Proc. Natl. Acad. Sci. USAVol. 92, pp. 10668-10672, November 1995Biochemistry

Submillisecond events in protein folding(barstar/relaxation kinetics/cold denaturation/protein engineering)

BENGT NOLTING, RALPH GOLBIK, AND ALAN R. FERSHT

Cambridge University Chemical Laboratory and Cambridge Centre for Protein Engineering, Medical Research Council Centre, Hills Road, Cambridge CB2 2QH,United Kingdom

Contributed by Alan R. Fersht, August 7, 1995

ABSTRACT The pathway of protein folding is now beinganalyzed at the resolution of individual residues by kineticmeasurements on suitably engineered mutants. The kineticmethods generally employed for studying folding are typicallylimited to the time range of 21 ms because the folding ofdenatured proteins is usually initiated by mixing them withbuffers that favor folding, and the dead time of rapid mixingexperiments is about a millisecond. We now show that thestudy of protein folding may be extended to the microsecondtime region by using temperature-jump measurements on thecold-unfolded state of a suitable protein. We are able to detectearly events in the folding of mutants of barstar, the polypep-tide inhibitor of barnase. A preliminary characterization ofthe fast phase from spectroscopic and 4D-value analysis indi-cates that it is a transition between two relatively solvent-exposed states with little consolidation of structure.

The pathway by which a particular protein folds will beresolved experimentally when the structures of all stable,metastable, and transition states adopted by the protein duringthe process have been characterized structurally and energet-ically. The only way to analyze the structures of transitionstates is by kinetics, and the details of their structure andenergetics can be gleaned from the kinetics of folding ofproteins whose structures have been carefully altered byprotein engineering (1-6). This protein engineering procedurehas been used to characterize transition states and interme-diates on a time scale of .1 ms (1-6), the time resolution ofthe stopped-flow and other rapid mixing techniques employedso far in these and most other studies (e.g., refs. 7-9). This istoo slow to allow detection of the early events that initiatefolding. To study faster events, kinetic techniques must beemployed that eliminate the time delays arising from rapidmixing techniques. Relaxation methods, by which a preexistingequilibrium between denatured and folded states is rapidlyperturbed by a change in physical or chemical conditions, maybe used to extend the time range. It is not easy, however, to findmethods that can readily cause a denatured state of a proteinto renature. Millisecond time resolution has been achieved byusing a repetitive pressure-perturbation method (10). Laserflash photolysis has been applied to dissociate CO fromCO-bound cytochrome c, with concomitant denaturation (11).However, rapid CO rebinding, binding of non-native ligands,and possibly aggregation prevented the complete transition tothe native state (11). A more generally applicable method forstudying fast events would be to raise rapidly the temperatureof a cold-denatured protein, since cold denaturation is acommon phenomenon of globular proteins under suitableexperimental conditions (12-15). Temperature jump (T-jump)by electrical discharge (16-20) and laser-induced heatingwould thus enable microsecond and nanosecond time resolu-tions, respectively.

The structure of barstar (21), the inhibitor of the ribonu-clease barnase from Bacillus amyloliquefaciens, has been solvedby NMR spectroscopy in solution (22), and the gross featuresof its folding pathway have been determined by rapid mixingmethods and equilibrium thermodynamics (23). The activebarstar mutant C40A/C82A/P27A (pseudo-wild-typebarstar), which was used in this study, contains no cysteineswhich may give rise to crosslinks in the denatured state andonly one proline residue.

METHODSProtein Expression and Purification. Site-directed mu-

tagenesis of the barstar mutant C40A/C82A (23) was per-formed with the Sculptor kit (Amersham). Mutant plasmids,expressed in the Escherichia coli strain TG2, were identified byDNA sequencing. Expression at 28°C and purification ofbarstar mutants were performed as described (22). Since theprotein is found in inclusion bodies, pellets of the lysed cellswere dissolved in 7 M urea and dialyzed against 50 mM Tris Clbuffer, pH 8/0.1 M NaCl before purification. Pseudo-wild-type barstar (C40A/C82A/P27A) and the mutants I5V, L16V,L34V, and L51V of the pseudo-wild type were prepared.

