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380 nature structural biology • volume 6 number 4 • april 1999

The information required for a protein to fold to its native struc-ture is contained within its amino acid sequence. How this struc-ture is attained efficiently given the countless possibleconformations that a protein in principle can adopt is one of themost complex as well as fascinating aspects of structural biology.Theoretical simulations and energy landscape models suggestthat progressive formation of native-like interactions is a majordriving force toward the final native state1,2. The extent to whichthere is a well-defined hierarchy in the formation of native con-tacts, however, and the relative importance for efficient foldingof the different types of interactions, for example, local and non-local contacts, remains to be established.

According to these conceptual models, the folding of a proteininvolves a reduction in the configurational entropy of thepolypeptide chain as stabilizing interactions within the develop-ing fold are formed2. Thus, the folding of a protein is expected tobe more efficient if the conformational space accessible to thepolypeptide chain during folding is more restricted, providedthat native-like conformations are favored over nonnative ones.However, the formation of excessively stabilizing interactionswithin a polypeptide chain may generate stable intermediateconformations that represent local minima on the energy sur-face. These can act as kinetic traps that prevent a protein fromproceeding efficiently to the native state. There has been muchdiscussion about the role of intermediates in protein folding3,4.Studies with ubiquitin have suggested that an intermediate statedetected during the folding of this protein aids the search fornative interactions5. On the other hand, a stable intermediatethat forms during the folding of CheY has been shown to be mis-folded and to require unfolding before the native conformationcan be formed6. Moreover, proteins with the most rapid foldingrates have been shown to fold cooperatively without the accumu-lation of intermediates7–12.

Among the proteins that have been shown to fold through atwo-state mechanism is acylphosphatase. This protein has 98residues and exists as two isoenzymes sharing about 50%sequence identity that are known as muscle and common-typeacylphosphatase (muscle and CT AcP)13. In both forms the

structure consists of two parallel α-helices packed against a five-stranded antiparallel β-sheet14–16. Studies using NMR and opticalspectroscopy have shown that both the proteins fold in a highlycooperative manner without significant accumulation of inter-mediates17,18. Both proteins contain two tryptophan residues atpositions 38 and 64 that allow the folding reaction to be easilymonitored by following changes of intrinsic fluorescence uponfolding.

In this study we investigate the effect of trifluoroethanol (TFE)and hexafluoroisopropanol (HFIP) on the folding of humanmuscle and CT AcP. Both TFE and HFIP are known to stabilizesecondary structure, particularly α-helices and β-hairpins, whileweakening hydrophobic interactions within a polypeptidechain19–21. As the latter can form from distant regions of thepolypeptide sequence, these solvents constitute valuable probesof the importance of short-range backbone hydrogen bonds rel-ative to long-range interactions between side-chain residues dur-ing the folding reaction. Several reports have shown that despiteits denaturing potential, small quantities of TFE can accelerateprotein folding22,23, although the origin of this effect has not beenexplored in any detail. Here we show how the addition of alco-hols can increase dramatically the folding rate of muscle and CTAcP and convert an apparently two-state folding into a processcharacterized by the accumulation of partially structuredspecies. The importance of local hydrogen bonding relative toother types of interactions in directing the folding process is discussed in light of the present findings.

TFE and HFIP promote α-helical structure in denatured AcPWhen CT AcP is titrated against TFE, monitored by far-UV cir-cular dichroism (CD), a considerable increase of ellipticityoccurs following a single highly cooperative transition (Fig. 1a).A single transition is also revealed when the TFE denaturation isfollowed by intrinsic fluorescence emission, where an abruptdecrease of fluorescence is observed at the same TFE concentra-tions as the CD changes are seen.

Far-UV CD spectra of CT AcP acquired under native and vari-ous denaturing conditions (Fig. 1b) are very similar to those of

Acceleration of the folding of acylphosphataseby stabilization of local secondary structureFabrizio Chiti1, Niccolo’ Taddei2, Paul Webster1, Daizo Hamada1, Tania Fiaschi2, Giampietro Ramponi2and Christopher M. Dobson1

The addition of trifluoroethanol or hexafluoroisopropanol converts the apparent two-state folding ofacylphosphatase, a small α/β protein, into a multistate mechanism where secondary structure accumulatessignificantly in the denatured state before folding to the native state. This results in a marked acceleration offolding as revealed by following the intrinsic fluorescence and circular dichroism changes upon folding. Thefolding rate is at a maximum when the secondary-structure content of the denatured state corresponds to that ofthe native state, while further stabilization of secondary structure decreases the folding rate. These findingsindicate that stabilization of intermediate structure can either enhance or retard folding depending on its natureand content of native-like interactions.

