Stabilisation of α-helices by site-directed mutagenesis reveals the importance of secondary...

doi:10.1006/jmbi.2000.3870 available online at http://www.idealibrary.com on J. Mol. Biol. (2000) 300, 633±647

Stabilisation of aaa-Helices by Site-directed MutagenesisReveals the Importance of Secondary Structure in theTransition State for Acylphosphatase Folding

NiccoloÁ Taddei1, Fabrizio Chiti1,2, Tania Fiaschi1, Monica Bucciantini1

Cristina Capanni1, Massimo Stefani1, Luis Serrano3

Christopher M. Dobson2* and Giampietro Ramponi1*

TtdtipmitedA

1Dipartimento di ScienzeBiochimiche, UniversitaÁ diFirenze, Viale Morgagni 5050134 Firenze, Italy2Oxford Centre for MolecularSciences, New ChemistryLaboratory, University ofOxford, South Parks RoadOxford, OX1 3QT, UK3EMBL, Meyerhofstrasse 1Heidelberg D-69012, Germany

etraoatcls¯miffi

Ka

E-mail addresses of the [email protected]®.it; chris.dobson

Abbreviations used: ACP, humanacylphosphatase; ADA2h, activationprocarboxypeptidase A2; BOC, t-butFmoc, ¯uoren-9-ylmethoxycarbonyl-hydroxybenzotriazole; OPfp, penta¯2,2,5,7,8-pentamethylcroman-6-sulfonphase; SPPS, solid-phase peptide syntri¯uoroacetic acid; TFE, tri¯uoroethtrinitrobenzenesulfonic acid; Trt, trit

0022-2836/00/030633±15 $35.00/0

he effects of stabilising mutations on the folding process of common-ype acylphosphatase have been investigated. The mutations wereesigned to increase the helical propensity of the regions of the polypep-

ide chain corresponding to the two a-helices of the native protein. Var-ous synthetic peptides incorporating the designed mutations wereroduced and their helical content estimated by circular dichroism. Theost substantial increase in helical content is found for the peptide carry-

ng ®ve mutations in the second a-helix. Acylphosphatase variants con-aining the corresponding mutations display, to different extents,nhanced conformational stabilities as indicated by equilibrium ureaenaturation experiments monitored by changes of intrinsic ¯uorescence.ll the protein variants studied here refold with apparent two-state kin-

tics. Mutations in the ®rst a-helix are responsible for a small increase inhe refolding rate, accompanied by a marked decrease in the unfoldingate. On the other hand, multiple mutations in the second helix result inconsiderable increase in the refolding rate without any signi®cant effect

n the unfolding rate. Addition of tri¯uoroethanol was found to acceler-te the folding of the acylphosphatase variants, the extent of the accelera-ion being inversely proportional to the intrinsic rate of folding of theorresponding mutant. The tri¯uoroethanol-induced acceleration is faress marked for those variants whose a-helical structure is ef®cientlytabilised by amino acid replacements. This observation suggests that tri-uoroethanol acts in a similar manner to the stabilising mutations in pro-oting native-like secondary structure. Analysis of the kinetic data

ndicates that the second helix is fully consolidated in the transition stateor folding of acylphosphatase, whereas the ®rst helix is only partiallyormed. These data suggest that the second helix is an important elementn the folding process of the protein.

# 2000 Academic Press

eywords: acylphosphatase; protein folding; transition state for folding;-helix stabilisation; tri¯uoroethanol
*Corresponding authors
ing authors:@chem.ox.ac.uk

common-typedomain of

hyloxycarbonyl-;; HOBt,uorophenyl-; Pmc,yl-; RP, reverse-thesis; TFA,

anol; TNBS, 2,46-yl.

Introduction

One of the most actively debated issues in the®eld of protein folding concerns the relativeimportance of local and non-local interactions infolding reactions (Goldenberg, 1999; Ionescu &Matthews, 1999). A variety of mechanisms of pro-tein folding have been suggested in the past, somebased on the early formation of native-like second-ary structure, followed by the organisation of ter-tiary contacts (Go, 1983; Kim & Baldwin, 1990;

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634 Role of �-Helices in Acylphosphatase Folding

Wetlaufer, 1990; Karplus & Weaver, 1994; Ptitsyn,1995), others based on the concept of a rapidhydrophobic collapse prior to the formation ofstable secondary structure (Levitt & Warshel,1975). Most of these models involve the presenceof intermediates have are observed experimentallywhen they are stable enough to accumulate duringthe folding reaction. Structural investigations ofsuch intermediates have shown that they are oftenrich in native-like secondary structure elements(Hughson et al., 1990; Jeng et al., 1990; Radford etal., 1992; Schulman et al., 1997; Dalby et al., 1998;Eliezer et al., 1998). The majority of small proteins,however, refold with two-state kinetics, and nointermediates are observable either in equilibriumor kinetic experiments (Jackson, 1998). The investi-gation of the kinetics of folding of such proteins,coupled with a protein engineering approach, hasled to the proposal of a mechanism in which thedevelopment of native structure follows a nuclea-tion-condensation mechanism, involving the for-mation of a nucleus of secondary structure whichguides the establishment of tertiary contacts which,in turn, expand and consolidate the initial nucleus(Fersht, 1999). Although it is unlikely that a singlesimpli®ed model can explain the folding behaviourof all proteins, it is becoming evident that there isan underlying uni®ed mechanism that describesthe fundamental process of protein folding(Dobson et al., 1998). The ``new view'' of proteinfolding, based on theoretical simulations and sim-pli®ed computer models, suggests that the pro-gressive formation of native-like interactions canoccur through a multiplicity of pathways(Bryngelson et al., 1995; Dill & Chan, 1997; Dobsonet al., 1998). On this view, the driving force forfolding is a statistical conformational search biasedby the fact that, on average, native-like interactionswithin the polypeptide chain are more stable thannon-native ones. In this scenario, the fundamentalmechanism of folding is common to all proteinsand the experimental differences shown by pro-teins in their folding behaviour is a consequence ofdifferent balances between entropic and enthalpiccontributions to the free energy of the system(Dobson et al., 1998).

An important step in the determination of thedetailed mechanism by which a given polypeptidechain folds is that the energetics and structures ofthe transition state region involved in the foldingreaction are fully characterised. A commonly usedand powerful approach to obtain structural infor-mation concerning the transition state for folding isthe f-value analysis of single point mutants of agiven protein (Matouschek et al., 1989; Fersht,1999). This method has been applied to a numberof proteins and has indicated that in most cases thetransition state possesses a signi®cant degree ofnative-like secondary structure as well as a highdegree of compactness (Jackson, 1998; Fersht,1999). Similar, but less detailed, conclusions havebeen drawn from other complementary

approaches, including solvent perturbationmethods (Chiti et al., 1998b).