Equilibrium Studies. Circular dichroism (CD) spectra ofbarstar in 50 mM Tris Cl buffer pH 8/100 mM KCl with ureaconcentrations and temperatures as stated were measured witha Jasco (Easton, MD) model J-720 spectrometer using 2-nmspectral bandwidth. The protein concentrations and path-lengths of the cell were 50 ,uM and 0.02 cm for the far-UV CDspectra, 30 ,uM and 1 cm for the near-UV CD spectra, and 20,uM and 0.1 cm for the temperature dependence of the CDsignal at 222 nm. Barstar concentrations were determined byusing an extinction coefficient at 280 nm of 22,690 M-1 cm-1(23). For measuring the temperature dependence of the CDsignal at 222 nm, the sample was equilibrated at 0°C for 1 hr,then heated at 20°C hr-1 to 25°C and then heated at 50°C-hr-'to 80°C. Equilibrium fluorescence experiments were per-formed with a Hitachi model F-4500 fluorimeter with excita-tion at 280 nm, using a 0.4 cm x 1 cm cell containing 3 ,uMprotein, 100 mM KCl, 50 mM Tris Cl buffer (pH 8), and 2 Murea. Samples were preequilibrated for 1 hr for experiments at2°C, and for 20 min for experiments at other temperatures.

Kinetic Measurements. A T-jump apparatus from DIA-LOG (Dusseldorf, Germany) was equipped with a 0.7 cm x 0.7cm cell of 0.8-ml volume and a 200-W mercury-xenon lamp.Fluorescence excitation was at 280 nm, and a cutoff filter at 295nm was usually used for emission. Noise levels of <0.01% rootmean square of the signal were achieved at a 5-,ts responsetime. The buffer was degassed and the cell and sample wereequilibrated at 2°C for 1 hr before the T-jumps. Signals ofseveral T-jumps separated by 3-min equilibration time wereaccumulated. For double T-jump experiments, the cell anddegassed buffer were precooled to 2°C prior to the addition ofan appropriate amount of 100 ,uM barstar stock solution, which

Abbreviation: T-jump, temperature jump.

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leads to rapid cooling of the protein within seconds. Thesample was then incubated in the cell at 2°C for various timesbefore the T-jumps. The conditions were 5-10 ,uM protein, 100mM KCl, 50 mM Tris Cl buffer (pH 8), and urea as stated.T-jumps were usually from 2°C to 10°C. For the temperaturedependence of the rate constants, 4°C T-jumps were per-formed. Pressure shock waves that sometimes arise in thesample during rapid heating are absent for small samplevolumes at temperatures around 4°C because the coefficient ofthermal expansion of water at this temperature approacheszero. The experimental error is ±20% for the rate constants.Changing the protein concentration from 5 to 30 ,uM at 0 Murea does not change the rate constants and relative ampli-tudes of the fast transition by more than 10%. Data wereprocessed by KALEIDAGRAPH [Abelbeck Synergy software(Reading, PA)].

4)-Value Analysis. The effects of mutations on the rateconstants were measured as described (4). The parent protein,"pseudo-wild-type," used in this study had the two cysteines,C40 and C82, mutated to alanine to avoid complications, andone proline, P27, mutated to alanine to destabilize the proteinand to reduce the number of slowly interconverting species inthe unfolded state. Additional mutations were made to desta-bilize the protein further. Free energies of unfolding forpseudo-wild type and mutants in 0 M urea, AGU-F, weredetermined by urea unfolding, with monitoring of the CDsignal at 222 nm and use of equations 9 and 10 in ref. 24. Sincethe baseline at low urea concentration has only a small slope,if any, its slope was fixed at zero. The difference in the changeof the free energy on folding between pseudo-wild type (wt)and mutant (mt), AAGF-U, was calculated by using Eq. 1. Thechanges in free energy of activation of folding, AAGt-u,between pseudo-wild type and the mutants were determinedfrom the kinetic data by using Eq. 2.