1Oxford Centre for Molecular Sciences, New Chemistry Laboratory, University of Oxford, South Parks Road, Oxford OX1 3QT, UK. 2Dipartimento di ScienzeBiochimiche, Universita’ degli Studi di Firenze, Viale Morgagni 50, 50134 Firenze, Italy.

Correspondence should be addressed to C.M.D. email: [email protected]

© 1999 Nature America Inc. • http://structbio.nature.com©

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nature structural biology • volume 6 number 4 • april 1999 381

the muscle protein. The CD spectra acquired for native muscleand CT AcP were processed to yield estimates of their secondary-structure content. Table 1 reports the results of such analysisshowing close agreement with the data obtained from X-raycrystallography and NMR spectroscopy. The spectra in the pres-ence of 10% (v/v) TFE were also analyzed, giving very similarresults and indicating that the overall fold of the protein is main-tained at low TFE concentrations before the unfolding transi-tion. The close agreement with the NMR and X-ray dataencouraged us to extend the analysis to the CD spectra of muscleand CT AcP denatured in 40% (v/v) TFE and 40% (v/v) HFIP. In40% TFE the α-helical content is estimated to be ~50%, com-pared to 23% for the native fold, and the remaining structureappears substantially disordered (Table 1). A difference spec-trum of CT AcP was obtained by subtracting from the experi-mentally measured CD spectrum in 40% TFE the spectrum of apure α-helix scaled by 0.51, the computationally determinedfraction of α-helix (Fig. 1b)24. The single negative band around197 nm that is evident in the resulting spectrum is indicative oflargely disordered structure, suggesting that little, if any, β-sheetstructure is present under these conditions. The CD analysis ofthe denatured protein in 40% HFIP reveals an even higher α-helical content, ~80%, for CT AcP. For AcP, therefore, theaddition of high concentrations of TFE or HFIP leads to a dena-tured protein characterized by the presence of α-helices that are,at least in part, nonnative in character.

These observations for AcP are generally in accord with theresults obtained with other proteins. In most cases the denatura-tion of proteins in TFE is accompanied by a global exposure of thenonpolar groups and by a concomitant formation of α-helicalstructure, the extent of which is often larger than that present inthe native state19–21,25. In lysozyme and α-lactalbumin the loca-tion of the helices has been identified by NMR, and in both casesthe α-helical structure stabilized by alcohol cosolvents has beenfound to involve portions that are α-helical in the native fold25,26.By employing a statistical analysis with a large number of pro-teins, Shiraki et al.21 showed that there exists a significant corre-lation between the α-helical structure present in theTFE-denatured state of a protein and the α-helical propensity ofthe sequence calculated by secondary-structure predictions. Wetherefore performed secondary-structure prediction analysis onboth CT and muscle AcP with the two algorithms AGADIR27,28

and PHD29,30. This analysis shows that the two polypeptide seg-ments that are α-helical in the native state have also the highestpropensity to form α-helical structure. It is therefore reasonableto assume that the large amount of α-helix present in the TFE-and HFIP-denatured states includes native α-helices.

TFE and HFIP accelerate the folding of AcPFolding of CT AcP is dramatically accelerated by TFE (Fig. 2a).Interestingly, the accelerating effect of this alcohol is not linearlycorrelated with its concentration but passes through a maxi-mum. At pH 5.5 the maximum folding rate, a factor of 20 higherthan in the absence of TFE, was found at ~10% (v/v) TFE.Further addition of TFE causes a deceleration of folding. A sec-ond slow phase of low amplitude (13%) is detected in the foldingof AcP and has been shown to arise from cis-trans proline iso-merism17, 18. This second phase is only very slightly accelerated byTFE, an effect persisting even at high alcohol concentrations(Fig. 2a), and expected for a reaction of this type31. This minorphase is not considered further in this paper. A dramatic acceler-ation of CT AcP folding was also observed as a consequence ofthe addition of HFIP (Fig. 2b). At similar concentrations thiseffect is much more marked than with TFE, but as with TFE amarked deceleration of the folding rate is observed at the higherconcentrations of HFIP; the optimum rate was found at 3.7%(v/v) HFIP and represents an acceleration by a factor of 30.

