PERSPECTIVE

The nature of protein folding pathwaysS. Walter Englander1 and Leland MayneJohnson Research Foundation, Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania,Philadelphia, PA 19104

Edited by Alan R. Fersht, Medical Research Council Laboratory of Molecular Biology, Cambridge, United Kingdom, and approved September 23, 2014 (received for review June24, 2014)

How do proteins fold, and why do they fold in that way? This Perspective integrates earlier and more recent advances over the 50-y history ofthe protein folding problem, emphasizing unambiguously clear structural information. Experimental results show that, contrary to prior belief,proteins are multistate rather than two-state objects. They are composed of separately cooperative foldon building blocks that can be seen torepeatedly unfold and refold as units even under native conditions. Similarly, foldons are lost as units when proteins are destabilized toproduce partially unfolded equilibrium molten globules. In kinetic folding, the inherently cooperative nature of foldons predisposes thethermally driven amino acid-level search to form an initial foldon and subsequent foldons in later assisted searches. The small size of foldonunits, ∼20 residues, resolves the Levinthal time-scale search problem. These microscopic-level search processes can be identified with thedisordered multitrack search envisioned in the “new view” model for protein folding. Emergent macroscopic foldon–foldon interactions thencollectively provide the structural guidance and free energy bias for the ordered addition of foldons in a stepwise pathway that sequentiallybuilds the native protein. These conclusions reconcile the seemingly opposed new view and defined pathway models; the two models accountfor different stages of the protein folding process. Additionally, these observations answer the “how” and the “why” questions. The proteinfolding pathway depends on the same foldon units and foldon–foldon interactions that construct the native structure.

protein folding | hydrogen exchange | protein structure

Proteins must fold to their active native statewhen they emerge from the ribosome andwhen they repeatedly unfold and refold duringtheir lifetime (1, 2). The folding process isdifficult (3, 4) and potentially dangerous (5).Biological health depends on its success anddisease on its failure. However, more than 50 yafter the formative demonstration that proteinfolding is a straightforward biophysical pro-cess (6), there is not general agreement onthe overarching questions of how proteins foldand why they fold in that way. Given thisuncertainty, one is not sure how to even thinkabout many related biophysical and biologicalproblems.Early in the history of the folding field,

experimentalists simply assumed that proteinsfold through distinct intermediate states in adistinct pathway (Fig. 1A), as seen for a classicalbiochemical pathways. Following Anfinsen’sdemonstration that proteins can fold all bythemselves without outside help (6), Levinthalperceived that no undirected folding processwould be able to find the native structure byrandom searching through the vast number ofstructural options (3, 4). Proteins must solvethe problem, he believed, by folding throughpredetermined pathways, although one hadno clue how or why that should occur.A realization of the inability to equilibrate

to a common structure (3, 4) and the en-semble nature of partially folded forms ledthe theoretical community to a very differ-ent more statistical “new view” (7–11). Itwas inferred that proteins must fold to their

unique native state through multiple un-predictable routes and intermediate con-formations. Another prominent inferenceconfigured the Anfinsen thermodynamichypothesis (6) in terms of a funnel-shapedenergy landscape diagram (Fig. 1B), whichpictures that proteins must fold energeti-cally downhill (the Z axis) and shrink inconformational extent (the generalized XYplane) as they go (9, 12–14). To fill out thelandscape picture, classical rate-determiningkinetic barriers are often replaced by qual-itative concepts such as ruggedness, frus-tration, and traps, and major species bydeep wells, all forming a kind of metalan-guage known as “energy landscape theory”(10, 15–17). The graphic funnel picture isa generic representation, independent ofstructural and thermodynamic detail andequally applicable to any protein, RNA, orother compact polymer. Although it pro-vides no constraints that would excludeany realistic folding scenario, even a definedpathway model, it has been widely inter-preted to require that proteins fold throughmany independent pathways.R. L. Baldwin took up the challenge and

led the field in a multiyear effort to exper-imentally define kinetic folding intermediatesand pathways (18–20). In a thoughtful pro-tein folding review 20 y ago, Baldwin consid-ered the disparate insights available at thetime from both theory and experiment(21). He highlighted uncertainties in the ex-perimental evidence for classical pathways.

Kinetic folding intermediates seemed to formasynchronously over a range of time scales.Equilibrium analogs of folding intermediatescalled molten globules yielded mixed results,sometimes agreeing with kinetic folding in-formation and sometimes not. Baldwin’s ar-ticle served to alert the experimental proteinfolding community to the new view of het-erogeneous folding and helped to establishthe current paradigm of a multipath fun-neled energy landscape.The distinction between the classical view

of a more or less single pathway throughdefined intermediates and the disorderedmany-pathway new view has broad signifi-cance for the understanding of protein bio-physics and biological function. The questioncould be resolved by determining experimen-tally the structure of the intermediate formsthat bridge between unfolded and nativestates in real proteins, but this effort hasturned out to be exceptionally difficult. Theusual methods, crystallography and NMR,cannot define partial structures that form anddecay in less than 1 s. Experimentalists havebeen forced to depend on spectroscopicmethods (fluorescence, CD, IR) that can fol-low kinetic folding in real time but are blindto the specifics of structure and so allow the

Author contributions: S.W.E. and L.M. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

1To whom correspondence should be addressed. Email: engl@mail.med.upenn.edu.