Acylphosphatase (AcP) is a small monomerica/b protein (Stefani et al., 1997) found in allvertebrate tissues in the form of two homologousisoenzymes, muscle and common-type AcP, whosethree-dimensional structures are known in detailand are closely similar (Pastore et al., 1992;Thunnissen et al., 1997). Human common-type AcPand muscle AcP have been shown to fold by anapparent two-state mechanism by both equilibrium(Taddei et al., 1994, 1999) and kinetic experiments(van Nuland et al., 1998; Taddei et al., 1999). Theyare among the most slowly refolding of all smallproteins studied so far (van Nuland et al., 1998;Jackson, 1998; Taddei et al., 1999). The structuralcharacterisation of the transition state for folding ofAcP has been performed by both protein engineer-ing and solvent perturbation methods (Chiti et al.,1998b, 1999c). These studies indicate that the tran-sition state for AcP folding is a relatively compactstructure, with elements of secondary structureformed in part, but lacking many of the persistentlong-range interactions typical of the native state.The addition of 2,2,2-tri¯uoroethanol (TFE) to therefolding buffer converts the apparent two-statefolding of AcP into a multi-state mechanism wheresecondary structure accumulates signi®cantly inthe denatured state prior to folding tothe native state (Chiti et al., 1999b). This isaccompanied by a marked increase in the refoldingrate which reaches a maximum when the second-ary structure content of the denatured state is simi-lar to that of the native state (Chiti et al., 1999b).

Here, we present a mutational analysis focusedon the two a-helices of the native state of humancommon-type AcP. A number of AcP variants hav-ing single or multiple mutations designed toincrease the stability of the a-helical segments ofthe enzyme have been produced. The thermodyn-amic and kinetic investigation of the folding ofthese AcP variants under different experimentalconditions has provided us with informationwhich contributes to the discussion on the import-ance of local interactions in protein folding and tothe nature of the transition states of folding reac-tions (Goldenberg, 1999; Ionescu & Matthews,1999).

Results

Design of mutations aimed at stabilisingaaa-helical structure

The native state of AcP has two a-helices, helix 1and helix 2, spanning residues 22-32 and 55-67,respectively. Mutations aimed at increasing thea-helical propensity of these two regions whilemaintaining the overall protein packing present inthe native fold are illustrated in Figure 1. Thesewere designed by replacing those residues that arefully exposed to the solvent in the native confor-mation on the basis of a theoretical prediction

Figure 1. Schematic representation of the structure ofAcP drawn by the program WebLab Viewer, version1.1. The mutated residues in the a-helices are shown ina ball and stick representation (Thunnissen et al., 1997).

Role of �-Helices in Acylphosphatase Folding 635

performed with the AGADIR algorithm (MunÄ oz &Serrano, 1994a,b), using its latest version AGA-DIR1 s-2 (Lacroix et al., 1998). The mutationschosen were:

helix 1 : F21S=A28E �1�

helix 1 : G30A �2�

helix 2 : S56A=K57L=H60K=E66K=T67A �3�The mutations are designed to increase the helicalpropensity of the selected residues (S56A, T67A),to optimise electrostatic interactions (A28E, K57L,H60K and E66K) and to introduce a favourable N-capping residue (F21S) (Lacroix et al., 1998). Whilehelix 2 was mutated at ®ve different sites toincrease its a-helical propensity, only those resi-dues of helix 1 that are fully exposed, residues 21

Table 1. Predicted changes in the helical content and stability

Peptide �Ga (kJ/mol)

Helix 1 WT 1.62Helix 1 F21S/A28E 1.74Helix 1 G30A 1.32Helix 2 WT 1.16Helix 2 multiple 0.52

�G is the free-energy difference for the coil-helix transition. The %a Residue 20 being the N-cap.b Residue 21 being the N-cap.

and 28, could be replaced without altering theother stabilising interactions within the native foldof the protein, i.e. the hydrophobic packing of theprotein. Although the replacement of Gly30 withan alanine residue is not expected to leave thehydrophobic core unaltered, the gain of a-helicalpropensity caused by such substitution is remark-able (Table 1), justifying its analysis in the presentstudy. Table 1 shows the predicted changes ina-helix stability following mutations.

The designed mutations stabilise the aaa-helicalstructure in model peptides

One peptide including the sequence of a-helix 1of wild-type AcP (helix 1 WT) and one correspond-ing to the sequence of a-helix 2 (helix 2 WT) weresynthesised and analysed with far-UV circulardichroism (CD) (see Materials and Methods fordetails). Three peptides similar to these but con-taining the mutations (1), (2) and (3) describedabove, named helix 1 F21S/A28E, helix 1 G30A,and helix 2 multiple, respectively, were also syn-thesised and subjected to CD analysis. Figure 2(a)shows the change in the CD spectrum of the helix2 WT peptide, as a consequence of the addition ofprogressive amounts of TFE. The peptide is sub-stantially unstructured in the absence of TFE, asrevealed by the strong negative ellipticity at ca.200 nm; the negative ellipticity at 222 nm could bedue to the presence of a tryptophan residue thatproduces a negative band in random coil peptides(Chakrabartty et al., 1993). However, it appears tobe essentially completely a-helical at concentrationsof TFE higher than 30 % (v/v); the spectra at highTFE concentrations exhibit strong negative elliptici-ties at 208 and 222 nm and positive ellipticitybelow 195 nm, features which are strongly indica-tive of a-helical structure. The isodichroic point at203 nm indicates that this helix-coil transition canbe described, to a good approximation, as a two-state process.

CD spectra were acquired for the ®ve peptidesstudied here at TFE concentration intervals of 2 %.Figure 2(b) describes the change of mean residueellipticity at 222 nm for the helix 1 WT, helix 1F21S/A28E and helix 1 G30A peptides. Analysiscarried out using AGADIR1s-2 predicts that only aminimal increase in a-helical structure can beobtained with the two mutations at positions 21

of AcP peptides

�Gb (kJ/mol) % Helix

1.70 0.711.34 1.051.40 3.33

0.9815.6

helix has been estimated at 28 �C.

Figure 2. Equilibrium conformational changes inducedby TFE on synthetic peptides corresponding to aminoacid sequence stretches of AcP. (a) Far-UV CD spectraof the helix 2 WT peptide in the presence of increasingTFE concentrations. Dependence of the CD signal at222 nm on TFE concentration: (b) helix 1 peptides: (&)WT; (^) F21S/A28E; (&) G30A. (c) Helix 2 peptides:(&) WT; (&) multiple. All the experiments were carriedout at 28 �C in 50 mM acetate buffer (pH 5.5).

Figure 3. Far-UV CD spectra of synthetic peptides ofAcP recorded at 28 �C, in 50 mM phosphate buffer(pH 7.0). (a) Helix 1 peptides: WT (Ð); WT in 40 % TFE( � � � � � � ); F21S/A28E (*); F21S/A28E in 40 % TFE (*);G30A (�); G30A in 40 % TFE (�). (b) Helix 2 peptides:WT as in (a), multiple (*); multiple in 40 % TFE (*).(b, inset) Helix 2 peptides: WT in 10 % TFE ( � � � � � � );multiple (*).


and 28 (Table 1). Indeed, only when the residue atposition 21 is considered as the N-cap, does thealgorithm predict an increase of a-helical propen-sity. The CD analysis con®rms this prediction, asthe a-helical content of the mutated peptides iswithin experimental error of that of the wild-typesequence. In the case of the helix 1 G30A peptide,the experimental data show a considerable increase

in negative ellipticity at 222 nm upon mutation, inagreement with the predictions made with AGA-DIR1s-2 (Table 1).