The folding of muscle AcP is significantly slower than that ofCT AcP. However, the behavior of muscle AcP following theaddition of fluorinated alcohols was found to be very similar tothat of CT AcP with maximum folding rates at 9.8% (v/v) TFEand 3.7% (v/v) HFIP, respectively (Fig. 2 a and b). Despite thesubstantial acceleration of the folding rate brought about by

Fig. 1 Equilibrium conformational changes induced by fluoroalcohols.a, TFE concentration dependence of the 222 nm mean residue ellipticityof CT AcP in 50 mM acetate buffer, pH 5.5. The mean residue ellipticity ofthe folded state and the position of the unfolding transition do notchange significantly in the range 0–2 M urea. (inset) Change of the 340nm fluorescence emission (p,P) and of the 222 nm mean residue elliptic-ity (l,L ) of TFE-denatured muscle (filled symbols) and CT (empty sym-bols) AcP upon the addition of urea. The changes are expressed aspercentiles of the initial signals in the absence of urea. The lines throughthe data represent the linear fits for muscle (dashed lines) and CT (solidlines) AcP. b, Far-UV CD spectra of CT AcP in the absence of TFE, in thepresence of 10% (v/v) TFE, 40% TFE, 40% HFIP and 8 M urea. The differ-ence spectrum was calculated as explained in the text. All spectra wererecorded at 25 oC in 5 mM phosphate buffer, pH 7.0. The CD spectra ofmuscle AcP acquired under the five conditions reported here were verysimilar to those of CT AcP shown in the figure.

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HFIP when present at very low concentrations, the maximumfolding rate induced by HFIP is similar to that caused by TFE athigher concentrations, and this applies to both muscle and CTAcP. The profile of the folding rate in the presence of HFIP dif-fers from that in the presence of TFE by a simple scaling factor ofthe alcohol concentration. From equilibrium experiments it hasbeen estimated that HFIP is 3.65 times more effective than TFEin causing conformational changes to proteins32. When such adifference is considered, the HFIP and TFE concentrationdependencies of the folding rate are very similar, an observationvalid for both isoforms of AcP. Therefore, the action of TFE isnot specific to this solvent and may indeed be emulated by othercompounds like HFIP that promote the same kind of structuralchanges within polypeptide chains.

Intermediate structure forms during folding of AcP in TFEThe folding of CT AcP was also studied in aqueous solutions inwhich TFE and urea were present in varying proportions. Inthese experiments, phosphate buffer at pH 7.0 was used to stabi-lize the native state of the protein and to enable study of the fold-ing reaction over wide ranges of urea and TFE concentrations.Inorganic phosphate is known to bind to the active site of AcP,causing a dramatic deceleration of the unfolding reaction with-out affecting the folding rate of the protein23. Addition of phos-phate therefore stabilizes the protein without altering the foldingreaction that is the main focus of our analysis. Preliminary equi-librium unfolding curves acquired in phosphate buffer show thatCT AcP is still fully folded in solutions containing 3 M urea and18% (v/v) TFE.

Plots of the natural logarithm of the folding rate constant versus urea concentration were determined for five different TFEconcentrations ranging from 3.6% to 18.2% (v/v) (Fig. 3). Thisanalysis was carried out by using fluorescence as a probe to mon-itor the folding reaction. In the absence of TFE this relationshipwas found to be linear up to zero urea concentration, an observa-tion indicating that intermediates do not accumulate significant-ly during folding17,18. The present data show that while thistwo-state behavior is maintained at 3.6% TFE, further increasein TFE concentration results in the appearance of curvature inthe plots at low urea concentrations. This curvature extends tohigher urea concentrations when the concentration of TFE isincreased and a linear region is no longer evident at the highest

TFE concentration tested. Curvature of this type, also known asrollover, is indicative of either the compaction of the denaturedstate or the formation of intermediates at the low denaturantconcentrations4,33. By contrast, the natural logarithm of theunfolding rate constant was found to be linearly related to ureaconcentration in the presence of TFE, suggesting that the addi-tion of TFE does not cause any significant change in the unfold-ing behavior (data not shown).