AAGF-U = (AGU-F)Wt - (AGU-F)mt [1]

AAGt-u = RTln(kwt/kmt), [2]

where R and T are the gas constant and absolute temperature,and kwt and kmt are the folding rate constants for pseudo-wildtype and mutants. kwt and kmt were approximated by using theobserved rate constants under strongly folding conditions-that is, in the absence of denaturant (see Discussion). The Fvalue of folding, (FF, is the ratio of the free energy changedetermined from the kinetic data relative to that determinedfrom urea equilibrium unfolding (i.e., FDF = AAG*_u/AAGF-U)(4-6).

RESULTSEquilibrium Studies. The pseudo-wild-type barstar C40A/

C82A/P27A displays cold unfolding at moderately low tem-peratures (=0°C; see Fig. 1A). Far-UV CD spectra in theabsence of urea are similar to those of wild-type barstar (Fig.1B). The amplitudes in the near-UV CD signal at 10°C (Fig.1C) are somewhat smaller and there are small differences inthe fine structure relative to wild-type barstar. This mayindicate slight differences in the dynamic behavior of themolecules. The equilibrium denaturation curves on addition ofurea are identical when monitored by fluorescence, whichfollows tertiary interactions, and far-UV CD at 222 nm, whichfollows the secondary structure (Fig. 1D). Both heat- andcold-induced denaturation are highly cooperative, in contrastto the behavior of many strongly destabilized proteins (e.g.,refs. 25-27). The m value (i.e., -a[AGU3F]/A[D], where AGU-Fis the free energy of unfolding at various concentrations ofdenaturant, [D]) of urea-induced unfolding is as high as thatfor wild-type barstar, at 1.2-1.3 kcal liter mol-2. This suggeststhat the population of any intermediates at equilibrium is small

-1C

,Eo -2C0E-J -3C

< -4C

E0

-

0

)QA

3M

)0 ZOxOM0 20 40 60 80Temperature (0C)

0

-2

.3

-4 N N . .1260 280 300

Wavelength (nm)

11

0.81

- 0.6004

U-

0.21

O r....e....(....M0 1 2 3 4 5

[urea] (M)

FIG. 1. Equilibrium studies of pseudo-wild-type barstar (C40A/C82A/P27A) in 100 mM KCl/50 mM Tris Cl, pH 8, with ureaconcentration as indicated (0, 2, 3, or 6 M). (A) Cold and heatdenaturation measured by CD at 222 nm. (B) Far-UV CD spectra:solid line, 25°C; dashed line, 2°C. (C) Near-UV CD spectra at 20°C.(D) Equilibrium unfolding at 2°C and 10°C: A and o, CD at 222 nm;A, fluorescence at 330 nm.

and so justifies the calculation of the free energy of unfoldingby using the equation for a two-state transition (see Methods).The free energy of unfolding is 3 kcal-mol-1 at 10°C. Itdecreases by 0.6 kcal mol-1 on lowering of the temperature to20C.

Kinetic Studies. There is a major folding transition at 32 s',which was monitored by stopped-flow fluorescence measure-ments of the refolding of C40A/C82A barstar at 25°C (23). Afast kinetic event with a rate constant of about 3100 s-1 wasresolved during the refolding of cold-unfolded pseudo-wild-type barstar on a rapid T-jump from 2°C to 10°C in the absenceof denaturant, in addition to the main folding transition at 8s-1, which is also clearly resolved (Fig. 2A). The increase ofrate constant with temperature is larger for the main transi-tion, suggesting a larger activation enthalpy (Fig. 2B). The fastphase occurs also in wild-type barstar but is technically moredifficult to resolve because of the lower temperature requiredfor cold denaturation. The presence of barnase stabilizesbarstar and so was found to suppress cold unfolding and, thus,these refolding phases. The rate constants for the refolding ofthe mutants ISV, L16V, L34V, and L51V are about 2500 s-1,2000 s-1, 2500 s-1, and 3300 s-1, respectively, at 10°C in theabsence of urea, compared with 3100 s-1 for the parent.

