intimate view of a kinetic protein folding intermediate...

Intimate View of a Kinetic Protein FoldingIntermediate: Residue-resolved Structure, Interactions,Stability, Folding and Unfolding Rates, Homogeneity

Mallela M. G. Krishna*, Yan Lin, Leland Mayne and S. Walter Englander

Johnson Research FoundationDepartment of Biochemistryand Biophysics, Universityof Pennsylvania Schoolof Medicine, PhiladelphiaPA 19104-6059, USA

A cytochrome c kinetic folding intermediate was studied by hydrogenexchange (HX) pulse labeling. Advances in the technique and analysismade it possible to define the structured and unstructured regions, equi-librium stability, and kinetic opening and closing rates, all at an aminoacid-resolved level. The entire N-terminal and C-terminal helices areformed and docked together at their normal native positions. They frayin both directions from the interaction region, due to a progression inboth unfolding and refolding rates, leading to the surprising suggestionthat helix propagation may proceed very slowly in the condensed milieu.Several native-like beta turns are formed. Some residues in the segmentthat will form the native 60s helix are protected but others are not,suggesting energy minimization to some locally non-native conformationin the transient intermediate. All other regions are unprotected, pre-sumably dynamically disordered. The intermediate resembles a partiallyconstructed native state. It is early, on-pathway, and all of the refoldingmolecules pass through it. These and related results consistently point todistinct, homogeneous, native-like intermediates in a stepwise sequentialpathway, guided by the same factors that determine the native structure.Previous pulse labeling efforts have always assumed EX2 exchangeduring the labeling pulse, often leading to the suggestion of hetero-geneous intermediates in alternative parallel pathways. The presentwork reveals a dominant role for EX1 exchange in the high pH labelingpulse, which will mimic heterogeneous behavior when EX2 exchange isassumed. The general problem of homogeneous versus heterogeneousintermediates and pathways is discussed.

q 2003 Elsevier Ltd. All rights reserved.

Keywords: protein folding; pulse labeling; hydrogen exchange; EX1;cytochrome c*Corresponding author

Introduction

Do proteins fold through a multiplicity ofalternative intermediates and parallel pathways,as envisioned by many theoretical1 – 3 and experi-mental studies,4 or through a small number ofdistinct intermediates on discrete pathway(s) tothe native state?5 – 7 To understand how proteinsfold, and why, it will be necessary to obtain

detailed information on the structure of kineticfolding intermediates. This has been difficult.Kinetic intermediates exist only briefly (, seconds)and cannot be isolated for structural studies.Spectroscopic methods fast enough to followkinetic folding yield essentially one-parameterdata that provide no structural definition and canconfuse intermediate formation with events suchas chain compaction and transient aggregation.Theoretical methods are not yet able to deal withthe complex structures, potential functions, andfolding time-scale of real proteins.

Detailed structural information has come mainlyfrom hydrogen exchange (HX) methods.7 An HXpulse labeling method, applied during kineticfolding, can provide residue-resolved information

0022-2836/$ - see front matter q 2003 Elsevier Ltd. All rights reserved.

E-mail address of the corresponding author:[email protected]

Abbreviations used: Cyt c, cytochrome c; HX,hydrogen exchange; NHX, native state hydrogenexchange; foldon, cooperative folding/unfolding unit;CD, circular dichroism; GdmCl, guanidinium chloride.

doi:10.1016/j.jmb.2003.09.070 J. Mol. Biol. (2003) 334, 501–513

on intermediates that accumulate transiently inthree-state kinetic folding.7 – 18 A native state HXmethod avoids the kinetic problem altogether bystudying the high-energy, partially folded formsthat proteins experience as they reversibly unfoldand refold under native conditions.7,19 – 21 ExtensiveHX results for cytochrome c (Cyt c; 104 residues;Figure 1) define a small number of discrete inter-mediates that form in a sequential pathway bythe stepwise addition of native-like structuralunits.19 – 27 However, HX pulse labeling results nowavailable for many proteins, including Cyt c, havemost often been interpreted somewhat differently,in terms of multiple alternative intermediates inalternative parallel pathways, similar to resultsfrom computer simulations of simplified foldingmodels.

The present work reconfigured the pulse label-ing experiment and the data analysis, taking intoaccount all contributing factors and the principlesof HX processes, especially at the high pH nor-mally used in the HX labeling pulse. The correctedpulse labeling experiment made it possible todefine the structure, stability, and dynamics of an

initial kinetic Cyt c intermediate at amino acidresolution. It represents a discrete partially formedreplica of the native protein. The entire proteinpopulation moves through this same early inter-mediate on the way to the native state. Whenpulse labeling experiments are interpreted withoutattention to the character of high-pH HX behaviorand appropriate data controls, the results willappear to show heterogeneous intermediates inalternative parallel pathways.

Results

Optimization

One wants to find conditions where refoldingCyt c enters some intermediate state rapidly andexits slowly so that a large fraction of the proteinpopulation occupies the intermediate and can beprobed by HX pulse labeling. The same infor-mation helps to choose optimum pulse labelingtimes.