To explore further the effect of TFE on the folding of AcP, thefolding reaction of CT AcP was followed by far-UV CD. As in theexperiments described already (Fig. 2), the protein was initiallydenatured in 8 M urea and refolding was initiated by dilutioninto solutions at pH 5.5 containing various concentrations ofTFE. The time course of the mean residue ellipticity change at222 nm during refolding (Fig. 4) reveals that in the absence ofTFE almost all the ellipticity is recovered during a relatively slowphase, as observed previously for muscle AcP17. At 7.2% TFE thisfolding event is preceded by a burst phase occurring within thedead time of the instrument, during which the acquisition of~50% of the final ellipticity is observed. At 10.9% TFE no changein ellipticity is observed in the experiment. Simultaneous detec-tion of fluorescence, however, revealed a normal folding reactionon a time scale well within the limits of the instrument. Hence,the unchanged CD signal during the folding reaction is a conse-quence of the fact that the denatured state has the same elliptici-ty as the final folded state. At 14.5% TFE the species formed inthe burst phase has an ellipticity even higher than that of thenative state. Subsequently, as the protein folds, the CD signaldecreases to the value of the native state. The folding rate con-stants obtained for the different TFE concentrations (Fig. 2a, X)are all in close agreement with those obtained from the fluores-cence traces acquired under corresponding conditions.

Conversion from two-state to multistate folding by TFEIn the absence of alcohols the denatured state of AcP in strongrefolding conditions and before initiation of folding is a highlyunstructured conformation without any significant nonrandomresidual structure17. Addition of TFE or HFIP changes the aver-age structure of such a state to an ensemble of conformationswith substantial secondary structure, in particular α-helical, asrevealed by the high 222 nm ellipticity. The acquisition of suchstructure is complete within the dead time of the stopped-flow

Fig. 2 TFE and HFIP concen-tration dependence of thenatural logarithm of theobserved folding rate con-stant for muscle (G , g ) andCT (P,p) AcP. The final refold-ing conditions were 0.72 Murea, 50 mM acetate buffer,pH 5.5, 25 °C. The solid linesthrough the symbols are thebest fit of the experimentaldata to equation (5). In thisanalysis the dependence ofthe observed rate constant offolding on urea concentra-tion has not been considered.a, TFE concentration depen-dence. Also shown is the TFEconcentration dependence ofthe second, slower phasedetected during folding of CT AcP and corresponding to a proline isomerization rate-limited event (r). In this case the data were fitted to a linear func-tion. b, HFIP concentration dependence . In each case the two folding rate constants were calculated by fitting the fluorescence trace acquired duringfolding to a double exponential function. The analysis for CT AcP in TFE was also carried out by CD (X).

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apparatus (<5 ms) and is thus extremely rapid compared to thefolding reaction, which occurs on the time scale of seconds.Despite the presence of secondary structure, this denatured statedoes not possess the characteristics of a compact state or moltenglobule. Such species, which form during the folding of manyproteins, are characterized by a rudimentary hydrophobic coreand a native-like fold34. A hydrophobic collapse does not occurfor AcP in aqueous solutions17, and TFE is expected to destabi-lize further any hydrophobic contacts in the denatured state.Moreover, an increase of fluorescence of 8-anilino-1-naphthalenesulfonic acid (ANS) was not detected during thefolding of AcP at low TFE concentrations. Hence, it is likely thatunder these conditions the denatured state of AcP consists of anensemble of noncollapsed conformations, though containing asignificant amount of secondary structure.

Several observations indicate that the α-helices stabilized inTFE or under other partially denaturing conditions form gradu-ally and independently of each other. The α-helical content ofAcP in 40% TFE, estimated to be ~50% from the analysis of theCD spectra, is reduced on addition of denaturants such as ureaor by increasing the temperature. This transition does not, how-ever, show any evidence for cooperativity (Fig. 1a, inset). Inaddition, after the main equilibrium transition, the ellipticitycontinues to increase (Fig. 1a), indicating further accumulationof secondary structure upon addition of TFE. Similarly, addi-tional α-helical structure can be generated by adding HFIP (Fig.1b). NMR studies have suggested that the α-helices of lysozymedenatured in an aqueous solution of TFE largely exist in theform of independent units that do not interact with one anoth-er26. Moreover, the α-helices present in the equilibrium moltenglobule of human α-lactalbumin can be disrupted independent-ly of one another upon individual alanine-to-proline substitu-tions35. All these observations suggest that the denatured stateformed in the presence of TFE before folding cannot be regard-ed as a single intermediate conformation. More likely it involvesan ensemble of conformations that differ in the number andextension of the α-helices and other elements of secondarystructure and the distribution of which is governed by the con-ditions and the alcohol concentration used.

