non-native local interactions in protein folding and stability: introducing a helical tendency in...

J. Mol. Biol. (1997) 268, 760±778

Non-native Local Interactions in Protein Folding andStability: Introducing a Helical Tendency in the Allbbb-Sheet aaa-Spectrin SH3 Domain

JesuÂs Prieto1, Matthias Wilmans1, MarõÂa Angeles JimeÂnez2

Manuel Rico2 and Luis Serrano1*

1EMBL, Meyerhofstrasse 1Heidelberg, D69012, Germany2Instituto de Estructura de laMateria, CSIC, Serrano 119Madrid, 28006, Spain

Abbreviations used: TFE, tri¯uorotype; NOE, nuclear Overhauser enhtwo and three-dimensional; TOCSY,spectroscopy; NOESY, NOE spectrocorrelated spectroscopy; ppm, parts

0022±2836/97/190760±19 $25.00/0/mb9

The relative importance of secondary structure interactions versus tertiaryinteractions for stabilising and guiding the folding process is a matter fordiscussion. Phenomenological models of protein folding assign an im-portant role to local contacts in protein folding and stability. On theother hand, simplistic lattice simulations ®nd that secondary structure ismainly the product of protein compaction and that optimisation of fold-ing speed seems to require small contributions of local contacts to thestability of the folded state. To examine the extent to which secondarystructure propensities in¯uence protein folding and stability, we havedesigned mutations that introduce a strong non-native helical propensityin the ®rst 19 residues of the a-spectrin SH3 domain. The mutant proteinshave the same three-dimensional structure as the wild-type, but they areless stable and have less co-operative folding transitions. There seems tobe a relationship between the non-native helical propensity and the com-paction of the denatured state. This suggests that in the denaturedensemble under native conditions there is a signi®cant proportion ofcompact structures with non-native secondary structures. Our resultsdemonstrate that non-local interactions can overcome strong non-nativesecondary structure propensities and, more important, that optimisationof folding speed and co-operativity requires the latter to be relativelysmall.

# 1997 Academic Press Limited

Keywords: a-helix; secondary structure; b-sheet; NMR; CD
*Corresponding author
Introduction

The relative importance of local versus non-localinteractions for stabilising the native state of a pro-tein and guiding its folding process is a matter fordiscussion. Local interactions are those between re-sidues close in sequence and could be involved inproducing the secondary structure propensities.Non-local interactions are involved in ®xing thesecondary structure elements and de®ning the ter-tiary structure. Local interactions have been con-sidered to play an important role in protein foldingand stability, in a variety of phenomenologicalmodels of protein folding (An®nsen & Scheraga,1975; Wetlaufer, 1973; Karplus & Weaber, 1976,

ethanol; wt, wild-ancement; 2D, 3D,total correlated

scopy; COSY,per million.

70984

1994; Harrison & Durbin, 1985; Wright et al., 1988;Kim & Baldwin, 1990), but not in all of them(Ptitsyn, 1993). The ®nding that very short proteinfragments could have signi®cant native-like sec-ondary structure in aqueous solution (for a review,see Dyson & Wright, 1993) strongly supportedthese models. More recently, statistical mechanicstheory and computer simulations of folding, haveallowed us to do experiments with the computerand analyse the relative role of local versus non-local interactions (for recent reviews, see Karplus &Sali, 1995; Dill et al., 1995; Bryngelson et al., 1995).More simplistic lattice simulations ®nd that sec-ondary structure is mainly the product of proteincompaction (Dill et al., 1995) and that optimisationof folding speed seems to require small contri-butions of local contacts to the stability of thefolded state (Govindarajan & Goldstein, 1995;Abkevich et al., 1995). On the other hand, off-latticesimulations have shown that inclusion of main-chain to main-chain hydrogen bonding is needed

# 1997 Academic Press Limited

Non-native Helical Tendency in All �-Sheet Protein 761

to obtain a secondary structure content akin to thatfound in proteins (Hunt et al., 1994). In the last fewyears, statistical mechanical models for proteinfolding (Bryngelson et al., 1995) have found a simi-lar contribution for local and non-local interactionson de®ning the energy gap between the nativestate and the rest of the conformations (Luthey-Schulten et al., 1995; Saven & Wolynes, 1996).Therefore, the role of local interactions in proteinfolding and stability is still an open question.

