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How does a polypeptide chain attain itsnative tertiary structure? This questionhas played on the minds of experimental-ists and theorists for the last half century.Clearly, many proteins can and do foldefficiently on their own. Among the goalsin understanding this process are: the effi-cient design of proteins with a particularfunction, the prevention of the proteinassociation involved in diseases caused byabnormal protein deposition in cells, suchas is found in Alzheimer's and theimmunoglobulin light chain depositiondiseases, and the elucidation of the con-formational changes involved in cellularprocesses, such as cell cycle control andsignal transduction. Indeed, some key players in signal transduction, includ-ing the anti-sigma factor FlgM and p21,only fold in the presence of their signaling partners1–3. A large barrier tocracking the protein folding code is the highcooperativity of the folding reaction atequilibrium. Thus, investigators haveturned to kinetic methods in order toidentify potential folding intermediatesthat could elucidate the process. While therole of folding intermediates as friend orfoe in the attainment of the native fold is apoint of lively discussion among investiga-tors4–16, it is clear that intermediates exist.Many proteins fold in a multiphasicprocess consistent with the population ofintermediate species. Frequently, a signifi-cant change in an optical signal occurs inthe dead time of the instrument beingused. These burst-phase signal changes arecommonly interpreted as a hallmark ofintermediate production. In addition, it isoften proposed that these signal changesreflect significant secondary structure for-mation.

While the coincidence of burst phasesignals with amide proton protectionargues convincingly for stable intermedi-ate formation17–19, Qi and colleagues nowpresent important new data on page 882of this issue which suggest that optical sig-nal changes alone may report solely on

transitions between differently biasedensembles of unfolded states that are dri-ven by changes in solution conditions.Thus, mechanistic arguments based onthe population of submillisecond burst-phase species must be reevaluated in lightof the possibility that some inferred inter-mediates may actually reflect the proper-ties of the unfolded protein in aqueoussolution. It is important to note that thisstudy does not dispute necessarily thepresence of partially folded intermediatesin some cases20–29, but rather stresses thepoint that ultrafast (submillisecond)events may more generally reflect thephysiochemical changes associated withrandom polymer collapse14,30,31.

Previous studies on the burst phasespecies formed in the dilution of both fulllength and truncated, folding-incompe-tent versions of cytochrome c into refold-ing conditions indicated that all formsproduced nearly identical optical signalchanges in the dead time of stopped-flowexperiments28,32. These results revealedthat significant signal changes can occurin the absence of stable structure form-ation. Thus, these optical changes may bean indicator of a shift in the populationsof species in the initial unfolded ensembleas a result of the differences in solutionconditions upon denaturant dilutionrather than stable structure formation.Indeed, this result is supported by theobservation that stable hydrogen bondformation is often absent or only weaklypresent in the burst phase species22,33,34.However, the absence of significant hydro-gen bond protection is not proof of theabsence of intermediates as side chainrather than main chain interactions maybe involved. In addition, the refoldingreaction of cytochrome c can be compli-cated by the formation of non-nativeheme ligation states in both the unfoldedand partially folded proteins16,32,35. Thus,the previous results could potentially beattributed to the particular peculiarities ofthe specific system rather than a general

phenomenon. Qi and colleagues nowreport their recent findings in a comparisonstudy of burst phase signals in the denatu-rant dilution experiments on folding com-petent and incompetent ribonuclease A(RNase A and r-RNase A respectively)36.These new results agree with their findingson cytochrome c — that the observationof submillisecond signal changes mayreflect the formation of an alteredunfolded ensemble that better models theinitial conditions for refolding in aqueous solution.

RNase ARNase A is one of the most thoroughlystudied protein folding model systemsand serves as a paradigm for mechanisticfolding studies of α/β proteins. The struc-ture of the native protein is known: pulselabeling NMR studies indicate the pres-ence of stable, hydrogen bonded sec-ondary structure prior to formation ofthe native protein37,38, and mutagenesisexperiments have been used to evaluatethe relative contributions of specific sidechain interactions during folding39–43. Inaddition, a reduced form of the protein, r-RNase A, has long been a standardreagent for understanding the propertiesof unfolded proteins in solution44–46. Byusing a combination of equilibrium andkinetic circular dichroism studies onRNase A and r-RNase A, Qi et al.36 havebeen able to compare directly the signalchanges associated with introducing theunfolded r-RNase A (which does not fold)or the unfolded RNase A (which will fold)into low denaturant concentrations. Theyfound that the two proteins yielded iden-tical changes in submillisecond burstphase amplitude in stopped-flow circulardichroism studies upon dilution from5.5 M denaturant to all guanidinehydrochloride concentrations between0.5 and 4.6 M. Since r-RNase A does notfold44–46 under the conditions of theexperiment, the signal change observed isnot attributed to productive intermedi-

846 nature structural biology • volume 5 number 10 • october 1998

Speeding along the protein foldinghighway, are we reading the signs correctly?Patricia A. Jennings

Detailed studies of submillisecond burst phase signals in non-folding proteins indicate that the observedoptical changes may reflect the population of different ensembles of unfolded polypeptides rather than thepresence of intermediate species.

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ates or stable secondary structure forma-tion. Indeed, r-RNase A shows no protec-tion of amide bonds from solventexchange under similar conditions44,47.The authors propose that the signalchanges observed reflect a shift in theensemble of states sampled by the unfold-ed protein because of changes in bothmain chain solvation and interactionsbetween apolar side chains as a function ofdenaturant concentration. Importantly,the ensemble of unfolded states populatedupon dilution of denaturant-unfoldedRNase A and in r-RNase A may betterreflect the conditions a protein experi-ences prior to folding in vivo.