Importance of local secondary structure for AcP foldingThe accelerating effect of fluorinated alcohols on the folding ofAcP is striking, especially when one considers the denaturing

potential of these compounds. Because TFE and HFIP weakenthe hydrophobic interactions within a polypeptide chain, theytherefore might be expected to hinder protein folding by destabi-lizing the compact transition-state ensemble of structures23.

Fig. 4 Folding of CT AcP monitored by 222 nm far-UV CD. The finalrefolding conditions were 0.72 M urea, 50 mM acetate buffer, pH 5.5, 25oC and various TFE concentrations as indicated. The signal-to-noise ratiodoes not allow the determination of the rate constant and amplitude ofthe slow phase arising from proline isomerization. The rate constant forthe major phase of folding was, however, determined, and the values atdifferent TFE concentrations are reported in Fig. 2.

Fig. 3 Folding rate con-stant of CT AcP, expressedas its natural logarithm,versus urea concentration.The plots were obtained atvarious TFE concentrationsranging from 3.6% to18.2% (v/v) by using fluo-rescence as an opticalprobe to monitor the fold-ing reaction. In all cases thefinal refolding conditionswere 50 mM phosphatebuffer, pH 7.0, 25 oC. Theplots are characterized byregions, at high urea con-centration, where the dataare well fitted to linearfunctions. This linearity isbroken by a curvature(rollover) at low urea concentration that is absent at the lowest TFE concentration studied. The data in the linear regions where a curvature is not evi-dent were linearly fitted, and the solid lines represent the best fitted straight lines.

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Other aspects of the protein–alcohol interactions have to beinvoked to explain the TFE- and HFIP-induced acceleration ofAcP folding. Among them is the effect of alcohols on the sec-ondary structure of proteins, particularly α-helices, β-turnsand β-hairpins. For both the isoenzymes, the marked accelera-tion of folding occurs concomitantly with the rapid formationof local hydrogen bonding in the denatured state at lowTFE/HFIP concentrations. The productive action of TFE andHFIP could originate from the ability of these alcohols to favorthe formation of native-like secondary structure. The stabiliza-tion of the denatured state through the formation of structuredoes not increase the energetic barrier between the denaturedstate and the transition-state if such structure is also present inthe transition state ensemble. On the contrary, the energeticbarrier is expected to decrease in such circumstances as a resultof the reduction of the entropic component of the energeticbarrier. Other possible explanations, such as a nonspecific vis-cosity effect of alcohols on the folding rate or the ability of fluo-rinated alcohols to allow the reorganization of the interactionsin the transition state, have also been considered. These non-specific effects, however, would be expected to result in theacceleration of folding for all proteins upon the addition ofalcohols. Recent findings that the folding of β-lactoglobulin, apredominantly β-sheet protein, and Fyn-SH3 domain, an all- βprotein, are little affected by the addition of low concentrationsof TFE (ref. 36 and D. Hamada et al., manuscript in prepara-tion) are not consistent with these suggestions. Rather, theyindicate that the acceleration observed for AcP is likely to arisepredominantly from the ability of alcohols to promote the for-mation of native-like secondary structure in the protein. Itcould be argued that the rollover found in plots of ln kobs versusurea concentration, originating from the compaction of the

denatured state due to the presence of α-helical structure, indi-cates that the formation of local hydrogen bonding slows downAcP folding. Nevertheless, a rollover of this kind is likely tooriginate from the fact that native-like structure, scarcely pop-ulated under weak folding conditions, approaches saturation atlow denaturant concentrations4.

As we have seen, however, the folding rate of AcP does notincrease monotonously with alcohol concentration. At concen-trations of TFE above 10% (v/v), the far-UV ellipticity of thedenatured state of AcP is higher than the value for the nativeconformation and the folding rate is lower than the maximumvalue (Fig. 5). Folding of muscle and CT AcP appears to beaccelerated as long as the secondary structure promoted byalcohols is within that present in the native state (Fig. 5). Anincreased rate of folding would be expected when a collapse orrelated process generates a reduction in the number of statesaccessible to the polypeptide chain, but before this processresults in stable structure that generates barriers to subsequentsteps. The introduction of local hydrogen bonding followingthe addition of small quantities of alcohol responds to bothrequisites. In contrast, at higher concentrations of alcoholsthese local interactions are more extensive than those existingin the native state, and the folding process requires that thisadditional structure be disrupted with the generation of anadditional energetic barrier to folding. A significant contribu-tion to the observed deceleration of folding at moderate fluo-roalcohol concentrations is also expected to arise from thepotential of such compounds to destabilize the hydrophobicinteractions that must form during the folding process(Hamada, D. et al., manuscript in preparation).

