailable and enable links to SNP databases to be made.We would certainly be pleased to see such a database existand be willing to share our data with our colleagues. Wouldanyone care to take up this challenge?

Editorial overview Clarke and Dobson 69