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possibility of alternative folding mechanisms.Theorists have attempted to avoid these dif-ficulties by simulating the folding process incomputers. Theory-based computer simula-tions can be remarkably powerful. For exam-ple, one can compute the path of a multitonrocket through 150 million miles of freespace to a pinpoint landing on Mars. Theequations that govern space flight are knownprecisely (22), computer power is ample, andthe track to be controlled is clear. Computingthe structural journey of minuscule proteinmolecules through submicrons of space hasproved to be more difficult. The computerpower required to track the folding processat the level of thermally driven residue-leveldynamics is immense. The forces that directprotein folding are delicately balanced, inter-locking, and not describable in exact terms.The reaction path(s) to be mined from themass of computer data are unknown.For both the classical and new view models,

Fig. 1 implies that the structure of foldingintermediates and their pathway connec-tions might be determined in three differentways: (i) as intermediates that reach signif-icant occupancy during kinetic folding; (ii)as conformationally excited states that existat their equilibrium Boltzmann level in thehigh free energy space above the native pro-tein; and (iii) as modified molten globuleforms made by destabilizing the native pro-tein so that higher energy states become thelowest free energy equilibrium form. Exper-imental advances have accomplished allthree of these approaches. At any time pointduring kinetic folding, a briefly present fold-ing intermediate can be marked in a struc-ture-sensitive way by hydrogen exchange(HX) pulse labeling and defined by lateranalysis. The structure of partially foldedstates minimally populated in the high free

energy space can be determined by HX la-beling, sulfhydryl labeling, and NMR meth-ods. Partially unfolded molten globules canbe labeled in a structure-sensitive way byhydrogen–deuterium (H-D) exchange andanalyzed later by site-resolved NMR in thereformed native state or directly by massspectrometry.These advances now make it possible to

determine the structure and properties ofintermediate protein folding states and theirpathway connections and so place the studyof folding pathways on the solid ground ofstructural biology. Experiment can now askwhether proteins fold through a limited num-ber of distinct obligatory intermediate struc-tures in an ordered kinetic sequence assuggested in Fig. 1A, or through a heteroge-neous collection of independent multiply par-allel forms and routes as in Fig. 1B, or throughsome other combination of conformations.

Intermediates During Kinetic FoldingHX Pulse Labeling and NMR Analysis. Itfirst became possible to obtain detailedstructural information on briefly presentprotein folding intermediates with the de-velopment of the HX pulse labeling meth-od (23, 24). The initially unfolded andD-exchanged protein is mixed into foldingconditions and then, at various times duringfolding, is subjected to a short, selective D toH exchange labeling pulse. The protein foldsto the native state, and D vs. H placement isanalyzed by NMR to identify amide sites thatwere already protected (still D-labeled) or notyet protected (H-labeled) at the time ofthe labeling pulse. The results provide a seriesof snapshots during the time course overwhich folding converts identifiable main chainamides to a protected H-bonded condition.The results will detect intermediates that

encounter a sizeable kinetic barrier and soreach significant population.Initial results obtained for cytochrome c

(Cyt c; Fig. 2) showed that approximately halfof the molecules form their sequentiallyremote but structurally contiguous N- andC-terminal helical segments early (12 ms),suggesting the formation of a specific on-pathway native-like intermediate. However,Baldwin’s review (21) emphasized the asyn-chrony in these kinetic results; some of themolecules protect their N- and C-terminalhelices early, whereas others do so at latertimes, along with other regions. Other proteinssimilarly studied have often yielded analogousresults. This heterogeneous behavior conflictswith a well-defined sequential pathway modelbut seems more consistent with the new viewof different routes, rates, and traps.One now knows that the heterogeneous

folding seen in kinetic HX pulse labelingexperiments can be due to previously un-recognized experimental issues. One prob-lem concerns the tendency of refoldingproteins to transiently aggregate, especiallyat the high concentrations used to facilitatethe preparation of samples for NMR analy-sis (25, 26). Another unexpected effect wasrevealed in a sophisticated analysis of theHX pulse labeling experiment, which showedthat intermediates populated during kineticfolding may repeatedly unfold and refold ona fast time scale. Sites that are already foldedand protected can nevertheless becomeH-labeled during the intense high pH in-terrogation pulse even with only a singlereversible unfolding during the pulse (theso-called EX1 HX regime; 50-ms pulse,back unfolding rate 12 s−1 for Fig. 2) (27,28). Other HX NMR pulse labeling studieshave been compromised by similar aggrega-tion and HX EX1 behavior, and also by theinability to differentiate mixtures of states dueto the ensemble averaging that occurs whenNMR is used to obtain a single measurementfor each individual residue.