Analysis of Kinetics. For a three state transition, composedof a slow and much faster transition,

klf k2f

U I F, [3]klu k2u

where U, I, and F designate the unfolded, intermediate, andfolded state, respectively, the observed slow rate constant,k2obs, is given approximately by

k2obs = k2f{K2u + 1/(1 + K1u)} [4]

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A

rK-

0L IA0 2 4 400 800

Time (ms)

1 0000r B1000 I

I--(a)

0Ies100-

10

1 I5 10 15Temperature (°C)

10000 DIi; ,

1000 F,CO)'co 100

n0

104

O..

0 50 100 150 200Time (min)

1 2[urea] (M)

FIG. 2. Kinetic studies of folding of pseudo-wild-type barstar in 100mM KCl/50 mM Tris Cl, pH 8, at 10°C unless stated otherwise. (A)Transients for the folding transitions in 0 M urea. (B) Temperaturedependence of the observed rate constants in 0 M urea. (C) Ampli-tudes of the kinetic events as a function of the time of preincubationat 2°C in 0 M urea prior to the T-jump (0, fast phase; *, slow phase).(D) Urea dependence of the observed rate constants (0, fast phase;*, slow phase) and relative amplitudes (A, fast phase; A, slow phase).Note that the observed rate constant for the fast phase increases withurea at higher urea concentrations because of a contribution from theunfolding rate, as discussed in the text in Analysis of Kinetics. Theamplitude of the fast phase correspondingly increases more slowly withurea concentration.

K2U= k2u/k2f [5]

Ku= klu/klf, [6]

where klf and klu are the rate constants of the fast foldingtransition, and k2f and k2u are the rate constants of the slowfolding transition (28). In a manner analogous to the exactsolution for a two-state transition, the observed fast rateconstant, klobs, is approximately the sum of folding andunfolding rate constants (28):

klobs kif + klu = kif (1 + Klu). [7]

At high urea concentrations, the observed rate constantincreases with urea concentration because of a contributionfrom the unfolding rate. The position of the minimum in theplot of klobs against concentration of denaturant, [D]min,reflects roughly the magnitude of the change in free energy ofthe observed transition. For the fast transition, [Dimin is atabout 0.5-1 M urea (Fig. 2D), suggesting a small free energychange in this stage. The minimum of the observed rate of themain transition is located at a significantly higher value of ureaconcentration, indicating a larger energy change.The peptidyl-proline 48 bond is in a cis conformation in the

native state (22), whereas the major population in a denaturedprotein is in the trans conformation (29). In order to allow theequilibration of cis and trans peptidyl-proline bonds in thedenatured state, we performed double T-jump experiments:the pseudo-wild-type barstar was rapidly cooled and incubatedat low temperature for various times before T-jump from 2°Cto 10°C for measurement of the kinetic events. The amplitudes

of the relaxations change only slightly with the time of incu-bation at 2°C in 0M urea at pH 8 and are still high at zero time(Fig. 2C). Thus, both folding transitions occur irrespective ofwhether the proline at position 48 is in the cis or the transconformation.The change in amplitudes of the two transitions during