Figure 2(c) shows that considerable stabilisationof the a-helical structure in the helix 2 WT peptideoccurs when the multiple mutations describedabove are considered. In the absence of TFE, the222 nm ellipticity of the helix 2 multiple peptide issigni®cantly higher than that of the wild-type con-struct. In a similar fashion, a shift towards thelower TFE concentrations for the helix-coil tran-sition is evident for the mutated peptide from thedata in the Figure.

Figure 3 shows the CD spectra acquired for thevarious peptides in the absence and presence of40 % TFE. The quantitative estimates of a-helicalcontent in the various cases are reported in Table 2.While the CD spectra of helix 1 WT and helix 1F21S/A28E are very similar under all conditionsinvestigated, the presence of the G30A mutationleads to a considerable increase in secondary struc-ture at all TFE concentrations, although the peptideremains largely unstructured when TFE is absent(Figure 3(a)). It is interesting to observe that the

Table 2. a-Helical content of peptides corresponding to wild-type and mutated a-helices

Peptide 0% TFE 10% TFE 40% TFE

Helix 1 WT 0% 4% 18%Helix 1 F21S/A28E 2% 4% 15%Helix 1 G30A 9% 15% 43%Helix 2 WT 17% 33% 97%Helix 2 multiple 38% 75% 98%

The content of a-helical structure, expressed as % of the total secondary structure, was determined from CD spectra as describedin Materials and Methods. CD spectra were acquired at 28 �C, in 50 mM acetate buffer (pH 5.5).


helix 1 WT peptide yields a CD spectrum of lowintensity, regardless of the TFE concentration used.

As far as a-helix 2 is concerned, stabilisation ofhelical structure after introduction of the ®vemutations is clearly apparent (Figure 3(b)). Thenegative ellipticity at 204 nm (intermediatebetween 198 and 208 nm) and the relatively lowintensity of the bands at 188 and 222 nm indicatethat the helix 2 multiple peptide is partially helicaleven in the absence of TFE (Table 2 andFigure 3(b)). The CD spectrum of the WT peptide,by contrast, is typical of a highly unstructured con-formation, taking into account the contribution ofthe tryptophan residues to the spectra of randomcoil peptides (Chakrabartty et al., 1993). The CDspectra in the presence of 40 % TFE are, however,very similar as both peptides are almost fully heli-cal (Table 2). The inset in Figure 3(b) underlinesthe very close similarity of the CD spectra of themutated peptide in the absence of TFE and of thewild-type peptide in the presence of 10 % TFE. Itappears that the presence of 10 % TFE reproducesclosely the effects of the stabilising mutations onthe a-helical content of the peptide.

The designed mutations stabilise the nativestate of AcP without affecting its activity

Variants of AcP containing the mutationsdescribed above were produced to assess whetheramino acid substitutions of this type are able tostabilise the overall fold of the protein as well asthe helical nature of the individual peptidesequences. In addition to the three variants con-

Table 3. Equilibrium urea-induced unfolding parameters and

AcP variant Cm (M) m (kJ molÿ1 M

Wild-type 2.68 6.30A28E 3.04 6.11F21S/A28E 3.10 6.24G30A 4.05 5.97Helix 2 multiple 3.91 5.92Helix 2 multiple � A28E 4.26 5.97Helix 2 multiple � F21S/A28E 4.55 5.74

All experiments were performed at 28 �C, in 50 mM acetate buffeof the protein molecules. �GH2O

UÿF is the Gibbs free energy of unfoldindenaturant concentration. The enzymatic activity was measured usinat pH 5.3 (Ramponi et al., 1966); one I.U. is de®ned as the enzymat25 �C. The average error for each m and �GH2O

UÿF value is approximate

taining mutations (1), (2) and (3), other variantswere produced, including one with the singleA28E substitution, one with the ®ve mutations onthe second a-helix and the A28E substitution, andone with the ®ve mutations on the second a-helixand the double F21S/A28E substitution. The con-formational stability of these AcP variants wasevaluated by means of equilibrium urea denatura-tion experiments as described in Materials andMethods. The urea denaturation curves of thewild-type protein and some representative variantsare reported in Figure 4 and the relevant thermo-dynamic parameters obtained from the analysis arelisted in Table 3. All the variants are stabilisedcompared to the wild-type protein, although thestabilisation caused by the mutations at positions21 and 28 is small. More pronounced stabilisationis obtained with the G30A substitution and withthe ®ve mutations on a-helix 2. The experimentalmeasurements of the extent of stabilisation by themutations correlate reasonably well with that pre-dicted using AGADIR1s-2, except for the helix 1F21S/A28E variant. In this case, a better agreementbetween predicted and experimental data isobtained when the residue at position 21 is con-sidered to maintain its role as the N-cap residuewithin the helix after mutation (Table 1). Indeed,AGADIR1s-2 predicts that the F21S/A28Emutations are stabilising or slightly destabilisingdepending on whether the newly introduced Ser21residue acts as the N-cap or not (Table 1). It ispossible that the structural constraints imposed bythe geometry of the a-helix within the native struc-ture of AcP do not allow an optimum capping

enzymatic activity of AcP variants

ÿ1) �GH2OUÿF (kJ molÿ1) ��GH2O

UÿF (kJ molÿ1)enz. activity(I.U. mgÿ1)

16.9 - 450018.6 �1.7 600019.3 �2.4 190024.2 �7.3 600023.2 �6.3 510025.4 �8.5 500026.1 �9.2 1500

r (pH 5.5). Cm is the urea concentration required to unfold 50 %g in the absence of urea and m is the dependence of �GH2O

UÿF ong saturating concentrations of benzoylphosphate as a substrate,

ic activity that hydrolyses 1 mmol of substrate in one minute atly 7 %, for Cm is 0.15 M and for the enzymatic activity is 10 %.

Figure 4. Equilibrium urea-induced denaturation ofAcP at 28 �C in 50 mM acetate buffer (pH 5.5), moni-tored by intrinsic tryptophan ¯uorescence changes. (&)Wild-type; (}) F21S/A28E; (&) G30A; (*) helix 2 mul-tiple; (*) helix 2 multiple � F21S/A28E. The raw datahave been converted to the fraction of folded protein byusing: ff � (yd ÿ y)/(yd ÿ yn), where yn and yd are thesignals of the native and denatured protein, respectively,extrapolated to the urea concentration under consider-ation.