HX Pulse Labeling and MS Analysis. Arecently developed variant of the HX pulselabeling experiment can produce a moreexplicit description of the kinetic foldingprocess. The new technology replaces NMRanalysis with a mass spectrometry technique(HX MS) that allows folding experimentsat 1,000-fold lower concentration and thusexcludes aggregation. As before, the un-folded and D-exchanged protein is mixedinto folding conditions and is subjected toa D to H exchange labeling pulse after var-ious folding times. The labeling pulse canbe adjusted to avoid or to study the back-unfolding behavior of transiently populated

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Fig. 1. (A) The classical view of a defined folding pathway, and (B) the new view of multiple routes through a funneledlandscape. Reprinted with permission from ref. 13. Dashed line in A illustrates the insertion of an optional error-dependentkinetic barrier, which can affect some population fraction and not others and thus mimic multipathway folding.

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intermediates. To terminate labeling andprepare for analysis, the selectively labeledprotein is plunged into slow HX conditions(low pH and temperature), then cleaved intoshort fragments, and the fragments areseparated and analyzed by fast HPLC andmass spectrometry. The two examples so farpublished, illustrated in Figs. 3 and 4, pro-vide detailed pathway information.When the large (370 residues) two-

domain and aggregation prone maltosebinding protein (MBP) is diluted intofolding conditions at <1 μM concentrationit does not aggregate, but it does rapidlycollapse into a dynamic polyglobular statewith heterogeneous low level HX protection(Fig. 3) (29). This condition might beexpected to spawn multiple folding routesas in the new view model, but it does not.The microsecond and millisecond timescales pass with no indication of native-likestructure formation, perhaps because con-formational searching in the collapsed stateis difficult. Ultimately, the entire proteinpopulation assembles sequentially remotesegments into a specific native-like interme-diate with a single exponential time con-stant of 7 s (blue in Fig. 3). Other peptidesthen report on later folding events thatmove to the native state over a broader timescale (60–120 s), suggesting several foldingsteps, but their kinetics are too compressedto allow clear resolution. These experimentslargely avoided the back-unfolding HX la-beling artifact by using a short labelingpulse (12 ms). Longer pulses (up to 42 ms)allowed the back-unfolding of the weaklyprotected regions in the initially collapsedform to be studied (29). Higher protectionseems to correlate with the amphipathicnature of different segments and their

tendency to form helical structure. The moreprotected segments (black curves in Fig. 3)are not the ones that form the emergent 7 snative-like foldon.The same technology was able to resolve

the entire folding trajectory of RibonucleaseH (155 residues; Fig. 4) in structural and

temporal detail (30). The overlapping peptideMS results allow transiently formed inter-mediates to be defined at near amino acidresolution. In each case they are composed ofsets of residues that form well-definedH-bonded elements in the native protein(foldons). The results display a stepwise as-sembly of the native structure, first helix A +strand 4 (blue in Fig. 4), then the neighboringhelix D + strand 5 (green), then the inter-acting B/C helix (yellow), and finally theterminal segments (red). The yellow foldondoes not reach complete protection becauseof some back-unfolding (∼20%) during the10-ms HX labeling pulse which, fortuitously,helps to distinguish the yellow and greenfoldons along with the small difference intheir formation rates seen in the renormal-ized kinetic phases (Fig. 4, Inset).We used the HXMS method to reexamine

the ambiguous kinetic folding results of Cytc measured before by HX NMR (Fig. 2).Low folding concentration (2 μM) avoidedthe previous transient aggregation problem,and a short labeling pulse (10 ms ratherthan 50 ms) minimized spurious labelingdue to back-unfolding during the pulse.The results confirm that all of the proteins

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Fig. 2. Initial HX NMR pulse labeling results for Cyt c (24). A brief D to H labeling pulse imposed after various foldingtimes was used to track the increasing protection (decreasing H-labeling) of individual residues and the segments thatthey represent. The results suggested early formation of a native-like N/C bihelical folding intermediate. Baldwin’sreview (21) noted the kinetic asynchrony, with the N- and C-terminal helical segments in different molecules folding atdifferent rates. Later work shows that the asynchrony is caused by protein aggregation and by HX pulse breakthroughdue to back-unfolding of the transiently populated intermediate during the H-labeling pulse (50 ms).