In exploratory experiments, Cyt c was initiallyunfolded in concentrated guanidinium chloride(GdmCl, p2H 7.5). At this pH (and denaturant),the normal Met80-S to heme iron ligand is replacedby a misligating peripheral histidine residue. Themisligation inserts a latent misfolding barrier thatbecomes obvious later in the folding process.Refolding was initiated by dilution into pH 6buffer (stopped-flow). Spectroscopic probeswere used to detect initial chain contraction(fluorescence; Figure 2(a)), helix formation (CD222;Figure 2(b)), and final native state acquisition(A695; Figure 2(c)).

An initial very fast burst phase represents, webelieve, the relaxation of the chain to a morecontracted but still unfolded form characteristicfor the lower concentration of denaturant, althoughsee Shastry & Roder28 for a different view. Perti-nent evidence has been analyzed in detail.29 Mostconvincingly, the fast continuous flow resultsreported by Akiyama et al.30 show that the entireCD spectrum of Cyt c, recorded long after theburst phase (at 400 ms), is identical with thethermally unfolded state and the acid low-saltunfolded state (compared in Figure 5 of Akiyamaet al.30 and Figure 6(F) of Krantz et al.29). This statehas been referred to as U0 29 or intermediate I.30

The protein starts to fold on a multi-millisecondtime-scale (Figure 2(a) and (b)). The histidine toheme misligation barrier is encountered, foldingpauses, and some intermediate accumulates.31,32

The native state is reached much more slowly(,one second; Figure 2(c)), rate-limited by themisligation-dependent “error repair” barrier.32,33

The detailed kinetics observed depends ondenaturant concentration. At 0.7 M GdmCl, mul-tiple phases are seen, perhaps due to the multipleconfigurations possible for the misligation-depen-dent barrier (His26 and His33; under and overthe heme, etc.). Lower denaturant concentration

Figure 1. Molecular Cyt c structure (1HRC.pdb80 andMOLSCRIPT81) color coded to indicate the folding unitsidentified by NHX experiments and ranked spectrally inorder of decreasing stability and apparent foldingsequence (blue to green to yellow to red to nestedyellow).19,20,22,23,27 The five foldons are the blue unit (Nand C-terminal helices), the green unit (60s helix and20s–30s V-loop), the outer yellow unit (a short anti-parallel b-sheet; residues 37–39, 58–61), the nestedyellow V-loop (residues 40–57), and the red unit (71–85V-loop). The histidine residues in the green loop canmisligate to the Met80 side of the heme Fe in theunfolded protein. This introduces an incipient barrierthat causes transient accumulation of the initial N/Cbi-helical intermediate (blue) by blocking the subsequentfolding of the green unit.

502 Residue-resolved Structure of a Kinetic Protein Folding Intermediate

(0.23 M GdmCl) speeds initial chain collapse(Figure 2(a)) and helix formation (Figure 2(b))because the transition states and intermediateformation are stabilized. Inversely, the lower con-centration of denaturant makes the native stateformation slower (Figure 2(c); reverse denaturanteffect) because the rate for overcoming the mis-folding barrier is limited by an unfolding reac-tion.24,32 – 37 Thus, lower denaturant concentrationincreases intermediate occupation.

The gray area in Figure 2 shows where theHX labeling pulse was applied in subsequentexperiments. Figure 2(c) shows that 15% of therefolding protein reaches N before or during theHX labeling pulse, while the remaining 85%occupies the blocked intermediate state during thelabeling pulse. These numbers must be known forthe data analysis. It is also important to note thatthe fraction already native at the time of the pulseshows the same kinetic behavior (Figure 2(c)) andtherefore has probably passed through the samecondition as the intermediate fraction that isprobed by the labeling pulse.

Final folding (Figure 2(c)) is not mono-expo-nential. A small fraction (,4%) folds more rapidlyto N, probably because it loses the His-hememisligation before or during the initial chaincollapse. In the 35% slower folding phase(s), 15%is due to proline mis-isomerization38 – 40 (checkedby us in double jump experiments). Much of therest is protein concentration-dependent, due atleast in part to intermolecular histidine to hememisligation.29,31,32,41 This kinetic heterogeneity hasno effect on the early N/C helix intermediatestudied here, apparently because the histidine toheme misligation and the four proline residues areremote from the N and C helices and can onlyinfluence later events.

HX pulse labeling

For pulse labeling experiments, Cyt c was

initially unfolded by denaturant in 2H2O (4.2 MGdmCl, p2H 7.5). All of the amides becomedeuterated and almost all of the molecules havethe histidine to heme iron misligation. Dilutioninto H2O buffer (0.23 M GdmCl (pH 6.0), 10 8C)initiates refolding. At this condition, 2H to Hexchange is slow (seconds). A folding time of100 ms was allowed in order to ensure uniformmixing and complete folding to the intermediateform (tf , 12 ms; Figure 2(a)). 2H to H exchangelabeling was then catalyzed by a brief high-pHpulse (50 ms, pH 7.5 and above where tex , ms).Amides that are still exposed in unstructuredregions of the intermediate are labeled at theirunprotected rate. Amides in already formed struc-ture are protected and are labeled more slowly ornot at all, as determined by the usual HX kineticequations. A third mix into low pH (pH 5.3) termi-nates the labeling pulse. The protein folds to itsnative state within several seconds, trapping theH– 2H labeling profile imposed during the shortpulse. Kept under slow HX conditions (,6 8C,reduced Cyt c, pH 5.3), the sample was then con-centrated (centrifugal filtration) and the amountand position of H-label was read out by 2D NMRof the native protein at amino acid resolution.