Figure 5. Urea concentration dependence of naturallogarithms of folding and unfolding rate constants, at28 �C, in acetate 50 mM (pH 5.5) for different AcP var-iants. (&) Wild-type; (}) F21S/A28E; (&) G30A; (*)helix 2 multiple; (*) helix 2 multiple � F21S/A28E. Thedata were linearly ®tted as described in Materials andMethods and the relevant parameters obtained by the®tting procedure are reported in Table 4.


along the sequence of the helix, thus explaining theobserved stabilisation of the helix 1 F21S/A28Evariant. It is interesting that the further increases inconformational stability obtained when themutation A28E or the two mutations F21S/A28Eare added to the ®ve substitutions on the a-helix 2are similar to the stabilisations obtained when suchsingle or double substitutions are made indepen-dently in the wild-type protein (Table 3). This indi-cates that the degree of stabilisation caused bythese amino acid replacements is roughly additive.

The AcP variants were also subjected to enzy-matic activity measurements in order to establishthe effects of the mutations on the overall architec-ture of the protein (Table 3). Previous studies havesuggested that enzymatic activity can be usedbroadly to probe the native-like conformation ofAcP variants provided the mutated residues arenot part of the active site (Paoli et al., 1999). Thenative-like structure of the protein appears to bemaintained in all AcP variants as remarkably highacylphosphatase activity is detectable in all cases(Table 3). An enzymatic activity comparable to thatof the wild-type protein is also observed for theG30A variant despite the potential disrupting effectof the amino acid replacement on the hydrophobicpacking of the protein. Only the two AcP variantsbearing the replacement of Phe21 (the F21S/A28Evariant and that containing the ®ve mutations ona-helix 2 and the F21S/A28E substitutions) werefound to be less active than the wild-type protein,a result attributable to the fact that the Phe21 resi-due is part of the catalytic loop of the protein(Taddei et al., 1997).

Acceleration of folding and deceleration ofunfolding of AcP resulting from the mutations

The rates of folding and unfolding of the variousmutants were studied in order to assess whetherthe observed stabilisation of each variant arisesfrom an acceleration of the folding reaction, adeceleration of the unfolding reaction, or from acombination of both effects. Figure 5 shows therates of folding and unfolding of the AcP variantsas a function of the urea concentration; the relevantkinetic parameters, determined as described inMaterials and Methods, are listed in Table 4. Allthe variants with amino acid substitutions ona-helix 1 have their folding process accelerated,and their unfolding process decelerated, relative tothe wild-type protein. By contrast, mutations ona-helix 2 cause only the folding process to bealtered, the unfolding rate being virtually the sameas that of the wild-type protein at all urea concen-trations investigated. The application of f valueanalysis yields f values of 0.20-0.45 for the var-iants regarding a-helix 1 and of 1.00 for thatregarding a-helix 2 (Table 4). Since the overallstabilisation of AcP resulting from these mutationshas to be attributed to the stabilisation of second-ary structure within the helices, the resulting fvalue analysis reports on the extent to which thebackbone hydrogen bonds of the helices areformed in the transition state relative to the nativeand unfolded states (Viguera et al., 1997). Theresults show that the two a-helices of AcP areformed to different extents in the transition statefor folding. While a-helix 1 appears to be partiallyformed, a-helix 2 appears to possess a native-likebackbone hydrogen bonding in the transition state.

Table 4. Analysis of the folding and unfolding kinetics of different AcP variants

AcP variant kH2Of (sÿ1)a kH2O

u (sÿ1)a mf (Mÿ1)a mu (Mÿ1)a m (kJ molÿ1 Mÿ1)b Cm (M)b �GH2OUÿF (kJ/mol)b fF

Wild-type 2.52 0.00236 ÿ2.04 0.73 6.93 2.52 17.5 -A28E 3.25 0.00173 ÿ1.84 0.69 6.33 2.98 18.9 0.45F21S/A28E 3.00 0.00119 ÿ2.00 0.63 6.58 2.98 19.6 0.20G30A 7.42 0.00036 ÿ1.96 0.57 6.33 3.93 24.9 0.36Helix 2 multiple 23.63 0.00239 ÿ1.59 0.74 5.83 3.95 23.0 1.00Helix 2 multiple � A28E 37.18 0.00172 ÿ1.65 0.66 5.78 4.32 25.0 -Helix 2 multiple � F21S/A28E 27.70 0.00107 ÿ1.59 0.62 5.53 4.60 25.5 -

All the experiments were performed in 50 mM acetate buffer (pH 5.5) at 28 �C. Experimental errors, given by the ®tting procedure, are as follows: kH2Of and kH2O

u 10 %; mf and mu 3 %; Cm

0.3 M; m and �GH2OUÿF 5 %.

a Parameters obtained by linear ®tting of the data reported in Figure 5.b Parameters obtained according to Jackson & Fersht (1991).

Figure 6. Dependence of natural logarithms of foldingrate constants on TFE concentrations, at 28 �C in 50 mMacetate buffer (pH 5.5). (&) wild-type; (}) F21S/A28E;(&) G30A; (*) helix 2 multiple; (*) helix 2 multi-ple � F21S/A28E. The experimental data in the accelera-tory region for each AcP variant have been linearly®tted in order to evaluate the extent of TFE inducedacceleration on the folding process, as described inMaterials and Methods, according to Hamada et al.(2000).


For each variant studied the relationshipbetween the natural logarithm of the rate constantfor folding (or unfolding) and the concentration ofurea was found to be linear (Figure 5). Moreover,good agreement was found between the thermo-dynamic parameters determined from equilibriumdenaturation experiments and those obtained fromthe kinetic analysis (cf. Tables 3 and 4). Theseobservations indicate that the two-state behaviourfound for wild-type AcP (van Nuland et al., 1998;Taddei et al., 1999) is also found for the AcP var-iants investigated here. The mf value, representingthe difference in solvent accessible surface areabetween the transition state and the denaturedstate under refolding conditions, is, however,slightly smaller for all the variants than for thewild-type protein (Table 4). A similar, but smaller,decrease is also observable in the mu value, repre-senting the difference in solvent accessible surfacearea between the transition state and the nativestate, and in the overall m value determined fromequilibrium experiments (Tables 3 and 4). Theobserved decrease in the m value might be attribu-ted to the presence of a-helical structure in thedenatured state under the refolding conditions,reducing the accessible surface of the residuesinvolved in such structure (MunÄoz & Serrano,1996). A reduction in the mf value has beenobserved previously for AcP refolding in the pre-sence of TFE (Chiti et al., 1999b), and in other pro-teins with stabilised native (MunÄ oz et al., 1996;Villegas et al., 1997) and non-native a-helices(Prieto et al., 1997). The presence of a-helical struc-ture in the denatured state of the AcP variantsunder refolding conditions at the level of the twoa-helices is indicated by the CD analysis of themodel peptides (Figures 2 and 3) and is alsoobserved experimentally for the wild-type proteinin the presence of TFE (Chiti et al., 1999b).