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Fig. 3. Pulse labeling HX MS results for maltose binding protein (29). (A) The time-dependent folding (HX protection)of 116 highest-quality MBP peptide fragments representing different protein regions. Black kinetic curves show theslow time course for folding of peptide fragments that are most protected in the initial collapse. (B–D) RepresentativeHX-labeled MS fragments from different protein regions (colored) define the separate folding steps, display theirconcerted two-state nature, measure their formation rates, and show that the entire protein population (>95%)experiences the same steps. (E ) The course of folding. On dilution from denaturant into folding conditions, MBPrapidly collapses into a heterogeneous polyglobular state (SAXS envelope reconstruction in gray) with widespread lowlevel HX protection, then slowly folds (kinetic curves in A) through an initial native-like intermediate (blue, τ = 7 s) andlater kinetically unresolved steps (green, gray, red; τ ∼60 s to 120 s; fastest green segments shown in C and E).Mutations known to greatly slow folding (stars) are all within the 7 s intermediate.

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fold and dock their two terminal helices ina single early step (∼12 ms), and the rest ofthe native structure folds later. A methodfor studying kinetic folding intermediatesat equilibrium, known as native state HX,described below, independently confirmsthis result and elucidates the entire sub-sequent folding pathway.Unlike all ensemble-level measurements

including HX NMR, these pulse labeling HXMS results provide snapshots that show thestructurally different populations that arealready formed and not yet formed at anytime point during kinetic folding rather thana potentially misleading population average.The HXMS data show that folding occurs ina stepwise manner and that each kinetic stepis individually two-state representing thecooperative formation of an additionalfolding unit (foldon) (MS data in Figs. 3 and4). The time-dependent MS data show that,once each foldon unit is formed, it remainsin place as subsequent foldons are added,demonstrating a stepwise buildup throughdistinct, progressively more folded forms.Essentially the entire refolding populationjoins synchronously in the same stepwisesequence of intermediate structures, in-dicating a single dominant folding pathway.The data show explicitly that less than 5%of the protein population folds throughany other pathway(s). However, other Cyt cresults do detect minimal branching inthe special case where the prior structurecan support two different but essentially

equivalent subsequent steps; either stepcan occur before the other (31).These results support a picture of protein

folding in which the entire protein pop-ulation folds through the same distinctintermediates and kinetic barriers in the samedefined pathway, as in Fig. 1A. A seminalobservation is that the intermediates formby assembling pieces of the native protein,called foldons.

Other Kinetic Studies. A large fraction ofthe protein folding literature is directed atfinding the determinants of folding rates.Prominent issues, highlighted by reviewers,concern the nucleation–condensation model,the φ value analysis method, and two-statefolding. Is the distinct pathway model con-sistent with current kinetic information?The nucleation–condensation model sug-

gests that folding is initiated by a nucleationevent that potentiates subsequent structuralconsolidation (32, 33). The φ value analysismethod attempts to define the parts ofa protein that gain structure in the initialrate-limiting transition state, the nucleatingevent, by measuring the effect of specificmutations on folding rate (34). The usualresult, that φ values are small and fractional(∼0.3 ± 0.2) (35), can be explained either bymultiple pathways or by the likelihood thatflexible partially folded structures can ac-commodate disruptive mutations more easilythan the rigid native state. Thus, implicationsfor the question of one pathway vs. manyare ambiguous. A related ψ value analysis

method, although much less used, is moredefinitive. It finds the same distinct partiallyformed native-like structure for the entirefolding population (36). These results favorthe distinct pathway hypothesis.Many proteins, especially small ones, tend

to fold and unfold in a kinetically two-statemanner, each with a single exponential rate.The same kinetic barrier is rate-limiting inboth folding and unfolding directions, andtheir ratio gives the correct equilibrium sta-bility constant. In this case, intermediates willnot be seen to populate either before or afterthe barrier, whether they exist or not, and theusual kinetic folding experiment simplycannot distinguish whether separate pathwaysteps do or do not occur. For example, thedefined pathway model in Fig. 1A will pro-duce two-state kinetic folding and unfolding(and linear chevron plots) in the absence ofthe inserted misfolding barrier noted. Un-fortunately, the absence of explicit evidencefor multiple kinetic steps is often taken, in-correctly, as evidence for their absence.However, again here one can note that theobservation of the same folding rate for thewhole protein population tends to favora single common pathway rather than mul-tiple independent paths.Thus, much of available kinetic infor-

mation is unable to distinguish alterna-tive pathway behaviors, although someobservations can be deemed supportive ofthe distinct pathway model.

Multiple Pathways and Misfolding. Someother optically measured kinetic results havebeen thought to support multiple pathways,although only a small number. The conflict isoften due to the chance occurrence of partialmisfolding, which inserts an optional kineticbarrier into the folding pathway, differentlyaffecting the folding of different populationfractions (37). In this case kinetic folding willappear heterogeneous and asynchronous,even when all of the molecules fold throughthe same sequence of intermediate structures.This barrier-based problem is common andhas greatly confused protein folding studies.Known optional errors include aggregation(26), partial proline mis-isomerization (38),incorrect disulfide pairing (39), nonnativehydrophobic clustering (40), and partialheme mis-ligation (24). (Note: The term“misfolding” has become associated withamyloid formation; we use it in a moregeneral sense.)In a prime example, folding experiments

on the large TIM barrel protein α-Trp syn-thase found several kinetically distinctpopulation fractions and intermediates, sug-gesting four parallel folding tracks (41).