This analysis, which takes hours, reveals theH– 2H labeling profile imprinted on a millisecondtime-scale, identifying the structure that wasformed and unformed at the time of the pulse.Repetition of the experiment, varying the pre-folding time or the pulse intensity (pH, time), canprovide site-resolved information on the time-course of structure formation8,9 and its stability10,11

and even on dynamics (below). We measured thelabeling obtained in multiple experiments with anincreasingly strong labeling pulse (pH 7.5–10), butwith constant pre-folding time (100 ms) and pulsetime (50 ms).

Figure 3(a) shows a typical H-labeling pattern(pulse pH 9). Figure 3(b) shows the residuesfound to have significant protection in the

Figure 2. Stopped-flow folding kinetics of Cyt c. Unfolded Cyt c (4.2 M GdmCl, p2H 7.5) was diluted into the foldingbuffer (pH 6.0, 10 8C) to final GdmCl concentrations of 0.23 M GdmCl (black) and 0.7 M GdmCl (cyan). Refolding wasmeasured by (a) Trp59 fluorescence relative to the unfolded state (chain contraction/collapse), (b) circular dichroism at222 nm (helix), and (c) absorbance at the 695 nm Met80-S to heme Fe charge transfer band (native state probe; finalvalue is 0.73; measured at 0.1 mM Cyt c as for the pulse labeling experiments). The gray shading indicates the timeduring which the HX pulse was applied in pulse labeling experiments.

Residue-resolved Structure of a Kinetic Protein Folding Intermediate 503

intermediate, evaluated as described below. Themore complete data are in Figure 4, which showsthe H-labeling at 45 amides that could be measuredwith good accuracy across the pulse pH range. Thecolor coding (compare with Figure 1) identifiesamides in the N and C helices (blue unit), thegreen loop and helix, the nested-yellow loop andouter yellow neck, and the red loop.

Data fitting

In Figure 4, the colored continuous curves fit thepH-dependent labeling data for each amide withthe complete HX equations given by Hvidt42

(equations (2)–(7)), with no assumption about HXmechanism (EX1 or EX2). The fitting parametersare kop and kcl, which also specify site-resolvedprotection (structure) and stability (Kop, DGop).

The broken curves in Figure 4 relate to labelingin the intermediate alone. The black broken curvesshow the labeling that is expected for each amide

when it is wholly unprotected (scaled to 0.85),calculated from kch as described.43,44† The coloredbroken curves show the labeling obtained at eachamide (HI,pulse), after correcting for labeling of thenative protein during the pulse (HN,pulse) andduring the sample workup (Hbkgd) (see Materialsand Methods). The corrections are nearly zero foramides in the blue and green units but are largerfor some faster exchanging amides in the yellowand red segments (compare the colored curves inFigure 4).

It can be noted that the brief high-pH pulse usedhere, at pH 10 and lower, is unlikely to seriouslydistort the parameters measured. Only the twohistidine residues are titratable below pH 10, bothare in the still unstructured green loop, and one isalready titrated and misliganded to the heme,which in addition protects against the usual

Figure 3. Labeling results. (a) Thedirectly observed fractional H-labelaccumulated by the various residueamides in HX pulse labeling experi-ments (Hobs in equations (1) and(2)), in this case at pH 9.0 beforecorrecting for the other factorsdescribed in Materials and Methods.The broken line indicates the frac-tional population of intermediate(15% is native). (b) The foldingequilibrium constant (kcl/kop) calcu-lated for each amide in the trappedintermediate, as detailed inMaterials and Methods. Only the Nand C helices are stably formed.Color coding relates to Figure 1.Residues 14, 15, and 18 (gray bars)are protected even in the unfoldedstate.

† http://hx2.med.upenn.edu/download.html


http://hx2.med.upenn.edu/download.html

alkaline transition.45 The pH 10 data point isslightly elevated for some residues, perhaps dueto the onset of some destabilization at elevatedpH,26 which can increase the HX labeling rateeven for EX1 exchange.46

Structure

The results shown in Figures 3(b) and 4 identifythe structured and unstructured regions of thetrapped intermediate and quantify their stabilityand kinetic parameters. Major protection in theintermediate is found for all of the amides that areH-bonded in the N and C helices of the nativeprotein, indicating that the entire length of bothhelices is formed. A similar conclusion wasinferred before on the basis of many fewerprotons.8,11

In native Cyt c, the amide hydrogen atoms ofresidues 64–70 are H-bonded in the 60s helix. Inthe intermediate, the core residues Leu64 andLeu68 and the more exposed Met65 have smallbut clear protection (Kop , 1; kop , 40 s21). Theother residues in the 60s segment are at mostmarginally protected. A possible interpretation ismarginal helix formation. A more likely option isthat the helix is not yet formed but exposed hydro-phobic residues tend to become buried. Energyminimization that forms non-native hydrophobicinteractions in a folding intermediate has beendemonstrated before.47,48

A number of amides that are H-bonded in b-turn

configurations in the native protein have HXprotection in the intermediate.49,50 His33 and theheme-related residues Cys14, Ala15, and His18, allknown to be protected in the unfolded state,19,23,31

are protected in the intermediate. Arg38, Gln42and Thr78, show small HX protection in the inter-mediate in the sense of a low EX1 plateau, buttheir equilibrium protection is within the noiselevel (Kcl , 0.2). Four other b-turns could not bemonitored due to weak cross-peaks and fastexchange during sample workup and analysis.