A simplified model of the folding behavior of AcPA mathematical interpretation of the folding behavior of AcP inthe presence of TFE can be approached through the followingsimplified model.

The denatured state of the protein under refolding conditionsis as an ensemble of conformations each with a certain propor-tion of native and nonnative secondary structure. For simplicitywe assume four possible conformations: D with no residual

Fig. 5 Relationship between secondary-structure content of the dena-tured state and folding rate. a, Equilibrium TFE titration of CT AcP moni-tored by far-UV CD (p). The mean residue ellipticity of the denaturedprotein under refolding conditions is also reported (L). b, Observed rateconstant of CT AcP folding, shown as its natural logarithm, as a functionof TFE and monitored by intrinsic fluorescence emission (P) and far-UVCD (X). The solid line represents the fitting of the fluorescence data toequation 5, and the dashed line indicates the TFE concentration atwhich the ellipticity of the denatured state equals that of the nativestate. In all cases the final conditions were 0.72 M urea, 50 mM acetatebuffer, pH 5.5, 25 °C.

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structure, Din and Dout with native (in place) or nonnative (out ofplace) secondary structure, respectively, and Din,out having ele-ments of both native and nonnative secondary structure. Everyelement of such structure is in equilibrium with the correspond-ing unstructured peptide through an equilibrium constant KH;all the equilibria are independent of each other and are estab-lished rapidly compared to the rate of folding. Folding to thenative state N is considered to proceed more efficiently from Din,through a rate constant kf, than from other conformations. Therate constants for the conversion of the remaining conforma-tions into the native state are therefore assumed to be negligible.In a complex system, such as a denatured polypeptide chain of98 residues in the presence of TFE, there are undoubtedly manymore states than those represented in the model and differentvalues of KH for each equilibrium. This model represents, how-ever, an analyzable case and includes the heterogeneous nature ofthe protein denatured in TFE. It is important to make it clearthat the network of conformations and equilibria depicted in themodel should not be interpreted to be present only at the earlystages of protein folding, but at any level in the progressivesearch for native contacts. Nor must the necessity to passthrough conformations containing exclusively native sec-ondary structure be considered to occur only at the early stepsof protein folding, but at one of the countless stages precedingthe transition state. Regardless of the stage at which a proteinforms the native structure during folding, this will be influ-enced by the favorable (or unfavorable depending on concen-tration) effects of fluorinated alcohols.

Taking into account all the assumptions described here, theequation showing the dependence of the experimentallyobserved rate constant on TFE concentration can be developedas described in equation 5 . The best fits of the data to equation(5) yielded the curves shown (Fig. 2), and the relevant parame-

ters are listed in Table 2. According to the model and the resultsof the fitting procedure, the acceleration of folding at low TFEconcentrations is caused by the stabilization of conformationscontaining native hydrogen bonding (that is, Din) relative to theunfolded conformations (D). In contrast, the deceleration atthe higher TFE concentrations derives from an overaccumula-tion of species containing both native and nonnative interme-diate structure (that is, Din,out), with consequent depletion ofthe Din species. The analysis indicates that the intrinsic foldingrate from the Din species decreases upon the addition of a fluo-roalcohol (negative value of mf

‡), suggesting that a contribu-tion to the reduction of the observed folding rate results fromthe ability of fluoroalcohols to weaken the hydrophobic inter-actions in the search of the native fold of the protein. Othermodels where folding is considered to proceed with similar effi-ciency from any of the conformations containing secondarystructure regardless of their native or nonnative nature, or fromany of the species of the scheme illustrated above fail to offer acomplete explanation of our kinetic and structural data.