cis-trans isomerization of the peptidyl-proline 48 bond (Fig.2C) can be explained as follows. In T-jump experiments, theamplitude in the observed kinetics reflects the change inoccupancy with temperature of the state preceding the tran-sition relative to the state following the transition. The relativeamplitude is maximal at about the midpoint of a two-statetransition. For conditions of high population of the folded stateand low population of the preceding state, the amplitudeincreases with increasing occupancy of the preceding state andthus with increasing stability of the preceding state. Also, fora multiple-state transition, the equilibrium populations de-pend on the stability of the unfolded and intermediate statesrelative to the folded state, while the transient populationsdepend also on the populations of the preceding states and thefree energies of the transition states. Since there is only a smallperturbation of the equilibrium in T-jump experiments, theamplitude of a transition reflects, to a first approximation, theequilibrium populations and population transfer with otherstates by faster transitions. Thus, to a first approximation, theincrease of amplitude with incubation time at low temperaturefor the fast phase (Fig. 2C) reflects a higher stability of thepreceding species containing trans-peptidyl-proline 48 (Ut orpreceding intermediate) relative to the cis species. The de-crease of the amplitude of the main transition with incubationtime may be caused by a smaller free energy change for thetrans species than for the cis species. However, the amplitudeof this transition is also affected by population transfer be-tween the fast exchanging species. A further, but unlikely,explanation is that the fluorescence signal of the trans species,Ft, differs from that of the cis species, Fc. However, nodifference between Ft and Fc has been found in C40A/C82Abarstar (23).

DISCUSSIONThe mutant barstar C40A/C82A/P27A is readily denatured bycooling. We observed a rapid phase of 3100 s-1 on T-jumpinga cold-denatured solution from 2°C to 10°C in about 10 ,us.This was followed by the major folding transition, at 8 s-1,which was detected previously from conventional stopped-flow studies on the millisecond time scale. The changes influorescence are very small, but they may be measured withprecision and are not the results of artifacts, since they vary

75~~~~~~~~~~C

CD 50i- CD

0

FL 25

300 350 400 450 300 350 400 450Wavelength (nm) Wavelength (nm)

FIG. 3. Fluorescence spectra of pseudo-wild-type barstar in 100mM KCI/50 mM Tris Cl, pH 8. (A) Emission spectra in 2 M urea from2°C to 70°C, as indicated. (B) "Kinetic difference spectra" in 0 M urea,determined from the amplitudes of the changes in fluorescence duringthe transitions at 10°C (0, fast phase; *, slow phase). The same

arbitrary scale was used for A and B.

1-0.3

CDC)C

'. 0.20

a)0C

C 0.10CD)

o

.-o

o-0)0-

U)

0)Co

(

U)C0CD)U)0

Ei

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Table 1. Kinetic and equilibrium analyses of pseudo-wild-type (pseudo-wt) barstar and its mutants

AGU-F, AAGF-U, AA&Gt-U,main, AAGt_U,fast,Protein kcal-mol-I kcal-mol-1 kcal-mol-1 kcal-mol-1 DF,main DF,fast

Pseudo-wt 3.015V 2.0 1.0 0.5 0.1 0.5-0.7 0-0.2L16V 1.9 1.1 0.9 0.2 0.7-0.9 0.1-0.3L34V 1.9 1.1 0.4 0.1 0.3-0.5 0-0.2L51V 2.4 0.6 0.2 0 0.2-0.5 0-0.2

The proteins were in the absence of denaturants at 10°C. AAGF-U = change in the free energy on foldingbetween pseudo-wild type and mutant (AAGF-U = (AGU-F)wt - (AGU-F)mt); AAG*-u = change in freeenergy of activation between pseudo-wild type and mutant (AAG*-u = RTln(kwt/kmt)), for main and fasttransitions; (D values of folding for the main and fast transitions are (DF,main and DF,fast, respectively.

from mutant to mutant and disappear when cold unfolding issuppressed.