Most of the amides in the red, yellow, and greenloops are labeled at very close to their expectedunprotected rates. This agreement provides a testof the experiment and the data analysis, and alsoof the previously calibrated rate constants forunprotected amides.43,44

Stability

Figure 5 shows equilibrium and kinetic para-meters obtained for the N-terminal and C-terminalsegments. For the C helix, maximum protectionoccurs where the hydrophobic side-chains of theN and C helices interact in the core of the nativeprotein (Figure 5(a)). Native-like N to C inter-actions were inferred before in a mutationalstudy.51 A fraying pattern appears to emanatefrom that region. The N helix may also fray butthis is less clear. (Note: this kind of fraying beha-vior may contribute to the dispersion of DGHX

Figure 4. Residue-resolved H-labeling results for the kinetic intermediate, which specify the position of protectedstructure and its equilibrium and kinetic parameters (color coded to match the foldons in Figure 1) The data pointsshow the experimentally observed pH-dependent labeling (Hobs; Materials and Methods). The continuous coloredcurves fit the equations given in Materials and Methods to the data, which yields kop and kcl, and therefore also Kop.The broken colored curves show the labeling in the trapped intermediate alone (HI,pulse) after correcting for theextraneous contributions (Hbkgd and HN,pulse). For comparison, the black broken curves show the labeling for eachamide in the intermediate that would be expected in the absence of protection (scaled to 0.85, the fraction of thepopulation in the intermediate at the time of the HX pulse).


Figure 5. Equilibrium and kinetic parameters through the N and C segments in the intermediate. Error flags are at^1s. DGHX in (a) is the residue-resolved stabilization free energy level of the unfolding reaction that allows exchange.The data are fit to Lifson–Roig helix-coil theory (continuous line). Also shown is the residue-resolved AGADIR helicalstability prediction for the isolated segments56,57 (broken line). (b) The approximately constant opening rate (continu-ous line at 14 s21). (c) Closing rates. The horizontal continuous line in (c) shows the folding rate measured by trypto-phan fluorescence (from Figure 2(a)). The fraying behavior in (a) appears to be due mainly to a progression inrefolding rates (c) but also in part to unfolding rates (b), as suggested by the dotted lines. Residues 14, 15, and 18,shown in gray, are protected even in the unfolded state.


values often seen within cooperative elements innative state HX experiments.)

The presence of helix fraying seems very reason-able. Its reality is supported by the patternobserved and the maximum apparent stabilitywhere the two helices normally interact, eventhough the error level for individual protons issizeable. If the fraying pattern is real, then a fit tothe Lifson–Roig equation52,53 leads to propensityvalues (w) of 0.6–0.8 for the peripheral residues,in the range normally seen,54,55† and much higher,4–50, for the middle helical residues, denotingexternal stabilization. The broken curves show theAGADIR56,57 prediction of helical stability forthese segments when unsupported‡. The N and Chelices in the intermediate are more stable thancalculated for the separately isolated segments by4 kcal/mol and 2 kcal/mol, respectively (theyhave negative stability when unsupported).

The maximum stability for the bi-helix in thekinetic intermediate, 1.4 kcal/mol, is less thanfound before by native state HX for the same inter-mediate at the same GdmCl concentration(2.6 kcal/mol at 0.23 M GdmCl). Given the differ-ences in conditions of measurement (10 8C at highpH for pulse labeling versus 30 8C at p2H 7 forNHX), the actual discrepancy is probably signifi-cantly less, since Cyt c goes through maximumstability at 20–25 8C and is more stable in 2H2Othan in H2O.

Kinetic parameters

The labeling pattern observed in Figure 4 indi-cates EX2 exchange at lower pH and EX1 exchangeat elevated pH. The EX2 to EX1 transition is deter-mined by the competition between each amide’sunprotected HX rate (kch) and the reprotection rate(kcl).

26 In the EX2 region (lower pH), HX labelingof the protected sites increases sigmoidally withpH, as expected. At pH 8.5 and 10 8C an averageunprotected amide HX rate is 100 s21, about equalto the measured rate for refolding to the inter-mediate. This is the condition (kch , kcl) whereEX1 exchange is expected to emerge, as is found.Every opening results in labeling, and the degreeof labeling reaches a pH-independent plateau.Different amides reach their plateau values athigher or lower pH, consistent with the knownintrinsic HX rate of each amide (kch). EX1 exchangeat high pH has now been observed in a number ofstudies.26,27,46,58 – 62

The plateau level for each amide (after correc-tions) shows the fraction that fails to label becauseit does not open even once during the labelingpulse. The fractional labeling obtained takentogether with the labeling time (50 ms) specifieskop. Figure 5(b) shows that almost all of the hydro-

gen bonds in the two helices open at about thesame rate, indicating that the two helices unfoldconcertedly (kop ¼ 14(^5) s21). Residues near thehelix ends unfold faster, as can be expected, as dothe protected b-turn amides. Figure 5(c) showsamide-resolved values for kcl. The calculatedreclosing rates match the rate for folding to theintermediate, independently measured by fluor-escence (from Figure 2(a)) (horizontal line), whichhelps to validate the HX result (see also Sivaramanet al.63).