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Fig. 4. Pulse labeling HX MS results for Ribonuclease H (30). (A and B) Kinetic curves for time-dependent HXprotection of peptide fragments that define the blue, green, yellow, and red foldons. (C–F ) HX MS pulse labelingresults for representative peptide fragments show the time course and two-state concerted nature of foldon foldingsteps, and that the entire protein population (>95%) experiences the same sequence of concerted steps in a singledominant pathway. The yellow foldon does not reach complete protection because of partial labeling due to back-unfolding during the 10-ms labeling pulse, which helps to distinguish the yellow foldon from the green foldon, alongwith the small difference in their formation rates seen in the renormalized kinetic phases (A, Inset).

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Subsequent work found that each additionaltrack could be suppressed, one at a time, bymutational replacement of one or more pro-line residues or by addition of a prolyl isom-erase (42), as expected for a defined pathwayinterrupted in some fraction of the foldingpopulation by optional mis-isomerized pro-line barriers. In similar work, multiple kineticfolding phases observed by optical methodsfor hen egg lysozyme (43, 44) and Staphylo-coccal nuclease (45) were also fit by theauthors to multiple pathway models, but itwas shown that the data can be fit at least aswell by a single pathway in which somefraction of the molecules experience an errorthat slows its folding (37, 46). In the absenceof structural information, it is not possible todistinguish between a multiple pathway in-terpretation and a given pathway with op-tional barriers. This can be seen intuitively byconsidering the insertion of an optional bar-rier at any step in a well-defined pathway asin Fig. 1A (dashed line).The common occurrence of on-pathway

optional errors has led to other incorrect sug-gestions: that well-populated kinetic inter-mediates are grossly misfolded artifacts ratherthan constructive on-pathway structures withsome particular misfolding error; that visibleintermediates hinder rather than promotefolding because visible intermediates andslowed folding occur together. Other literature

results have been interpreted in terms ofmultiple pathways, either during unfolding atconditions far from native, or during foldingbut potentially confounded by ensemble av-eraging, or by complex spectroscopic phasesthat allow different interpretations, as well asby spurious barriers due to optional errors. Inall of these cases, the structural informationthat is necessary to support a definitive con-clusion is absent.More definitive information comes from

the kinetic HX MS experiments illustratedabove, which do document a distinct path-way, and from a number of equilibrium-based methods described in the following,which have been able to reveal multiplenative-like partially folded on-pathway inter-mediates, even when simple folding seems tobe kinetically two-state.

Intermediates Observed at EquilibriumIntermediates as Conformationally Ex-cited States. An experiment called equilib-rium native state HX, explained in Fig. 5, firstdetected and described cooperative foldonunits (2, 47). The experiment uses low con-centrations of denaturant (or other destabi-lant) to promote sizeable unfolding reactionsto the point where they come to dominatethe H-exchange of the amides that they ex-pose. The results, reproduced in Fig. 5A,showed that specific structural elements of

Cyt c (Fig. 6) repeatedly unfold and refold,accessing partially unfolded high energystates with ΔGo of 4–13 kcal/mol above thenative state, corresponding to steady-statepopulations between 10−3 and 10−9 of thetotal protein. These results identify foldonunfolding units in terms of their detailedresidue composition, specify the free energyof the partially unfolded states relative to thenative state, and can measure unfolding andrefolding rates.However, these results do not fully identify

the different partially unfolded forms (PUFs).At each intermediate state, sites that havealready exchanged to D are invisible (NMR);one cannot tell whether they are structuredor not in the given intermediate. Therefore,one cannot tell whether the different foldonssimply unfold independently or in a pathwaysequence, as posed in Fig. 5E. This is unlikethe kinetic HXMS experiments in Figs. 3 and4, where the pulse labeling approach directlyprovides a snapshot of the folded conditionof all of the residues during the folding pro-cess. Ultimately, a series of “stability labeling”experiments showed that the high energystates seen for Cyt c do represent a quantizedstepwise series of progressively more un-folded PUFs, as pictured in Fig. 5E (48). Inthe unfolding direction, the transition to eachhigher energy PUF unfolds one more foldonin a sequential pathway manner. Becausethese experiments were done under equi-librium native conditions (pD 7, 30 °C),each uphill unfolding step must be matchedby an equivalent refolding step. The down-hill sequence defines a stepwise sequentialfolding pathway.In detailed confirmation, these equilibrium