Structural Events During the Two Transitions. We havemade preliminary investigations of the nature of the eventsthat occur during the two processes. There are two simple,overall probes: the dependence of the rate constant on theconcentration of urea, and the spectral changes. At low ureaconcentrations, the rate constant for the main folding transi-tion decreases significantly with increasing urea concentration(Fig. 2D). This is diagnostic of a significant decrease in thesolvent-exposed surface area of the protein (30), since thesensitivity of the reaction to denaturant depends on the amountof surface area buried. There is only a small dependence,however, of the rate of the fast transition with urea at lowconcentrations, and then an increase at higher concentrations.This behavior is complicated because of the complex kinetics,whereby the rate constant is dominated by that for unfoldingat higher urea concentrations (see Results), but is indicative ofonly a modest change in the time-averaged solvent-exposedsurface area. It is indicated from the kinetic data that the mvalue of the fast transition is only roughly half of that of themain transition. The amplitudes of both transitions peakaround 1.5 M urea (Fig. 2D). The broader distribution for thefast transition may be further indication of a smaller m valuerelative to the main transition. One important reason for thelow value of urea concentration at the peak positions relativeto that of the urea concentration at the 50% transition inequilibrium experiments (Fig. 1D) is a lower stability of thefolded state with trans conformation of the peptidyl-proline 48bond relative to that with the cis conformation.There is evidence from an analysis of spectral changes for

the burial of hydrophobic side chains in the slow transition.The fluorescence spectrum of pseudo-wild-type barstar showsa characteristic blue shift upon cold and heat renaturation(Fig. 3A), indicative of the burial of the side chains oftryptophan on folding. The significant increase in fluorescenceaccompanied by a blue shift during the slow kinetic event (Fig.3B) indicates a decrease in solvent exposure of the aromaticside chains. In contrast, the shape of the kinetic differencespectrum of the fast transition, derived from monitoring aseries of kinetic measurements between 300 and 350 nm,resembles that of the equilibrium spectrum of the cold un-folded protein, although there is a small increase in amplitude.At longer wavelengths, however, the amplitude of the kineticspectrum is negative, suggestive of a small blue shift. Theseobservations suggest a small increase in hydrophobic burial ofaromatic side chains for the fast phase, too. The position of thewavelength maximum in the kinetic spectrum, however, indi-cates that the fast transition occurs between highly solvent-exposed states. Water can have access to aromatic side chainsin the early-formed structure over the few-nanoseconds timeaverage.¢-Value Analysis of a Three-State Transition. Specific

probes of structural changes come from monitoring the effectsof mutations on the rate constants, as described for barnase(4-6) and the barley chymotrypsin inhibitor 2 (3). We have

introduced some additional mutations that reduce the hydro-phobicity of side chains; ISV, L16V, L34V, and L51V. The (Fvalues of folding, (F (1-6), for the main transition of thosemutants are 0.2-0.9 (see Table 1; a OF value of 0 means thatno structure was formed at the site of mutation; a (DF value of1 indicates complete formation). Thus, many stabilizing inter-actions are formed during the main folding transition. Con-sistent with this picture, the OF values for the fast phase ofthose mutants are -0.3, suggesting little formation of stablehydrophobic interactions. Considering that the value of K1u forthe mutant is equal to or larger than that for the pseudo-wildtype, according to Eqs. 4-7, the (FF-values may be underesti-mated for the fast transition and overestimated for the maintransition by using the observed rate constant as the foldingrate constant in the absence of denaturants. The small devi-ation from zero of the (F value for the fast phase of L16V issignificant and so shows that there is formation of structure atthis position in the fast phase.The events monitored during refolding of the cold-unfolded

state of barstar clearly include the consolidation of hydropho-bic interactions, including those of the aromatic side chains. Asignificant amount of structural consolidation occurs duringthe main transition state. The fast folding phase, however,involves only a small change in free energy, modest structuralconsolidation in the transition state, and a high degree ofexposure to solvent of the species involved. The presentexperiments show that it is, in principle, possible to map out indetail the structures of the transition states, and the interme-diates, by studying the folding kinetics of a series of mutantsby T-jump, as performed for barnase (5) and chymotrypsininhibitor 2 (3, 6) on slower time scales using stopped flow.

Note Added in Proof. Huang and Oas (31) have reported monitoringthe submillisecond folding of A repressor by NMR line broadeninganalysis.

B.N. is supported by a European Union Human Capital andMobility fellowship.

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