Stabilisation of native-like aaa-helical structureby mutagenesis reduces the accelerating effectof TFE on folding

TFE is known to accelerate the folding of AcP(Chiti et al., 1998b, 1999b) as well as of other pro-teins (Hamada et al., 1996; Lu et al., 1997; Kentsis &Sosnick, 1998; Main & Jackson, 1999; Hamada et al.,2000). In the case of AcP, the acceleration of fold-ing promoted by TFE has been attributed to theability of this alcohol to stabilise native-like a-heli-cal structure, therefore reducing the entropic costassociated with the search for native-like contacts(Chiti et al., 1999b; Hamada et al., 2000). Other fac-tors have, however, been suggested to explain theaccelerating effect of TFE on protein folding inspeci®c cases (Lu et al., 1997; Kentsis & Sosnick,1998; Main & Jackson, 1999). In order to shed lighton this complex issue we have studied the foldingkinetics of the AcP variants with stabilised a-helices in the presence of TFE (Figure 6). For all themutational variants of AcP, the relationshipbetween the natural logarithm of the observed

folding rate constant and the concentration of TFEyields a characteristic plot with an acceleratory anda deceleratory region (Chiti et al., 1999b; Hamadaet al., 2000). The maximum rate of folding is foundfor the proteins in solutions containing between 5and 10 % TFE, depending on the variant con-sidered (Table 5). For the variants with a-helix 2stabilised by mutations, the maximum acceleratingeffect occurs at lower TFE concentrations and,under these conditions, the folding rate is increasedfar less than is the case for the wild-type protein(Figure 6 and Table 5). Indeed, for these variantsthe accelerating effect of TFE is very small, close tothat expected from the non-speci®c effects of thechange in solvent conditions (Hamada, et al., 2000).

The data points in the acceleratory regions of theplots shown in Figure 6 were ®tted to linear func-tions in order to determine the macR

ÿ1Tÿ1 value,the slope of the plot of lnk against TFE concen-tration (Table 5). This parameter has been found tobe a reliable measure of the potential of TFE toaccelerate the folding process of a protein(Hamada et al., 2000). As shown in Figure 7 thereis a remarkable correlation between the macR

ÿ1Tÿ1

value and the intrinsic folding rate in the absenceof TFE for the AcP variants. The high linear corre-lation coef®cient (r � 0.99) and the small p value(p < 0.001) indicate that the observed correlation isstatistically very highly signi®cant. This resultre¯ects the fact that the folding of the AcP variantspossessing an intrinsically higher folding rate isless effectively accelerated by TFE.

Table 5. Effects of TFE on the rate of AcP folding

AcP variantkH2O

f(sÿ1)a

kfmax

(sÿ1)a % TFEmaxa

Rateenhancementa

m{acRÿ1Tÿ1

(%ÿ1)b

Wild-type 2.11 20.09 9.9 9.5 0.280A28E 3.22 27.39 9.4 8.5 0.270F21S/A28E 3.18 28.22 10.0 8.9 0.285G30A 6.62 38.86 9.6 5.9 0.230Helix 2 multiple 21.33 31.19 5.4 1.5 0.107Helix 2 multiple � A28E 36.23 50.40 5.1 1.4 0.090Helix 2 multiple � F21S/A28E 32.14 56.25 6.8 1.7 0.105

% TFEmax is the TFE concentration at which the folding rate is at maximum. The rate enhancement is de®ned as kfmax/kH2O

f . Allexperiments were performed at 28 �C, in 50 mM acetate buffer (pH 5.5). Experimental errors are as follows: kH2O

f and kmaxf 8 %;

m{acRÿ1Tÿ1 5 %.

a Parameters obtained by ®tting the experimental data reported in Figure 6 to equation (5) of Chiti et al. (1999b).b Parameters obtained by linearly ®tting the data in the acceleratory region of Figure 6, according to Hamada et al. (2000).


Discussion

In contrast to the large number of mutationsdescribed previously for AcP designed to investi-gate the enzymatic mechanism of the protein andthe nature of the transition state for folding (Taddeiet al., 1995, 1996, 1997; Chiti et al., 1998a, 1999c;Paoli et al., 1999), all the mutations used in thiswork cause the native state of AcP to be stabilised.These ®ndings, together with similar resultsobtained with the activation domain of procarboxy-peptidase A2 (ADA2h) (Viguera et al., 1997), CheY(MunÄ oz et al., 1996), and GCN4-p1 (Zitzewitz et al.,2000), indicate that protein stabilisation can be suc-cessfully achieved by rationally designingmutations which increase a-helical propensities,provided that such mutations are located in regionsthat are in an a-helical conformation in the nativestate. In addition to stabilising the native fold, themutations studied here have been found to acceler-ate the folding process when the mutated a-helix isat least partially formed in the folding transitionstate, in agreement with previous results forADA2h and GCN4-p1 (Viguera et al., 1997;

Figure 7. Linear correlation between the acceleratoryeffect of TFE on folding, expressed by m{

acRÿ1Tÿ1 and

lnkobs for different AcP variants. r � 0.99, p < 0.001.

Zitzewitz et al., 2000). The G30A mutation and the®ve mutations on a-helix 1 and a-helix 2 have simi-lar effects on the overall conformational stability ofAcP, but accelerate the folding process to differentextents. This suggests that these helices be formedto a different extent in the transition state forfolding.

aaa-Helix 2 forms early in the folding of AcP

The f value analysis presented here suggests thatthe backbone hydrogen bonds that stabilisea-helix 2 are fully formed in the transition state.Mutations involving residues located on the twoa-helices of AcP have also been studied previouslyin the muscle form of the protein (Chiti et al., 1999c),a protein closely related structurally to the com-mon-type protein studied here, and sharing 55 %sequence identity. All the residues in a-helix 2 ofthe muscle protein were found to have f valueslower than 0.35, indicating that the native-like struc-ture associated with these residues is only partiallyformed in the transition state. The residues mutatedin the previous study were, however, part of thehydrophobic core of the protein and the substi-tutions were not designed, or expected, to increasethe a-helical propensity of the polypeptide chain inthe mutated region. Rather, these probes assess theextent to which the side-chain hydrophobic inter-actions formed by the mutated residues havenative-like structure in the transition state. Themutations described in the present study, by con-trast, report on the extent to which local main-chaininteractions are formed in the transition state interms of the stability of the helical conformation.They do not probe the side-chain contacts becausethe replaced residues are fully exposed to the sol-vent. The rather lower f values found for the resi-dues in the helices that contribute to the stability ofthe hydrophobic core are therefore likely to re¯ectthe differential degree of formation in the transitionstate of the interactions associated with the hydro-phobic side-chains and backbone polar groups atthe level of a-helix 2, a ®nding consistent with themore qualitative conclusions drawn from a pre-

Figure 8. (a) Highly schematic diagram of the nativestructure of AcP illustrating the regions with high fvalues (�1) which are assumed to form native-like inter-actions in the transition state for folding. Tyr11 andPhe94 are represented in blue, Pro54 and the a-helix 2are in red. (b) Folding for AcP. The starting point of thefolding process is an ensemble of relatively stable andhighly unfolded structures (U). Folding proceedsthrough the formation of an ensemble of relativelyunstable and transiently populated structures (I) inwhich a-helix 2 is formed to a large extent. In the tran-sition state for folding (TS) a-helix 2 appears to be com-pletely formed while a-helix 1 is only partiallystructured. At this stage of the folding reaction the over-all topology of AcP is established. Most of the hydro-phobic and other long-range interactions are present,but these are signi®cantly weakened compared to thenative state. Once the folding transition state is attainedthe achievement of the native structure (N) is rapid. Thespecies labelled I are in rapid equilibrium with U, suchthat the overall kinetics are effectively two-state. See thetext for further details.


vious study based on a solvent perturbation meth-od (Chiti et al., 1998b).