results identified the same N/C bihelicalfoldon (blue) as did the kinetic pulse label-ing experiment. The pulse labeling experi-ment places this state as first in the foldingsequence; the native state HX experimentplaces it as last in the unfolding sequence.The initially folded N/C bihelical PUFaccumulates in Cyt c kinetic folding when itencounters a histidine to heme mis-ligationbarrier; both peripheral histidines of Cyt care placed on and therefore block formationof the green foldon segment, which is pro-grammed to fold next. An independentkinetic mode native state HX experimentshowed that the various foldons unfold inthe kinetic order shown in the rising ladderin Fig. 5E. The unfolding rate for a firstunfolding step (by EX1 HX) accuratelymatches the independently measured Cyt cglobal unfolding rate in two-state unfoldingconditions (49).Distinct native-like pathway intermediates

have been found for other proteins by HX

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Fig. 5. Initial equilibrium native state HX NMR results for Cyt c (47). (A–D) HX rates of many individual Cyt c residues,measured by NMR as a function of low levels of added denaturant far below the melting transition, are plotted interms of the free energy of the exposure reaction that determines each amide HX rate. HX governed by a small localfluctuation is insensitive to denaturant and produces a horizontal curve. HX determined by a large unfolding reactionis sharply promoted by denaturant and can come to dominate the exchange of the residues that it exposes. Theresidues that join each cooperative unfolding (large slope) specify the identity of that unfolding unit. The intercept ofeach HX isotherm defines the free energy level of each PUF at zero denaturant; the slope relates to its surface ex-posure. These data identified four large unfolding units (foldons), coded as blue, green, yellow, and red. The lessdefinitive red foldon and the infrared foldon not seen here (gray in Fig. 6) were better defined in later work. (E ) Thefree energy levels of the PUFs produced by the individual cooperative unfolding reactions place them on a free energyladder. The data in A–D specify the identity of each foldon unfolding unit but do not specify the complete PUFproduced by each unfolding. Therefore, one cannot tell whether the foldons unfold independently or sequentially or insome other manner. A series of stability labeling experiments defined the PUFs shown (far right). They constitutea stepwise unfolding and refolding pathway, as in Fig. 1A.

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pulse labeling, native state HX, and non-HXmethods. Silverman and Harbury (50) de-signed a proteomics method to measure thereactivity of 25 cysteine SH side chains intriose phosphate isomerase, analogous to thenative state HX experiment. The experimentidentified three partial unfolding reactions,and stability labeling experiments showedthat they stack up in an unfolding/refoldingsequence, as for Cyt c. Sekhar and Kay (51)used NMR relaxation dispersion to identifyindividual partially unfolded forms in severalsmall, supposedly two-state proteins, andsupported their role as defined foldingpathway intermediates.All of these results are fully consistent with

a classical folding pathway with each in-termediate PUF separated from its neigh-bors by the folding or unfolding of onemore foldon.

Molten Globules as Lowest Energy Fold-ing Intermediates. In his 1995 new viewpaper (21), Baldwin considered the then-current status of the molten globule hy-pothesis. Earlier thinking shaped by proteindenaturation studies had supposed that pro-teins are highly cooperative two-state struc-tures and can only occupy, at equilibrium,either their fully native or fully unfoldedcondition (although see ref. 52). Ptitsyn andcoworkers and others found that certaindestabilizing conditions, especially low pH,could induce a new, more dynamic, andsomewhat expanded protein form, with loosetertiary structure but often considerable sec-ondary structure, which came to be called themolten globule (53–55). Ptitsyn suggestedthat molten globules represent equilibriumanalogs of kinetic folding intermediates. Insome cases, HX NMR connected the sec-ondary structure with native-like helical

elements, consistent with the Ptitsyn hy-pothesis. However, Baldwin (21) comparedthis proposal with expectations from classicaland theoretical models, and again here am-biguity prevailed. For example, the pH 4equilibrium molten globule of apomyoglobincontains the very same native-like A, G, andH helices found by HX pulse labeling duringkinetic folding, in line with the Ptitsyn hy-pothesis, but a Cyt c molten globule containsall three of its native helical segments and notjust the N/C bihelical intermediate observedin kinetic HX pulse labeling.Later work connects the enigmatic struc-

tural character of molten globules with thefoldon construction of native proteins. Anumber of partially structured proteins havenow been prepared by synthesis or mutation,whether guided by foreknowledge of kineticfolding intermediates or not (56). They mim-ic natively structured pieces of the native pro-tein. Most incisively, Bai and coworkers (57)used native-state HX to define partially un-folded intermediates of apoCyt b562, and theninserted mutations that selectively destabilizethe native state. In the present terms, one ormore of the less stable foldons, on the lowerrungs of the energy state ladder (as in Fig.5E), were made to remain unfolded, whichcaused some higher energy partly unfoldedstate to become the dominant lowest energyform. Feng et al. solved the structures of twore-engineered versions by NMR and foundthat both are close mimics of the foldingintermediates indicated by native state HX.They are both partially folded and clearlynative-like, although they energy minimizeto produce some nonnative distortionsthat shield otherwise exposed hydrophobicside chains.