The analysis indicates that if native and nonnative structuralelements have similar intrinsic stabilities and are populated tothe same extent upon the addition of fluoroalcohols, the popu-lation of species with local interactions of native character will,however, be higher in the presence of small amounts of alcoholrelative to the remaining conformations (that is, the populationfraction of Din is higher in the presence of low concentrations ofTFE than in its absence). Consequently, the characteristic plotswith a maximum folding rate (Fig. 2) do not necessarily arisefrom the fact that native secondary structure is the most popu-lated in the denatured state of AcP upon the addition of alcoholrelative to the nonnative structure. The calculations show thatby assuming a much higher intrinsic stability for the elementsof native relative to nonnative structures (that is, a much higher

Table 1 Estimate of secondary-structure content of native and alcohol-denatured AcP from CD spectra1

0% TFE 10% TFE 40% TFE 40% HFIP NMR and X-ray structuresMuscle AcP CT AcP Muscle AcP CT AcP Muscle AcP CT AcP CT AcP Muscle (NMR) CT AcP (X-ray)

α-helix 22% 23% 22% 24% 50% 51% 79% 24% 24%Antiparallel β-sheet 37% 34% 38% 31% 9% 8% 4% 34% 34%Parallel β-sheet 4% 4% 4% 4% 6% 5% 6% 5% 4%β-Turns 11% 12% 11% 13% 13% 13% 14% (12%) 15%Disordered 25% 27% 25% 29% 22% 22% 11% (25%) 22%

1The secondary-structure content was estimated by applying the variable-selection method to the CD spectra39, as described in the Methods section.The disordered secondary structure estimated by the algorithm includes both random-coil regions and extended loops. For a compa rison, we alsoshow the secondary-structure content estimated from X-ray crystallography16 and NMR spectroscopy14,15. For the NMR data, the numbers in bracketsare estimates that are subject to a large experimental error.

Table 2 Kinetic parameters for the TFE- and HFIP-induced acceleration of AcP folding1

kf(H2O) (s−1) mf‡ KH(H2O) mH % TFE or HFIP Maximum rate of maximum acceleration enhancement

CT AcP, TFE 41.5 -102 0.0133 1,032 10.4 21Muscle AcP, TFE 5.32 -79 0.0205 976 9.8 15CT AcP, HFIP 59.3 -287 0.00767 3,279 3.7 31Muscle AcP, HFIP 4.26 -240 0.0189 2,648 3.7 14

1Parameters were obtained by fitting the data shown in Fig. 2a and b to equation (5). This analysis has been carried out at a constant urea concen-tration of 0.72 M. In this mathematical approach we have considered the potential dependence of kf on TFE concentration (that is, the mf‡ parame-ter). A negative value of this parameter could reflect the ability of TFE to inhibit the formation of hydrophobic interactions. On the contrary, apositive value might arise from the favorable action of TFE to allow the reorganization of these interactions in the transition-state ensemble. The fit-ting procedure leads to values of mf‡ that are small compared to mH. Nevertheless, these are significantly negative, suggesting that TFE has a desta-bilizing effect on the formation of hydrophobic interactions.


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value of KH(H2O) for the formation of Din), the kf values are~25% of the values reported in Table 2, that is, of the sameorder of magnitude.

ConclusionThe addition of fluoroalcohols converts the two-state foldingof AcP into a process in which partially folded conformationswith local hydrogen bonds form before the major phase offolding. This supports the view that the distinction betweentwo-state and multistate folding is not a fundamental one, butdepends on whether the stabilities of intermediate states underrefolding conditions are sufficiently large for experimentaldetection. Theoretical simulations suggest that efficient fold-ing can be generated by restricting the conformational searchthrough the denatured configurations to a limited number ofstructures, provided that the stabilization of these conforma-tions does not raise kinetic barriers for the reorganization ordisruption of preformed contacts2. The addition of fluoroalco-hols to the solvent can increase the stability of local hydrogenbonding without introducing hydrophobic contacts, hencewithout coupling the formation of local secondary structure tohydrophobic collapse where long-range interactions need tobe reorganized within a compact state. Our experimentalresults show that the stabilization of local secondary structurecauses a substantial acceleration of the folding process, where-as the presence of additional nonnative-like hydrogen bondsresults in its retardation. Although the partially folded statesformed in fluorinated alcohols are very different from thecompact states formed during the folding of many proteins inaqueous solution, this study shows that intermediate structurecan act either to facilitate or to retard the folding process of aprotein depending on its content of native-like interactions.