The present results can be brought together withthose obtained previously (Chiti et al., 1998b,1999a,b,c; van Nuland et al., 1998; Taddei et al.,1999) to describe in further detail the folding mech-anism of AcP. When urea-denatured wild-typeAcP is transferred from denaturing to refoldingconditions, no signi®cant structure forms withinthe ®rst few milliseconds prior to the major phaseof folding (van Nuland et al., 1998). Indeed, the ¯u-orescence and CD signals, as well as the 1H NMRspectrum, do not change detectably as a result ofthe removal of denaturant (van Nuland et al.,1998). Moreover, the CD analysis presented hereshows that the two a-helices do not possess stablestructure without assistance from the stabilisinglong-range interactions of the native conformation.Hence, folding of AcP into its ®nal native confor-mation is not preceded by formation of a burst-phase intermediate with signi®cant secondarystructure. Folding of AcP is rather a slow processwhere all the interactions present in the nativestate, including the local hydrogen bonds of thea-helices and the long-range interactions of thehydrophobic core, form through a single coopera-tive process, if we exclude the small fraction ofmolecules whose folding is rate determined by pro-line isomerisation (van Nuland et al., 1998; Chitiet al., 1999a; Taddei et al., 1999).

The various interactions that stabilise the nativefold of AcP, however, are present in the transitionstate to different extents (Figure 8(a)). The analysisreported here indicates that a-helix 2 (residues 55to 67) is highly structured in the transition state. Ithas been established that the interactions formedby Pro54 (f � 0.98) and the hydrophobic inter-actions involving Tyr11 (f � 0.92) and Phe94(f � 0.76) are also important in the folding process(Chiti et al., 1999c). The residue Pro54 forms a tightturn connecting a-helix 2 with the precedingb-strand. Moreover, its backbone carbonyl group ishydrogen bonded to the backbone amide group ofthe residue at position 58, which gives Pro54 therole of acting as the N-cap of a-helix 2. The fvalue of 0.98 measured for the P54A mutation, adestabilising replacement that undermines the deli-cate role that the residue at this position plays inthe secondary structure of the protein, indicatesthat the high level of secondary structure presentin the transition state at the level of a-helix 2extends to the residue at its N-cap. The high fvalues of Tyr11 and Phe94 indicate a native-likeclose packing of hydrophobic groups around theirside-chains. This region is likely to be the mostimportant in the consolidation of the hydrophobiccore during folding and in the establishment of theoverall topology (Chiti et al., 1999c; Vendruscoloet al., unpublished results).

On the basis of all the experimental data avail-able, a-helix 2 appears to be a region of structurethat forms early in the folding process relative toa-helix 1. Since the polypeptide chain forming this


helix in the native protein is not structured in itsisolated state, as indicated by the study of the syn-thetic peptides described here, and is not yetformed in the denatured state of AcP under refold-ing conditions, as indicated by previous studieswith stopped-¯ow CD (van Nuland et al., 1998), itsstabilisation is likely to be associated with the con-densation of the rest of the polypeptide chain inthis region of the structure. A possible scenario isthat folding of AcP proceeds through the formationof an ensemble of partially folded conformations inwhich the local hydrogen bonds stabilising a-helix2 are present despite the absence of speci®cinteractions involving particular side-chains(Figure 8(b)). These conformations are unstablerelative to the unfolded ensemble and are, there-fore, only transiently populated in the folding pro-cess. The condensation of the remainder of themolecule to produce the native structure, drivenby interactions such as the hydrophobic contactsaround Tyr11 and Phe94, results in the stabilisationand consolidation of the helix enabling rapid fold-ing to the native state.

At the level of the transition state for folding thehelix appears to be fully formed, mainly for thepartial formation of long-range interactions formedby the side-chains of its residues. It might be poss-ible that, even in the ensemble of conformationscontaining a-helix 2 and formed earlier than thetransition state for folding, partially formed inter-actions other than local hydrogen bonds areimportant for the stabilisation of the helix. In thisscenario the formation of the helix and the conden-sation of the remainder of the polypeptide chainare coupled events, rather than representing twodistinct fully independent steps. Moreover, theyare both rate-determining as indicated in thescheme of Figure 8(b).

The formation of a-helix 2 has also been foundan important feature of the folding of ADA2h, anon-homologous protein displaying an overall top-ology similar to that of AcP (Viguera et al., 1997;Villegas et al., 1998). Indeed, the folding transitionstates of AcP and ADA2h appear extremely similarwhen interactions other than hydrogen bonds arealso considered (Chiti et al., 1999c). The foldingprocess of these proteins appears to be stronglydependent on the overall topology of the nativestructure, rather than by the speci®c details ofinteractions stabilising the structure as it develops(Chiti et al., 1999c; Goldenberg, 1999). Given thesimilarity in the folding mechanism, it has beensuggested that the difference of folding rate inthese two systems arises from the different averagedistance between residues interacting in the nativestate (i.e. the ``contact order'') (Chiti et al., 1999c).

Early formation of secondary structure has alsobeen revealed in other protein systems such as inbarnase (Serrano et al., 1992) and, more recently, inthe folding/association reaction of GCN4-p1, anhomodimeric coiled-coil system (Zitzewitz et al.,2000). In these studies the importance is underlinedof the consolidation of part of the native a-helical

elements in the transition states for the foldingreactions. Thus, the important role of a-helix 2in the transition state of AcP appears consistentwith a variety of evidence from studies of otherproteins.

aaa-Helix stabilising mutations or addition ofTFE generate similar effects on the rate offolding of AcP

The results described in this paper show thatstabilisation of a-helical structure of AcP by site-directed mutagenesis causes the folding reaction tobe accelerated to an extent proportional to theincreases in a-helical propensity resulting from theamino acid replacements and to the extent of for-mation of the a-helix in the folding transition state.The acceleration of folding resulting from additionof TFE has also been correlated to the ability ofthis alcohol to stabilise local secondary structure(Chiti et al., 1999b; Hamada et al., 2000). Here, TFEhas been found to be less effective in acceleratingAcP folding when the native a-helices are stabil-ised by amino acid substitutions (Figure 6). Thisresults in the remarkable correlation found in theplot shown in Figure 7, which shows that theeffects on the folding rate of AcP resulting fromstabilisation of a-helical structure by mutations orby addition of TFE are essentially identical.Although the extent of secondary structure for-mation in the transition state of protein foldingreaction appears dif®cult to quantify from kineticstudies in the presence of TFE (Kentsis & Sosnick,1998; Main & Jackson, 1999), the present resultsshow clearly that the acceleration of folding pro-moted by TFE is strongly correlated to the abilityof this alcohol to stabilise local secondary structurewithin the polypeptide chain. These observationscomplement the previous ®nding that the maxi-mum folding rate of AcP occurs at the TFE concen-tration at which the denatured state possesses asecondary structure content comparable to that ofthe native state (Chiti et al., 1999b). They are alsoin agreement with the ®nding that the TFE-induced acceleration of folding for proteins show-ing two-state kinetics correlates well with the num-ber of local hydrogen bonds present in their nativestructure (Hamada et al., 2000).