Submillisecond eventsThe findings of Qi et al.36 are especially

nature structural biology • volume 5 number 10 • october 1998 847

significant in light of the current discus-sion of the importance of fast reactions inprotein folding20–29. Energy-landscapetheory suggests that folding reactions arebest understood in terms of the progres-sive reduction in the entropy of thepolypeptide chain and the number ofstates allowed in the ensemble of speciesas the reaction proceeds towards the finalfolded form. The ruggedness or smooth-ness of the respective landscape willreflect the particular properties of theprotein and the conditions under that itwas studied. Proteins that undergoapparent two-state folding reactions(usually fast but occasionally slow48) willbe characterized as a smooth funnel withan average energy barrier between theensemble of unfolded species and the

native fold. Proteins that undergo multi-state reactions with the observa-tion of stable intermediates will bereflected in a more rugged energy land-scape. Whether the ruggedness of thelandscape reflects artifactual traps or nec-essary species is a point of muchdebate5,8,10–13,16–18,23,29,32,33,35,49–51. The sub-millisecond burst phase signal in RNase Arefolding characterized by Qi et al.36,which is quantitatively reproduced with r-RNase A, is proposed to reflect the initialcollapse of the unfolded protein as aresult of the drastic changes in solutionconditions. An initial collapse of this sortwould be akin to a glass transition andwould not be expected to follow simplefirst-order exponential kinetics14,29.However, if the burst phase signal reportson the formation of a stable intermedi-ate, the early landscape would be morecomplex. It is convenient to look at thecharacteristics of the folding landscape interms of two dimensional reaction coor-dinate diagrams (Fig. 1). In the first sce-nario, the transition between U and U' isakin to a slippery slope while in the sec-ond scenario the submillisecond eventsreflect first order transitions between sta-ble and/or metastable states. Qi et al. nowpropose that the early landscape of pro-tein folding may not be as rugged as sug-gested from fast kinetic studies. Inaddition, their data leads them to pro-pose that, in general, the initial events inprotein folding consist of a long rangeconformational search for a transitionstate nucleus and requires at least a mil-lisecond time scale36.

Fast and ultrafast foldingThe folding of many small proteins (lessthan ~100 amino acids) occurs in theabsence of detectable intermediates in anapparent two-state reaction in the multi-millisecond time range. In one intriguingcase, Oas and colleagues have found thata monomeric variant of the λ repressorprotein folds and unfolds in microsec-onds21. Thus, significant structure for-mation of some proteins can and doesoccur in the submillisecond time regime(reviewed in ref. 52). It may be that suchproteins are ultrafast folders because theconformational preferences of theunfolded state are maintained and thatenergetically significant non-nativeinteractions are absent during fold-ing53–56. Qi et al. additionally proposethat these ultrafast folders may result insituations where the initial nucleationevent involves only short range confor-mational searches36 . The characteriza-

Fig. 1 Schematic free energy diagrams illustrating the point that burst phase signals may reflectvery different transitions. a, Possible reaction coordinate diagrams for the refolding of RNase A,where each symbol represents an ensemble of states and U represents the unfolded protein, U'represents a different state of the unfolded species after dilution from high denaturant, I is theburst phase intermediate and N is the native protein. b, Schematic plot of the relative energies ofthe ensembles of populated states in the two mechanisms. The view on the left suggests that thesubmillisecond kinetics reflect a continuum of changes in the unfolded state in a second ordertransition14 while the view on the right suggests the rapid formation of distinct structural inter-mediates in a first order process. In each case, the respective species can be thought of as anensemble of states. The allowed ensemble narrows as the protein folds.

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tion of the specific interactions present inthe unfolded protein at the start ofrefolding would directly address thishypothesis. Structural characterizationof a large ensemble of interconvertingspecies, as is found for unfolded proteins,is difficult because of conformationalaveraging over φ, ψ space. Nevertheless,some studies have indicated the presenceof specific interactions in proteins understrongly unfolding conditions57,58. Indeed,detailed solution NMR studies on theconformational preferences in the acid-unfolded, molten globule and nativestates of apomyoglobin55 demonstratethe presence of specific native interac-tions in the unfolded peptide chain thatare preserved throughout folding19,59–61.

The quantitative reproduction of theoptical properties of the burst phase inRNase A and r-RNase A in stopped-flowcircular dichroism studies calls into ques-tion the widely held assumption that burstphase signal equals intermediate forma-tion. However, since circular dichroism isonly one probe of structure and amideproton protection probes only regionswhich are stable in the native protein, thiswork also raises the following questions.Are there conformational preferences inthe ensemble of unfolded states in lowdenaturant prior to folding? How col-lapsed is the unfolded protein from thatobserved in high denaturant? Does dilu-tion from high denaturant simply shift thepopulation of states as a result of changesin solvent conditions or are some specificinteractions formed? Are the conforma-tional states of r-RNase A and RNase A atthe start of folding identical? Do unfavor-able interactions in the unfolded stateslow down folding? How do we differenti-ate unfolded proteins from unstable,weakly folded intermediates? The currentwork indicates that care must be taken in interpreting submillisecond opticalchanges and suggests that the RNase A/r-

RNase A system is ideal for further muta-genic and high resolution studies that canaddress these important questions. Inaddition, characterization of the unfold-ed protein ensemble under native solu-tion conditions will facilitate comparisonof in vitro and in vivo folding reactions.

Patricia A. Jennings is in the Department ofChemistry and Biochemistry, 0359,University of California San Diego, 9500Gilman Drive, La Jolla, California, 92093-0359, USA. email: pajennin@ucsd.edu

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