These results provide a clear picture of thestructure of an authentic folding intermediate.They also explain the molten globule ambi-guities described in Baldwin’s new view article(21) and elsewhere. A molten globule mayemulate, as a free-standing equilibrium spe-cies, any one of the quantized intermediatePUFs seen by the kinetic and equilibriummethods just described, depending on howlower energy states are destabilized. Evidentlythe foldon concept has broad applicability forunderstanding the range of protein structures.

Protein Folding in SilicoThe ability to simulate protein folding hasbeen hampered by the immense computerpower necessary, by incompletely adequateforce fields, and by the difficulty of discerninga meaningful course of events (reaction co-ordinate) within the vast data files generated.Until recently most efforts have attempted toevade the computational problems by usingsimplified nonphysical force fields andmodels. They have not found coopera-tive foldons and discrete foldon-dependentpathways.In one exception Weinkam et al. (58)

simulated the folding course of a Cyt c mimicwithout side chains using a modified G�omodel. The computer was initially told whatthe target native structure looks like, thecalculation was instructed to assign morefavorable energy as the mock residues drawcloser to their normal partners, a multiatomcooperativity term was added, and outsizedinfluence was given to the heme. The pre-sumed shape and properties of the foldinglandscape did not enter the calculation exceptfor the energetically downhill tendency.These instructions caused foldon units toemerge and associate to produce a stepwisefolding pathway, resembling the Cyt cexperiments. This success was considered toshow that the experimental Cyt c resultdepends especially on the influence of theheme group, but other proteins with noprosthetic group are now known to foldthrough distinct intermediates and pathways.The significance of the Cyt c calculation isthat it tends to identify the factors that de-termine the foldon-based behavior. As forany mathematical derivation, the factors thatdetermine the output result must be codedinto the initial premises. In the mock Cyt csimulation, the important factor seems to bethe added cooperativity term, as emphasizedin the foldon hypothesis.A new generation of theoretical analysis

with real proteins in realistic force fields andenhanced computer capabilities is over-coming the calculational difficulties in otherways (59–61). Gathering results from these

N term

C term

ABC

E

1

2

3

4

5

D

N-helix

C-helix

W59

H33

60’s-helix

H26

M8 0

cemorhcotyCHesaelcunobiR

Fig. 6. The foldon construction of Ribonuclease H and Cyt c. The order of folding is blue, green, yellow, and red, andfinally gray for the large bottom Cyt c loop.

15878 | www.pnas.org/cgi/doi/10.1073/pnas.1411798111 Englander and Mayne

approaches tend to emulate the foldon-baseddistinct pathway picture.

DiscussionThis article considers the fundamental ques-tions of protein folding, previously answeredso differently by the classical and new viewmodels. How do proteins fold, and why dothey fold in that way? Extensive experiencewith the folding problem over a 50-y periodhas shown that clear structural informationon the intermediate states that bridge be-tween the unfolded and native states will berequired. Experimentation has developed threeuseful approaches. Folding intermediates canbe studied as significantly populated formsduring kinetic folding, or as conformationallyexcited forms present at equilibrium undernative conditions, or as equilibrium moltenglobule forms. Structural results from thesedifferent approaches converge on the sameconclusions.

The Foldon Hypothesis. In all of theseobservations, cooperative foldon units playa pivotal role. Foldon units were first dis-covered and characterized in the initial nativestate HX experiment (2, 47). The experimentshowed that native Cyt c at equilibrium un-der native conditions repeatedly unfolds andrefolds. A series of experiments showed thatthe foldon unfolding reactions occur in a se-quential pathway-like manner (48) ratherthan independently (Fig. 5). That chain ofresearch was rather complex; it developedover a period of years and has evidently beendifficult for most investigators to follow.However, reversible partial unfolding andrefolding steps have now been seen in variousways for many proteins, and they have oftenbeen connected to the protein folding pro-cess. Most pointedly, a recently advanced HXMS capability made it possible to observematching behavior as it occurs during kineticfolding for MBP (29), RNase H (30), and Cytc, as just described. In all cases one sees thatunfolding and refolding proceed in steps thatsubtract or add one more native-like co-operative foldon unit. The detailed foldonconstruction of Cyt c and RNase H is illus-trated in Fig. 6. Both fold by first formingtheir blue foldon, then an immediately adja-cent foldon to form the blue + green PUF,and so on.The centrally important point is this: con-

trary to previous belief, proteins are multistateobjects built from separately cooperativefoldon units. This fundamental insight leadsto a foldon-based hypothesis that suggeststhe “how” and the “why” of protein folding.The cooperative foldon construction of pro-teins predisposes them to unfold and refold

through foldon-determined steps. The dis-crete steps produce an ordered repeatablemacroscopic folding pathway because pre-viously formed foldons tend to guide andstabilize the formation of incoming foldonsthat they are designed to interact with in thenative protein.