While computer models can easily alter the ratiobetween local and non-local interactions, until veryrecently there has not been a clear experimentalstrategy to do the same. The development of ahelix/coil transition algorithm tuned for heteropo-lypeptides (AGADIR; MunÄ oz & Serrano, 1994,1995a), allows the design of speci®c mutations thatincrease the helical propensity of a polypeptide se-quence. This permits, once residues in a-helicesthat are solvent-exposed and making only localinteractions are identi®ed, us to mutate them inorder to speci®cally enhance the helical tendencyof the protein segment (MunÄ oz et al., 1996). Thistype of experiment has been done for all the helicesof the chemotactic protein from Escherichia coli,CheY (MunÄ oz et al., 1996) and of the activation do-main of procarboxypeptidase A (Ada2h) (Villegaset al., 1996). In both cases it was found thatenhancement of native local interactions makesproteins less sensitive to thermal and chemical de-naturation, but the folding transition became lessco-operative. These results indicate that native-likelocal interactions affect the denatured state produ-cing its overall compaction, suggesting that innatural proteins the partition function of the de-natured ensemble presents a signi®cant number ofconformations that have residual native-like sec-ondary structure. An interesting question is: areconformations possessing non-native secondarystructure also statistically abundant in the de-natured state of a protein? One way to ®nd out ifthis is the case is to do the opposite experiment,which means introducing non-native local inter-actions that will be made only in the denatured en-semble of conformations, not in the folded state.

The protein we have chosen for our work is thea-spectrin SH3 domain. The three-dimensionalstructure of this domain has been obtained byX-ray diffraction (Mussachio et al., 1992) and NMR(Blanco et al., 1997). The a-spectrin domain is a 60residue protein with no disulphide bridge, whichfolds as a ®ve-stranded orthogonal b-sheet sand-wich. The kinetic analysis of this protein is simpli-®ed by the fact that only the denatured and nativestates are signi®cantly populated and thereforechanges in the unfolding and/or refolding ratescan be easily assigned to any of these two states, aswell as to the transition state (Viguera et al., 1994).The ®rst antiparallel b-sheet is composed of strandsb5, b1 and b2 and the second b-sheet is composedof strands b2, b3 and b4. Strand b2 participateswith its N-terminal residues at the ®rst b-sheet andwith its C-terminal residues at the second b-sheet.

The SH3 fold contains four distinct loops that are,in part, named by their functions (Musacchio et al.,1992). Strands b1 and b2 are connected by the RTloop that includes 19 residues. This loop wrapsaround one face of the second b-sheet. The con-formation of this loop is established by severalintra-loop hydrogen bonds that form an irregularb-sheet-like network. The remaining three loopsare referred to as the N-src loop, the distal loopand the 310-loop.

An interesting feature of this protein is that the®rst and last b-strands are quite solvent-exposedand in the former case there are ®ve residues pre-ceding it which adopt a random conformation(Musacchio et al., 1992). This allows a greater free-dom in the design of mutations to enhance helicalpropensities, since an important requirement inthese type of studies is to modify only local inter-actions and not contacts with other parts of theprotein. Here, we have selected the ®rst 19 residuesof the SH3 domain, containing the ®rst b-strandand part of the RT-loop, to alter its secondarystructure propensities. The wild-type and the mu-tant fragments have been analysed by far-UV CDand NMR in aqueous solution. We have mutatedthe same residues in the protein, determined theX-ray structures of the two mutants that possesshigher helical tendency, and analysed the effectof enhancing through local interactions non-nativehelical propensities on the stability and folding ofthis domain.

Results

Design of the mutants

Increasing the helical propensity of a peptide se-quence is easy in principle if we have total freedomto mutate any residue. This is not the case here,since we want to introduce non-native secondarystructure propensities in the SH3 domain by dis-turbing only local interactions and not tertiary con-tacts. Also, we cannot mutate polar to large apolar(L, I, V, M, F, Y and W) residues, since we canmodify the structure of the unfolded state undernative conditions. Therefore, the number of resi-dues that we can mutate is small. Secondary struc-ture prediction algorithms like those of Chou &Fassman (1978) predict several helical regions forthis mainly all b-protein, thus indicating the exist-ence of some helical propensity in the protein(Figure 1A). The helix/coil transition algorithmAGADIR1s (a version of AGADIR: MunÄ oz &Serrano, 1994, 1995a), which uses the classical one-sequence approximation (MunÄ oz & Serrano, 1995b,1997), predicts several regions in the protein tohave some helical tendency (Figure 1A). NMRstructural characterisation of peptides spanning thedifferent b-hairpins and the three-stranded b-sheetof this protein, showed that in the presence of 30%TFE some of the b-strands became a-helices(Viguera et al., 1995; Figure 1A). More interestingly,the three independent approaches, in general,