The helix fraying (Figure 5(a)) appears to be dueto a progression of both unfolding and refoldingrates, moving from the bi-helix contact region andnot from the heme attachment region. Most sur-prising, the helices appear to propagate (closingrate) on a millisecond time-scale, a million-foldslower than the nanosecond time-scale seen forindependently stable helices in free solution. Thisresult suggests slow phi–psi rotation in thecondensed milieu, presumably due to side-chainhindrance and interactions.64 – 67 The observation offraying (Figure 5(a)) and its dependence on thehelix propagation (reclosing) rate (Figure 5(c))depends on data with significant error levels, butis supported by the persistent pattern of the dataand the likelihood of the fraying result.

Homogeneity

Is the intermediate population homogeneous?The results in Figure 4 show that all intermediateforms present must have all amides in the N andC segments well protected, and many otherdefined sites essentially unprotected. In agreement,the CD222 measured at this stage in kinetic folding(Figure 2(b)) indicates that all of the molecules inthe intermediate population must have essentiallyall of the N/C bi-helix formed (CD parametersfrom Chin et al.68). The intermediate populationtherefore appears to be homogeneous in astructural sense.

Can the intermediate population be hetero-geneous in a stability sense, with differing N/Cprotection? The labeling curve for any givenamide in Figure 4 would then consist of differentcomponents with different stabilities and openingrates. Heterogeneous intermediates would producea superposition of curves like those in Figure 4,with the component curves variably displaced inthe rising EX2 portion of the labeling curve (notethat a one-component labeling curve is identical inform to the standard one proton pH titrationcurve). In fact, the data in Figure 4 fit well tothe equations used, which assume a singlehomogeneous intermediate and use only two freefitting parameters. The goodness-of-fit obtainedfor many protons does not allow for the presenceof significant heterogeneity in the measuredlabeling.

These considerations still do not rule out thepresence of another population with only the N/Cresidues protected but with protection so large, or

† ftp://cmgm.stanford.edu/pub/helix‡ http://www.embl-heidelberg.de/Services/serrano/

agadir/agadir-start.html


ftp://cmgm.stanford.edu/pub/helix

http://www.embl-heidelberg.de/services/serrano/agadir/agadir-start.html

http://www.embl-heidelberg.de/services/serrano/agadir/agadir-start.html

kop so small, that it does not label measurablyunder the conditions used. What fraction couldthis population represent? The data in Figure 4show that at least 60% of each protected site iswell accounted for by a single fitting curve. Thisfraction is limited at high pH by an EX1 plateau,indicating that the major intermediate character-ized here represents a proportion of the populationthat is larger than 60%. Any more stable popu-lation must be less than 40%. In the regions nearerthe helix ends and elsewhere, a single fitting curveaccounts for a homogeneous fraction close to100%. Also, the matching of the kcl value withthe folding rate measured from fluorescence(Figure 5(c)) is against the possibility that somevery different fraction exists.

These results leave no room for significantheterogeneity.

Equilibrium behavior of the kinetic intermediate

The kinetic parameters found lead to an interest-ing perspective. The trapped intermediateunfolds and refolds repeatedly, , ten times(1/kop , 70 ms; 1/kfold , 800 ms), before the barrierthat blocks its forward progress is overcome andfolding can proceed to N. Implications are notedin Discussion.

Discussion

The N/C intermediate: distinct and native-like

The present experiments reconfigured the HXpulse labeling experiment and analysis. Theintermediate population was maximized, controlexperiments corrected for labeling that occursbefore and after the pulse and for protection dueto a population of already native molecules, andthe data analysis allowed a role for high pH EX1behavior. These advances made it possible toportray the structure, energetics, and dynamics ofthe populated intermediate in some detail.

The residue-resolved pattern of HX protectionshows that the entire N and C-helices are formedand they appear to interact as in the native protein.A small number of protected amides elsewhere inthe protein suggest several native-like b-turns,which are known to be marginally stable even inthe unfolded state. Some small protection is seenfor other amides, perhaps due to the burial ofhydrophobic residues in some non-native butenergy minimized48 format. The unstructuredregions of the partially folded intermediateundoubtedly represent a broad dynamic ensemble,as emphasized by theoretical studies, but it is clearthat fairly extensive native-like structural elementsare also present.

In summary, the kinetic folding intermediatestrongly resembles a partially constructed nativeprotein.

On or off pathway: the initial nucleation barrier

Is the trapped N/C intermediate on or off of theCyt c folding pathway? A demonstration comesfrom studies of the folding barriers.

A previous study showed that the rate-limitingbarrier for two-state Cyt c folding (U to N) isidentical with the rate-limiting barrier for for-mation of the N/C intermediate in three-statefolding (U to I). They exhibit the same rate (DG ‡

and pre-exponential), the same temperaturedependence (DH ‡ and DS ‡), and the same denatur-ant dependence (m ‡).35 It is obvious that the barrierthat limits the rate of two-state folding is on thefolding pathway. Since the very same barrier leadsdirectly to the N/C intermediate in three-statefolding, this intermediate must also be on themain pathway (U to I to N) (the misfolding barrierthat traps the N/C intermediate, producing three-state folding, is placed afterwards). In support,Krantz et al.69,70 found evidence that the N andC helices are formed in the initial rate-limitingtransition state in two-state folding.