Contribution of aaa-helix formation to the freeenergy barrier of AcP folding

From the plot shown in Figure 7 the maximumfolding rate that can be achieved by optimising thestabilisation of the local secondary structure can beobtained by extrapolation to the point at which noacceleration of folding results from addition of TFE.Hamada et al. (2000) have shown that such a situ-ation occurs at a value of macR

ÿ1Tÿ1 of 0.05 %ÿ1, avalue that takes into account the contribution ofnon-speci®c solvent effects to the folding accelera-tion promoted by TFE. The value of the folding rateobtained from such an extrapolation for AcP is


58.6 sÿ1, corresponding to the valueof kobs where the continuous line ®tted to the exper-imental data intersects the broken line in Figure 7.From a simpli®ed model derived from the exper-imental data on the effects of TFE on AcP describedpreviously, we calculated a folding rate constant of50 sÿ1 when folding is initiated from an hypotheti-cal conformation possessing a native-like content oflocal secondary structure (Chiti et al., 1999b). Thisvalue is in excellent agreement with that obtainedfrom the extrapolation shown in Figure 7. By apply-ing transition state theory (Fersht, 1999) one can cal-culate that the reduction of the activation freeenergy of folding derived from an optimised stabil-isation of overall local secondary structure (i.e. the��GH2O

zÿU value) is 7.9(�0.5) kJ molÿ1 under the con-ditions studied here (i.e. 28 �C, pH 5.5). The totalfree energy of activation of AcP folding can beobtained from transition state theory using a valueof 107 sÿ1 as an estimate of the pre-exponential fac-tor for a protein folding reaction (Bieri et al., 1999).The calculated total free energy for AcP under theconditions studied here is ca. 40 kJ molÿ1. This indi-cates that the activation energy required for the for-mation of native-like a-helical structure is only asmall part of the activation energy for AcP folding,a result consistent with the fact that even the var-iants of AcP with highly stabilised helices are stillrelatively slow folding proteins compared to manyothers (Hagen et al., 1996; Jackson, 1998). The for-mation of interactions other than local hydrogenbonds appears, therefore, to provide a large contri-bution to the free energy barrier of the folding ofAcP.

Conclusions

The ability to design rationally mutations thatstabilise the native state of a protein has importantimplications in the search for protein moleculeswith the ability to resist denaturation in adverseenvironments (Imoto, 1997). This work, and pre-vious studies (MunÄ oz et al., 1996; Viguera et al.,1997), suggest that the rational design of mutationsdirected to the stabilisation of native a-helices canrepresent a strategy of general value to increasethe stability of proteins without signi®cantly affect-ing their function.

The kinetic investigation of the AcP variantswith stabilised a-helices has allowed the foldingnucleus of AcP to be characterised in more detailthan was possible by single point mutations. Thisapproach reveals the importance in folding of theformation of local interactions within a-helix 2,including the residue at its N-cap that connects thehelix to the preceding b-strand. The combination ofkinetic experiments performed on a-helix stabilisedmutants with those performed in the presence ofTFE has allowed the fraction of free energy barrierassociated with the formation of overall local sec-ondary structure to be estimated. The results onAcP indicate that although the folding process is

initiated with the formation of persistent local sec-ondary structure, the consolidation of hydrophobicand other non-local interactions is the primarydeterminant of the folding rate of this protein.

Materials and Methods

Fmoc OPfp amino acid residues were obtained fromNovabiochem (LaÈufel®ngen, Switzerland); the PAL-PEG-PS resin, HOBt, DMF, TFA and piperidine from Persep-tive Biosystems (Wiesbaden, Germany). All otherreagents and solvents were analytical grade.

Peptide synthesis

Five N-acetylated, amide peptides were chemicallysynthesised by Fmoc SPPS using a MilliGen 9050 peptidesynthesiser. Two peptides have sequences correspondingto a-helix 1 (AcNH-VFFRKYTQAEGKKLG-NH2) anda-helix 2 (AcNH-GPASKVRHMQEWLETKG-NH2) ofAcP and are named helix 1 WT and helix 2 WT, respect-ively. Two other peptides, named helix 1 G30A (AcNH-VFFRKYTQAEAKKLG-NH2) and helix 1 F21S/A28E(AcNH-VSFRKYTQEEGKKLG-NH2), contain the corre-sponding substitutions in the a-helix 1, and a peptidecorresponding to a-helix 2, named helix 2 multiple,contains ®ve amino acid replacements (AcNH-GPAALVRKMQEWLKAKG-NH2). Peptides were syn-thesised on a PAL-PEG-PS resin (overall substitutionlevel of 0.176 mmol/g) functionalised with the PALlinker (5-(40Fmoc-aminomethyl-30,50-dimethoxyphenoxy)valeric acid). Peptide synthesis was carried out by thestandard ¯uoren-9-ylmethoxycarbonyl (Fmoc) protocolusing a fourfold excess (four equivalents) of each Fmocamino acid penta¯uorophenyl (OPfp) ester in the pre-sence of one equivalent of hydroxybenzotriazole (HOBt).Coupling cycles of one hour were used throughout thesynthesis; when necessary, double coupling cycles wereperformed. Side-chain protection was accomplished by t-butyl ether or ester for Asp, Glu, Ser, Thr and Tyr, bythe t-buthyloxycarbonyl (BOC) group for Lys and Trp,by the trityl (Trt) group for His and the 2,2,5,7,8-penta-methylcromon-6-sulfonyl (Pmc) group for Arg. Fmocgroup deprotection was carried out with 20 % piperidinein DMF. At the end of the synthesis, the last Fmoc groupwas removed and the free N terminus of each peptide-resin was acetylated by incubation with 20 % aceticanhydride in DMF for 30 minutes. Acetylation ef®ciencywas assessed by the 2,4,6-trinitrobenzenesulfonic acid(TNBS) test. Peptide cleavage and deprotection were car-ried out on the dried resins by incubation with deprotec-tion cocktails and times depending upon the amino acidcontent of each peptide. Each crude peptide was precipi-tated with cold ether, centrifuged, washed four timeswith an excess of cold ether and dried under vacuum.The dry powder was dissolved in 10 % (v/v) aceto-nitrile/water and puri®ed by semipreparative RP-HPLCon a C18 Vydac (20 mm � 250 mm) column. Peptidepurity and concentration were determined by mass spec-trometry and amino acid analysis, respectively.