Figure 1. A, Secondary structureprediction of the spectrin SH3domain, helical propensities andconformational shifts of the Ca pro-tons. In boxes we show the seg-ments predicted to be helical by theChou±Fassman algorithm (1978).The broken line shows the helicalpopulation at a residue level pre-dicted by the helix/coil transitionalgorithm AGADIR1s (MunÄ oz &Serrano, 1995a, b). The continuousline with ®lled circles presents theconformational shifts of the Ca pro-tons corresponding to protein frag-ments encompassing the wholeSH3 sequence, analysed in 30%TFE (Viguera et al., 1995). B, Aminoacid sequence and secondary struc-ture elements in the a-spectrin SH3domain. The primary sequence ofthe wild-type protein (WT) and ofthe S, AE, AE-S and AE-K mutantsare shown below the Kabsch &Sander (1983) secondary structureassignment. The length of the pep-tides analysed here is shown belowby an arrow. C, AGADIR1s predic-tion of the helical content at a resi-due level of the wild-type anddesigned mutant peptides. The ex-perimental conditions introducedinto the program were pH 7.0 and278 K. Filled circles, WT peptide;open triangles, S peptide; open cir-cles, AE peptide; ®lled triangles,AE-S peptide; continuous line, AE-K peptide.

762 Non-native Helical Tendency in All �-Sheet Protein

agree on the regions that have non-native helicaltendency. Logically, we should use one of these re-gions for our experiments, since the amount of en-ergy that we need to provide to a sequence withsome helical propensity, to increase its helical con-tent, is small. This is because of the co-operativenature of the helix/coil transition (MunÄ ox &Serrano, 1995a,b).

Inspection of the three-dimensional structure ofthe protein obtained in solution (Blanco et al., 1997)or in the crystal form (Mussachio et al., 1992),shows that in SH3 the ®rst ®ve residues are notstructured (Figure 2). These ®ve non-structured re-sidues are followed by a protein region (residues 6

to 18) that has some helical tendency (Figure 1A).Therefore, we chose this N-terminal region as ourtarget (Figure 2). The mutations were designed toenhance a-helix propensity as indicated by AGA-DIR1s and only considering polar to polar or polarto Ala changes.

In the non-structured region, Thr4 and Gly5were simultaneously substituted by Ala and Glu tocreate a capping box motif (Harper & Rose, 1993;Zhou et al., 1994) to nucleate the helix (AE pep-tide). In the RT loop following b-strand 1 there isan Asp residue ¯anked by two Tyr residues(Figure 2), which is often Ser in other SH3 do-mains. Since Asp14 is a very bad residue close to

Figure 2. Ribbon representation of the mutated regionin the a-spectrin SH3 domain. In green we show themutated 1 to 19 region. The target Asp14 is shown inatom colour code. The other two target residues, Thr4and Gly5, are not shown, since the ®rst ®ve residues arenot structured in solution or in the crystal.

Table 1. Percentage helical population and helix lengthin the wild-type and mutant peptides, determined byfar-UV CD and NMR

HH2Oa (CD) HH2O

b (NMR)Peptide (%) (%) AGADIRc H. lengthd

WT 4.1 (5.5) 0 2.2 n.d.AE 6.4 (8.6) 7.0 (6.2) 9.2 3-12S 4.5 (6.2) 3.2 (2.3) 3.6 7-12AE-S 11.0 (14.3) 10.0 (10) 14.6 3-14AE-K 18.0 (24.0) 16.0 (20) 34.0 3-17

a Helical content determined from the ellipticity at 222 nmusing the method of Chen et al. (1974). In parentheses is thehelical content after correction for the aromatic contribution(Chakrabartty et al., 1994).

b Helical content calculated from the conformational shifts ofthe Ca protons (Jimenez et al., 1993). The values in water wereobtained after correcting for the aromatic contribution in therandom-coil state (Viguera et al., 1995). Only the values for theestimated helix length are considered (N and C-caps are notincluded), and then averaged for the whole peptide. In parenth-eses is the helical content determined after subtracting the con-formational shifts of the wild-type from the correspondingvalues in the mutants.

c Helical content predicted by AGADIR1s (MunÄ oz et al.,1995a, b) with the standard one-sequence approximation.

d Helix limits determined using the conformational shifts ofthe Ca protons and the i, i�3 NOEs (see Materials and Meth-ods). n.d., Non-determined, since the conformational shifts inwater are very close to the random coil.


the C-cap of a-helices (Huyghues-Despointes et al.,1993), we mutated it to Ser, which can make thesame side-chain to main-chain H-bond as the Aspresidue (S peptide). The triple peptide mutantsAla4/Glu5/Ser14 (AE-S) and Ala4/Glu5/Lys14(AE-K) were also made. The Asp residue was mu-tated to Lys, because it is very favourable at C ter-mini of helices due to its interaction with the helixdipole (Serrano et al., 1992; MunÄ oz & Serrano,1995a). Figure 1B shows the SH3 sequence, to-gether with its secondary structure assignmentusing the Kabsch & Sander (1983) criteria and thesequence of the ®ve peptides analysed here.Figure 1C shows the prediction by AGADIR1s ofthe helical content at a residue level for these pep-tides. AGADIR1s predicts a very small helical ten-dency for the wild-type peptide in the regioncomprising residues Lys6 to Leu12. Mutation ofAsp14 to Ser eliminates the unfavourable inter-action of the negative charge with the helix dipole,and the helical content is predicted to increaseslightly in the same region, with the helix extend-ing to Ser14. The substitution of Thr4 and Gly5 byAla and Glu, respectively, increases the helical con-tent of the previous region and, interestingly, ex-tends the helical length at the N terminus by onehelical turn. In this mutant, Asp2 is the N-cap resi-due. Incorporating the Asp14Ser mutation on theAE peptide further increases the helical content.Finally, in the triple peptide mutant AE-K there is