Thus, it appears that Cyt c folds through thesame initial on-pathway N/C intermediatewhether it becomes apparent in three-state foldingor remains kinetically hidden in two-state folding.The same conclusion comes from a quantity ofnative state HX data,19,20,22 – 27 which places theN/C intermediate at the beginning of the foldingpathway, even when no kinetic trapping occurs,and shows that all of the molecules movethrough it.

On or off pathway: the I $ U ! N issue

The on-pathway question is raised also by theobservation that the trapped N/C intermediateunfolds and refolds repeatedly (perhaps all theway to U), before the misligation-dependentbarrier is overcome and folding to N can proceed.

When refolding is initiated by denaturantdilution, an unfolded protein may initially relax tosome form that is not on the productive foldingpathway (chain contraction, aggregation, etc.), in asequence usually written as I $ U ! N. Thisdescription is often taken as the definition of anoff-pathway species. An off-pathway form musttravel back through the U state in order to go for-ward. However, it should be noted that the reverselogic does not hold. A trapped intermediate that ison-pathway may still re-equilibrate with U beforegoing forward (U $ I (blocked) ! N). This willoccur if the barrier that blocks forward folding ishigher than the I to U barrier. The trapped N/Cintermediate is one example. Other trappedon-pathway intermediates may do the same.

In the trapped condition, the repeated unfoldingenters the rate for final folding to N as a pre-equi-librium step, as does the heme deligation step,which may occur only when the collapsed inter-mediate transiently unfolds. The need to unfold inorder to repair the misfolding and fold to N


explains the reverse dependence of folding rate ondenaturant concentration24,32,34 – 37 and it duplicatesa suggested mechanism for GroEl action.71

In summary, the N/C intermediate is clearlyon-pathway, best described as a U $ I $ N situa-tion, with a large inserted error-repair barrierslowing the I ! N rate.

Homogeneity of the intermediate population

The pH-dependent pattern of labeling seen inFigure 4 has been observed in previous HX pulselabeling studies with Cyt c and other proteins.Some fraction of any given amide is labeled whileanother fraction appears altogether resistant tolabeling, even at high pulse pH. The commoninterpretation, based on assuming EX2 HXbehavior, has been that different fractions of therefolding population have different protection,indicating alternative folding intermediates withdifferent structure and/or stability, supposedly inalternative parallel pathways.

The present work identifies a different reason.At pH above 9 or so, the chemical HX rate oftransiently exposed amides becomes faster(,1 ms) than reclosing. This is the condition forEX1 exchange (kch . kcl). A plateau level of labelingis reached because amides have reached theirmaximum labeling rate, equal to kop. Amides thatopen at least once during the brief pulse arelabeled. Those that do not open even once are notlabeled, no matter what their stability may be.When EX2 exchange is assumed, the resistantfraction appears misleadingly to represent a morestable form. Another contribution to the protectedfraction can come from molecules that reach thenative state before the labeling pulse. These factorshave generally not been considered in previouspulse labeling work.

Analysis shows that the N/C bi-helical inter-mediate observed here is a distinctly structuredform with defined stability that is homogeneouslyshared by essentially all of the refolding popu-lation. One appreciates that conformation inunstructured regions must be grossly hetero-geneous from one chain to another, but thestructured regions are essentially the same inconformation and stability. Some folding modelspicture that the heterogeneous nature of multipletrapped intermediates is most pronounced earlyon the funnel-like folding landscape where con-formational freedom is large, and much morerestricted near the bottom of the funnel whereconformations merge to the native form. Thepresent intermediate is distinctly structured,homogeneous, and native-like even though it is anearly one.

Pathway heterogeneity and optionalmisfolding barriers

Other results in the literature seemed to showheterogeneity in folding pathways. Different popu-

lation fractions are often seen to refold at differentrates, taken to imply that proteins fold throughmultiple alternative pathways.

A different possibility32 is that all of themolecules fold by way of the same sequence ofstructure formation (intermediates), but that somefraction of the population encounters an optionallyinserted barrier (misfold). When optional mis-folding barriers are inserted into a pathway withprobability between 0 and 1, different fractions ofa refolding population will encounter a limitingbarrier at different points along the pathway,populate different intermediates, and reach N atdifferent rates.

This occurs in Cyt c where a misligation-depen-dent barrier can be inserted into the foldingsequence of some variable fraction of the popu-lation simply by adjusting the pH of the initiallyunfolded protein, even though folding is alwaysdone at the same low pH value.32 Molecules withthe misligation encounter the misfolding barrier,populate the N/C intermediate, and fold in a slowthree-state manner. The rest of the populationfolds in a fast two-state manner.4,32,72,73 Foldingappears to be heterogeneous. However, a greatdeal of data now support a single dominantpathway that can be variably interrupted by anoptionally inserted error repair barrier.7,25,33

Analogous examples in other proteins dependon the fractional mis-isomerization of prolineresidues,4,73 alternative disulphide bond forma-tion,4 alternative docking modes for differentfolding domains,4 and formation of non-nativehydrophobic clusters.66 These kinds of proba-bilistically inserted barriers have often been dis-cussed in terms of parallel pathways. The sameresults might be better described as single path-ways that are variably interrupted by optionallyinserted barriers.

Implications

The present experiments trap and characterize afolding intermediate during kinetic folding. Theintermediate resembles a partially constructednative state. The entire refolding population passesthrough it on the way to N. In agreement, a quan-tity of native state HX results indicate that Cyt cand some other proteins fold in a stepwise mannerthrough a small series of native-like intermediates,sequentially adding cooperative native-like elementsto progressively assemble the final native structure.These results point to a classical, stepwise, foldingpathway.