The results presented here also reveal the importance oflocal interactions that stabilize α-helices, β-turns or β-hair-pins in directing the folding process of AcP. Analysis of ourdata indicates that any hypothetical conformation with well-formed elements of native secondary structure will fold with arate constant of ~50 s–1 and 5 s–1, for CT and muscle AcP,respectively (Table 2). This estimate is derived on the assump-tion of similar intrinsic stabilities for native and nonnativeconstituents of secondary structure, and the rate constant val-ues appear four times lower if native elements of secondarystructure are considered to possess a higher stability. Thesefolding rate constants are much smaller than those of thefastest folding proteins so far characterized, which fold withrate constants in the range 1,000–10,000 s–1 (ref. 37). Thisindicates that the rate of folding of small proteins of the size ofAcP is not dependent only on the search for, and relative sta-bility of, local secondary structure, but also of long-rangeinteractions. Such a conclusion supports the picture emergingfrom an increasing range of theoretical and experimental stud-ies where the transition-state regions of folding reactionsinvolve the development of partially folded states containingmany of the elements of native-like structure2,3.

MethodsMaterials. Human CT and muscle AcP were purified as reportedelsewhere38. For muscle AcP, the C21S mutant was used to elimi-nate complexities in folding kinetics associated with a free cys-teine residue17. For simplicity this mutant is referred to as muscleAcP throughout the analysis.

Circular dichroism spectra. Far-UV CD spectra at equilibriumwere acquired using a Jasco J-720 spectropolarimeter and tem-perature-controlled cuvettes with 1 mm path length. Protein con-centration was determined spectrophotometrically using ε280values of 1.42 and 1.25 ml mg–1 cm–1 for muscle and CT AcP,respectively. AcP was diluted to a final concentration of 0.1 mgml–1 in 5 mM phosphate buffer, pH 7.0 containing 0%, 10% or40% (v/v) TFE or HFIP. Before spectral acquisition, the instrumentwas calibrated with a solution of d-10-camphorsulfonic acid, theconcentration of which was optically determined24. Secondary-structure analysis was performed by the variable-selectionmethod starting with a set of 33 proteins39.

Stopped-flow kinetics. The kinetics of folding of AcP werestudied at 25 oC by mixing one volume of AcP denatured in either8 M urea or 40% (v/v) TFE with 10 volumes of solutions containingvarious concentrations of urea, TFE or HFIP. Applied Photophysicsand Biologic stopped-flow instruments were used to follow thechanges in the intrinsic fluorescence emission and 222 nm elliptic-ity, respectively, until after equilibrium was attained.

Data analysis. From the model and the assumptions described inthe text, the experimentally observed rate constant of folding isdetermined by:

kobs = kf f(Din) (1)

where f(Din) is the fraction of Din (that is, [Din] / ([D] + [Din] + [Dout] +[Din,out])). By expressing the concentrations of D, Dout and Din,out as afunction of the concentration of Din and of KH we obtain:

kobs = kf / [( 1 + KH ) ( 1 + 1 / KH )] (2)

We assume that the natural logarithm of the equilibrium or rateconstant of a single step i → j in the folding process is linearly corre-lated with the TFE concentration, as has been documented for com-mon denaturants4:

ln Kij = ln Kij(H2O) + (mij / RT) C (3)

ln kij = ln kij(H2O) + (mij‡ / RT) C (4)

where Kij(H2O) and kij(H2O) are the equilibrium and rate constants inwater, respectively, whereas mij and mij‡ express their dependencieson denaturant concentration C. By substituting equations (3) and(4) in equation (2), and by taking the natural logarithm, we obtainthe dependence of the apparent rate constant of folding on TFEconcentration:

ln kobs = ln kf(H2O) + (mf‡/RT) [%TFE] - ln {1 + KH(H2O)

exp(mH [%TFE] / RT)}) - ln {1 + 1 / [KH(H2O) exp (mH [%TFE] / RT)]} (5)

where mf‡ and mH are the dependencies of kf and KH on TFE concen-

tration, kf(H2O) and KH(H2O) the corresponding values in water.

AcknowledgmentsWe are grateful to L. Serrano and M. Buck for useful discussions. F.C. wassupported by a grant from the European Community. D.H. was supported by JSPSPostdoctoral Fellowships for Research Abroad. This is a contribution from theOxford Centre for Molecular Sciences, which is funded by BBSRC, EPSRC andMRC. The work has also been supported by funds from the Italian CNR (TargetProject Biotechnology), from MURST (Project Structural Biology) and from theEuropean Community (Biotechnology Unit). The research of C.M.D. is supportedin part by an International Research Scholars award from the Howard HughesMedical Institute and by The Wellcome Trust.

Received 7 July, 1998; accepted 7 December, 1998.


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