Peptide analysis

The synthesised peptides were analysed with far-UVCD to evaluate their content and stability of a-helicalstructure. In each case, 25 solutions containing the pep-tide at 0.075 mg mlÿ1, 50 mM acetate buffer (pH 5.5) andvarious TFE concentrations ranging from 0 to 50 % (v/v)


were prepared and their CD spectra acquired at 28 �Cusing a Jasco J-720 spectropolarimeter and 1 mm cuv-ettes. At three representative TFE concentrations (0 %,10 % and 40 % TFE) the CD spectra were acquired in50 mM phosphate buffer (pH 7.0) to reduce the noisebelow 195 nm arising from acetate and obtain reliablemeasurements down to 185 nm. The a-helix content wasdetermined as [y]222/[ ÿ 42500 (1 ÿ 3/n)], where [y]222 isthe mean residue ellipticity at 222 nm and n is the num-ber of residues in the peptide (Myers et al., 1997). Spectrain acetate or phosphate buffer yielded identical estimatesof helix content.

Site-directed mutagenesis

The mutant containing the ®ve substitutions ona-helix 2 (S56A, K57L, H60K, E66K and T67A)was obtained by the use of the polymerase chainreaction (PCR). Brie¯y, two synthetic oligonucleotides(50-GTAAGATGCAGGAATGGCTTAAAGCAAGAG-GAAGTCC and 50-GCCATTCCTGCATCTTACGCACCAAGGCGATGGGACC), containing the mutations H60K,E66K, T67A and S56A, K57L, H60K respectively, andpartially overlapping, were synthesised. These primerswere used in two different PCR reactions together withthe oligonucleotides BamHIACPER (50-TTTGGATCCG-CAGAGGGAAACACCCTG) and EcoRIACPER (50-CTTAGAAAACTTGAATTCAGG), complementary to the 50and 30 ends of the cDNA, respectively. The completecDNA of AcP was obtained by a ®nal PCR reaction uti-lizing the two PCR fragments and the oligonucleotidesBamHIACPER and EcoRIACPER. The cDNA wasinserted in the unique restriction sites BamHI and EcoRIof the prokaryotic expression vector pGEX-KT, down-stream and in frame with glutathione-S-transferase. Theremaining mutants were obtained from the cDNAs ofeither wild-type AcP or the mutant containing the ®vesubstitutions of a-helix 2, using the unique site elimin-ation method (USE method) according to Deng &Nickoloff (1992).

Protein purification and enzymaticactivity measurements

Cloning, expression and puri®cation of wild-type andmutated AcP were performed as described by Taddeiet al. (1996). The presence of the desired mutations wascon®rmed by DNA sequencing and electrospray massspectrometry. Protein purity was checked by SDS-PAGE,showing that the ®nal content of AcP was higher than95 %. The acylphosphatase activity of the AcP variantswas measured by a continuous spectrophotometricmethod at 283 nm using benzoylphosphate as a sub-strate (Ramponi et al., 1966). Protein concentration wasdetermined by UV absorption using an e280 value of1.25 ml mgÿ1 cmÿ1.

Equilibrium experiments

Equilibrium unfolding curves for wild-type AcP andits mutants were obtained by measuring the intrinsic¯uorescence of 25-30 equilibrated samples containingvarious concentrations of urea in 50 mM acetatebuffer (pH 5.5), 28 �C. A Shimadzu RF-5000 spectro-¯uorophotometer, equipped with a Pharmacia LKBMultiTemp II water circulator, was used with excitationand emission wavelengths of 280 and 335 nm, respect-ively. Reversibility of unfolding was determined by

measuring the ¯uorescence of a diluted urea-denaturedsample. Each urea titration curve was analysed accord-ing to the method of Santoro & Bolen (1988) to yield thefree energy of unfolding in the absence of denaturant(�GH2O

UÿF) the dependence of �G on denaturant concen-tration (m value) and the urea concentration of half dena-turation (Cm).

Kinetic analysis

Unfolding and refolding reactions were followed on aBio-Logic SFM-3 stopped-¯ow ¯uorimeter using an exci-tation wavelength of 280 nm and monitoring the ¯uor-escence emission change over 335 nm. A NesLab RTE-200 water circulator was utilised to maintain the tem-perature at a constant value throughout the instrument.All the experiments were performed at 28 �C in 50 mMacetate buffer (pH 5.5), at a ®nal protein concentration of0.02 mg/ml. The refolding experiments were carried outby a 30-fold dilution of AcP samples denatured in 7 Murea into solutions containing various concentrations ofurea ranging from 0 to 3 M. The unfolding experimentsat ®nal urea concentrations up to 8.1 M were performedby a tenfold dilution of urea-free AcP samples. Therefolding experiments in the presence of TFE were car-ried out by a 30-fold dilution of AcP samples denaturedin 40 % TFE into solutions containing various concen-trations of TFE ranging from 0 to 16.5 %. In some cases,a 40-fold dilution of AcP samples denatured in 30 % TFEwas used in order to reach a lower ®nal TFE concen-tration (e.g. 0.75 %). The results obtained under bothexperimental conditions were comparable. The ¯uor-escence traces were ®tted to exponential functions for thedetermination of the folding and unfolding rate con-stants, kf and ku, respectively. Plots of lnkf and lnku

against urea concentration were ®tted linearly to yieldthe natural logarithm of the folding and unfolding ratesin water (ln kH2O

f and ln kH2Ou , respectively) and their

dependences on urea concentration (mf and mu, respect-ively). The m, �GH2O

UÿF and Cm values were calculatedfrom these kinetic data as shown elsewhere (Jackson &Fersht, 1991). f values were calculated as described byChiti et al. (1999c).

Acknowledgements

The authors thank Daniele Veggi for technical supportand Daizo Hamada and Tobin Sosnick for valuable dis-cussions. We are also indebted with Giuseppe Pieraccinifor performing the mass spectrometry experiments at theCentro di Spettrometria di Massa of the UniversitaÁ diFirenze. F.C. is supported by the Accademia dei Lincei.The stopped-¯ow ¯uorimeter was a generous gift fromthe Cassa di Risparmio di Firenze. The research of theDipartimento di Scienze Biochimiche is supported, inpart, by funding from the Ministero dell'UniversitaÁ edella Ricerca Scienti®ca e Tecnologica (project ``Folding emisfolding di proteine'', fondi ex 40 %). The OxfordCentre for Molecular Sciences is supported by the Bio-technology and Biological Sciences Research Council, theEngineering and Physical Sciences Research Council andthe Medical Research Council. The research of C.M.D. issupported, in part, by an International Reasearch Scho-lars award from the Howard Hughes Medical Institute,and by the Wellcome Trust.


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Edited by C. R. Matthews

(Received 3 March 2000; received in revised form 11 May 2000; accepted 12 May 2000)

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