From all of these experiments some principleshave emerged. The unit folding steps are deter-mined by the intrinsically cooperative nature ofthe secondary structural elements of the nativestructure or pairs thereof, termed foldons. Thepathway sequence is determined by the arrange-ment of the foldons in the native protein; priornative-like structure guides and stabilizes thesubsequent formation of adjacent units (sequential


stabilization25,74). In this process, easily reversiblenon-native interactions may commonly serve tominimize the energy of transient forms.48 Lessrapidly reversible interactions may insert kineticbarriers that slow folding and cause intermediateaccumulation. These principles can be seen as anatural consequence of thoroughly establishedprotein structural behavior that seem unlikely todiffer fundamentally in other proteins.

However, many studies have been interpreteddifferently, in terms of multiple heterogeneousintermediates in multiple parallel pathways. In thecase of HX pulse labeling experiments, the presentwork shows that these conclusions have dependedon incorrect assumptions about HX behaviorand some other experimental problems. We notethat other studies often interpreted in terms ofheterogeneous pathways might be reconsideredin terms of optionally inserted error-dependentbarriers.

Materials and Methods

The purity of WT equine Cyt c (type VI; SigmaChemical Co.) was checked and further purified byreverse phase HPLC when necessary.75 The pH buffersused were of highest quality available from Sigma.2H2O was from Isotec or Aldrich. Deuterated GdmCl(d-GdmCl) was prepared by dissolving in 2H2O andvacuum evaporating three times.

Folding kinetics were measured at 10 8C usingstopped-flow Biologic SFM-400 apparatus (for 695 nmabsorbance and tryptophan fluorescence) or an Avivstopped-flow module attached to an AVIV CD spectro-meter (model 202). Buffers and pH values were thesame as those in HX pulse labeling experiments.

The following HX pulse labeling procedures wereused. Cyt c was unfolded in 4.2 M d-GdmCl in 10 mMphosphate, (p2Hread 7.5), 2H2O buffer. Refolding wasinitiated by diluting 18 times into the folding buffer(0.23 M GdmCl (pH 6), 10 mM Mes, H2O). Protein con-centration during folding was about 100 mM. Foldingwas continued for 100 ms. A high-pH pulse (pH 7.5–10)was applied for 50 ms. Pulse buffers were (after mixing):50 mM Hepes (pH 7.5), EPPS (pH 8), Bicine (pH 8.5),Ches (pH 9 and 9.5) and Caps (pH 10) in H2O. HXlabeling was stopped by mixing with the quenchbuffer (63 mM citrate, 35 mM ascorbate, H2O (pH 5.3)).Ascorbate was used to reduce Cyt c and pH 5.3 mini-mizes background labeling during the sample workup.

Final samples were concentrated (15 ml–0.5 ml; 4 8C;Amicon centriprep YM-10), then buffer-exchanged(Sephadex spin columns pre-equilibrated with NMRbuffer; 100 mM deuterated acetate, 12 mM ascorbate,2H2O, p2Hread 5), transferred to an NMR tube filled withargon, and frozen at 280 8C pending NMR analysis.Typical workup time was two hours. Duplicate sampleswere run at each pH value and the data were averaged.

Data analysis

The 2D COSY NMR spectra were recorded on a500 MHz Varian INOVA spectrometer in magnitudemode (24 scans of 2048 complex data points for 512increments; no water suppression, 20 8C). Spectra were

processed using Felix 2.3 (Biosym/MSI). NH–CaHcross-peak volumes for each amide were calculated toobtain the fractional H labeling, Hobs, as in equation (1):

Hobs ¼V=Vref

VC=VrefC

ð1Þ

The cross-peak volume after background subtraction,V, is normalized to the volume for a non-exchangingreference cross-peak (Vref, heme bridge 4). VC and VC

ref

are the analogous values in a native protein controlsample equilibrated (pH 7.5, 55 8C, three hours) to thesame H/2H ratio as during the pulse, representing 100%labeling. Hobs and the analogous terms consideredbelow vary from 0 to 1.

Hobs includes the labeling that occurs in all of the pro-tein forms (unfolded, intermediate, and native) at allstages of the experiment (before, during, and after thepulse and during the sample workup). We want toremove the extraneous contributions and extract theinformation, structural, equilibrium, and kinetic, that iscontained in the way that the intermediate alonebecomes labeled during the pulse. The two extraneouscontributions, Hbkgd and HN,pulse, were evaluated incontrol experiments.