Time and Energy.A successful folding modelmust resolve major questions concerningfolding time and energy. Levinthal pointedout that the vast array of protein con-formations in unfolded space cannotsimply reequilibrate and reach the uniquenative state by an undirected random searchin any reasonable time (3, 4). Early the-oretical work therefore focused on thedownhill energetic drive and the manyindependent routes that heterogeneity andmicroscopic thermal searching alone seemedto require. The new view answer to the“why” question is that, from the micro-scopic point of view, there seems to be noother viable choice.Experimental work recounted here reveals

an emergent macroscopic behavior thatprovides a previously unrecognized mecha-nism. Random search does not have to carrythe protein all of the way to the native state. Itonly needs to accomplish the formation ofa first native-like foldon. This process isthermodynamically downhill and is guidedby the inherent cooperativity of native foldonunits. Present information indicates that thefirst-formed foldon tends to be stable in thecontext of the rest of the protein (27, 62). Thestill-unfolded regions can shield and energyminimize unfavorably exposed groups, as inthe molten globule situation described before.The time scale for forming a first foldon unitby an unguided search, perhaps two seg-ments ∼20 residues in length, is shorter byfar than for a reference 100-residue protein[3100/(2 × 320) ∼ 1040]. The formation ofsubsequent foldons must proceed by way ofsimilar microscopic searching but in a moreguided way analogous to the process of“folding upon binding.” The concept thatproteins start folding by forming a native-like structural nucleus has been widely ac-cepted (33). This minimal structure can besufficient to seed subsequent foldon–foldoninteraction steps in a sequence of moreguided searches that follow through,rapidly, to the native target.Does this process have the energetic bias

necessary to select specific folding steps anddrive folding to completion in a short time?Zwanzig et al. (63) calculated that a freeenergy bias of 2 kT toward correct inter-actions is necessary for a folding sequenceto complete on a time scale of seconds. It

should be appreciated that this degree ofbias, more than 1 kcal/mol, is unreasonableat the individual residue level. A singleresidue has very low probability for findingits correct native partners in a sea of non-native alternatives. Certainly, microscopicthermal searching must underlie any struc-ture formation process. However, given therequired energy bias computed by Zwanziget al., it seems that microscopic-levelsearching alone cannot swiftly reach thenative state.By contrast, in a more macroscopic fol-

don-based scenario each correct native-likechoice is driven by the collective energy ofmany interaction sites held stereochemicallyin a native-like geometry in partner foldons.This mechanism has been described be-fore as sequential stabilization (48). It isanalogous to the well-known folding uponbinding process, except that here theincoming disordered segment is advan-tageously tethered to its already struc-tured partner. The macroscopic foldon-levelfactors provide both the qualitative structuralbasis and the quantitative energetic biasrequired to rapidly and repeatably selectdiscrete determinate pathway steps incompetition with all of the other possiblealternatives.

ConclusionsThe supposed conflict between the classicaland new views can be resolved by the re-alization that they touch on different butequally essential parts of the folding mecha-nism. Laboratory experiment is able to dis-cern macroscopic molecular behavior, but itis blind to the microscopic thermally drivenamino acid-level searching behavior thathas been the domain of theoretical analy-sis. The disordered microscopic multitracksearch envisioned in the paradigmatic newview model describes the initial stage aminoacid-level search to form cooperative native-like foldon structures, but not the final na-tive state. Experiment displays an emergentfoldon-based macroscopic behavior that pro-vides the structural guidance and free energybias for the ordered stepwise formation ofdiscrete native-like intermediates in a foldingpathway that leads to the native state.Folding in moderately small, separately

cooperative units may be necessary forproteins to fold at all. A much larger stepsize would confront the Levinthal time scaleproblem; much smaller steps cannot as-semble the energy bias required by theZwanzig criterion for fast folding. Thus, asbefore for the microscopic view, it may bethat there is no other viable choice. Efficientfolding may well require foldon-based

Englander and Mayne PNAS | November 11, 2014 | vol. 111 | no. 45 | 15879

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protein folding pathways. However, herea related constraint enters. Because theessential folding intermediates closelyduplicate native structure, as perhaps theymust in a reasonable pathway sequence,it seems that the same requirement has

reciprocally shaped the foldon-basednature of native protein structure. Inrespect to foldon-based folding and fol-don-based native structure, it seems thateach necessitates the other, and that protein-based biology may require both.

ACKNOWLEDGMENTS.We thank K. A. Dill, M. F. Gellert,S. Lund-Katz, V. S. Pande, M. C. Phillips, G. D. Rose,T. R. Sosnick, A. Szabo, A. J. Wand, and members ofour laboratory for helpful contributions. This workwas supported by National Institutes of HealthGrant RO1 GM031847, National Science Founda-tion Grant MCB1020649, and the Mathers Charita-ble Foundation.

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15880 | www.pnas.org/cgi/doi/10.1073/pnas.1411798111 Englander and Mayne