A no-pulse control was used to determine thesummed background labeling at each amide during theentire experiment except during the pulse. In this controlexperiment, the stopped-flow mixing and sampleworkup were done exactly as before but with thelabeling pulse omitted (100 ms folding at pH 6, nopulse, quench to pH 5.3, workup at pH 5.3). The frac-tional labeling obtained as in equation (1) is Hbkgd,which is dominated by the labeling that occurs duringthe sample workup at sites that exchange significantlyin the native protein. Hobs for the total experiment canthen be expressed as in equation (2):

Hobs ¼ Hpulse þ ð1 2 HpulseÞHbkgd ð2Þ

Hpulse includes mainly the fractional labeling of theintermediate during the pulse (HI,pulse), which is theexperimental parameter of major interest. It also includesthe labeling in the 15% of native protein ( fN) presentduring the pulse (HN,pulse), as in equation (3):

Hpulse ¼ ð1 2 fNÞHI;pulse þ fNHN;pulse ð3Þ

To evaluate HN,pulse, pre-deuterated native protein wascarried through the entire pulse labeling procedure (in0.23 M d-GdmCl at each pH) to obtain HN,obs (likeequation (1)). A no-pulse control experiment was runwith the native protein to obtain HN,bkgd. These para-meters determine HN,pulse (like equation (2)). HN,pulse

values were close to zero for amides in the three helicesat all pulse pH values. Some labeling occurred at highpH for amides in the three omega loops but this correc-tion is multiplied by fN (equation (3)). (Note: this controlexperiment is identical with the kinetic native state HXexperiment described elsewhere,26 which measured thestability and kinetic parameters for unfolding of theloops in the native protein).

Equations (2) and (3) together with the independentlyevaluated parameters, Hbkgd, HN,pulse, and fN, connectthe measured Hobs to the desired (1 2 fN)HI,pulse. Theequations described below relate HI,pulse to opening andclosing (deprotection and reprotection) rates. Theserelationships allow experimental pulse labeling data for


Hobs versus pH to be fit in order to obtain amide-resolvedopening and closing rates in the intermediate. Accuracyimproves as the term HI,pulse becomes the dominantcontributor to Hobs and the other contributions decrease.This condition was promoted by the experimentalmanipulations described above.

HX equations

The Linderstrøm-Lang scheme for opening dependentHX was assumed:76

NHðclosedÞYkop

kcl

NHðopenÞ!kch

exchanged ð4Þ

As usual, kop and kcl are the opening (unfolding) andclosing (refolding) rates of the protecting structure,and kch is the unprotected chemical exchange rate calcu-lated for the ambient conditions from model peptidecalibrations.43,44

Analysis of the present HX pulse labeling datarequires the complete equations given by Hvidt42

(equations (5)–(7)), rather than the usual steady-staterate equation.76,77 The parameters tf and tp are the folding(0.1 second) and pulse (0.05 second) times, and[NH(open)]tf

is the population fraction in the open formduring the pulse, after folding for time tf. Equation (7)expresses [NH(open)]tf

in these terms (assuming noprotection in the unfolded state):

HI;pulse ¼ 1 2kch½NHðopenÞ�tf

2 l2

l1 2 l2

� �e2l1tp

2l1 2 kch½NHðopenÞ�tf

l1 2 l2

� �e2l2tp ð5Þ

where:

l1;2 ¼kop þ kcl þ kch ^

ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiðkop þ kcl þ kchÞ

2 2 4kopkch

q2

ð6Þ

½NHðopenÞ�tf¼

kop

kop þ kclþ

kcl

kop þ kcle2ðkopþkclÞtf ð7Þ

When kcl q kop, kch, and tf is sufficiently long, equation(5) reduces to the usual steady-state rate equation.When these conditions are not met, the additional firstbracketed term in equation (5) is necessary in order toaccount for the fraction of the population, NH(open),that is exposed when HX labeling is initiated. As inequation (7), this term is negligible when structure isvery stable (kcl q kop), in which case [NH(open)] andl2 , 0, and/or when the initially exposed sites arerapidly protected before labeling can occur (kcl q kch).This term is significant in the present work because thestability of the intermediate is low. Therefore, a signifi-cant fraction of the intermediate is unfolded whenlabeling is initiated and it can become labeled withouthaving to wait for opening (neither EX1 nor EX2).

These equations are written without any assumptionsabout the relative magnitudes of kop, kcl and kch and there-fore about the role of EX1 HX (kop, kcl p kch; kex ¼ kop) orEX2 HX (kcl q kch, kop; kex ¼ Kopkch; Kop ¼ kop/kcl). Formore discussion about the application of these equations,see Krishna et al.78

To implement these equations, the basic experimentaldata for each amide (Hobs versus pulse pH; as in Figure

4) were fit to equation (2), substituting equation (3) forHpulse and equations (5)–(7) for HI,pulse. The kch valueswere calculated from peptide models43,44 using the Excelspreadsheets†. The numerical parameters, Hbkgd andHN,pulse, for each amide were fixed at the values deter-mined in the control experiments. There are only twoindependent fitting parameters, kop and kcl. Figure 4 alsoshows these fitted curves after correction for the back-ground contributions (colored broken lines). For eachamide, the corrected plateau level (EX1 region) deter-mines the opening rate (kop); the offset from the expectedunprotected curve (black) at lower pH (EX2 region)largely gives the equilibrium stability (Kop). Data fittingused Sigma Plot 2001.

The residue-resolved unfolding free energy of protect-ing structure was then obtained using (8):

DGHX ¼ 2RT lnkop

kcl

� �ð8Þ

The errors in DGHX (Figure 5(a)) and kcl/kop (Figure3(b)) were calculated from the individual errors in kop

and kcl obtained from data fitting using standard errorpropagation formulas.79

Acknowledgements

This work was supported by research grantsfrom the NIH and the Mathers Foundation. Wethank Yawen Bai for sharing his manuscript48

before publication.

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Edited by P. Wright

(Received 19 June 2003; received in revised form 26 September 2003; accepted 26 September 2003)


intimate view of a kinetic protein folding intermediate...

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