a signi®cant increase in the helical content and thehelical population comprises Glu3 to Glu17. Table 1shows the average helical content predicted for the®ve peptides by AGADIR1s.

Far-UV CD analysis of the wild-type andmutant peptides

Figure 3 shows the far-UV CD analysis of the®ve peptides in aqueous solution. The spectra inwater solution (Figure 3A) indicate a signi®cant in-crease in the helical population for peptides AE,AE-S and AE-K, as determined from the change inellipticity at 222 nm. In the case of the S peptide,this increase is very small (see Table 2) and withinexperimental error. In none of the cases can we ob-serve double minima at 208 and 222 nm, typical ofthe helical conformation. This could be because thehelical content of the peptides is low, but also dueto the presence of two Tyr residues at position 13and 15, which could have a large effect on the far-UV CD spectrum band at 222 nm (Chakrabarttyet al., 1993). Subtraction of the far-UV CD spectrumof the wild-type peptide from those of the otherpeptides (Figure 3B) shows very clearly that in thecase of peptides AE-S and AE-K, the change in theshape of the spectrum is due to the appearance ofa helical population (see Table 1). The results arenot so clear for peptides AE and S, but we mustconsider the aromatic contribution that could ob-scure small conformational changes. Addition of30% TFE (Figure 3C) results in a CD spectrum forthe ®ve peptides typical of an a-helix, thus indicat-

Figure 3. Secondary structure characterisation of peptides corresponding to residues 1 to 19 of the wild-type andmutant sequences. A, Far-UV CD analysis of the ®ve peptides in aqueous solution at pH 7.0 and 278 K. From less tomore negative ellipticity at 222 nm: open circles, WT; open triangles, S; ®lled circles, AE; ®lled triangles, AE-S; opensquares, AE-K. B, Difference spectra obtained from subtracting the WT spectra from those of the other four peptides.CD spectra of the ®ve peptides in aqueous solution at pH 7.0 and 278 K: open triangles, S peptide; ®lled circles, AEpeptide; ®lled triangles, AE-S peptide; open squares, AE-K peptide. C, CD spectra of the ®ve peptides in 30% TFEsolution at pH 7.0 and 278 K. From less to more negative ellipticity at 222 nm: open circles, WT; open triangles, S;®lled circles, AE; ®lled triangles, AE-S; open squares, AE-K. D, TFE titration of the ®ve peptides: open circles, WT;open triangles, S; ®lled circles, AE; ®lled triangles, AE-S; open squares, AE-K.


ing that even the wild-type peptide has some heli-cal tendency as predicted by AGADIR1s (seeFigure 1C). Interestingly, the value of the mean re-sidue ellipticity at 222 nm is not the same for allthe peptides. These differences could be due to thefact that we have not ®nished titrating with TFE,or because the length of the a-helix segment isdifferent in the peptides, as predicted by AGA-DIR1s. Figure 3D shows the results of the TFEtitration for the ®ve peptides. In all cases the pep-

tides have reached a plateau around 30% TFE andtheir helical content is different. The wild-type pep-tide (WT) and the S peptide have similar helicalcontents (�45%). The same happens for the AE-Sand AE-K peptides (�68%), while the AE peptideis between both groups (�60%). Assuming that thehelical region is populated to 100% in the differentpeptides, and using the helical content of the pep-tides obtained after correcting for the aromatic con-tribution (Chakrabartty et al., 1993), we ®nd that in

Table 2. TFE titration for the wild-type and mutantpeptides

ma �GH2Oa ��Gb ��Ga

Peptide (kcal/mol) (kcal/mol) (kcal/mol) (kcal/mol)

WT 15 � 1.5 2.8 � 0.3 ± ±S 17 � 2.8 2.9 � 0.5 0.1 0.2AE 14 � 0.8 2.2 � 0.2 0.6 0.4AE-S 12 � 1.1 1.4 � 0.1 1.4 1.2AE-K 12 � 1.8 0.9 � 0.2 1.9 1.9

a These values have been obtained by ®tting equation (3) to theTFE titration curves.

b Differences in free energy calculated with AGADIR1s, asindicated in Results.


peptides WT and S the helical segment is �11 resi-dues, in the AE �15 and in the AE-K and AE-S,�17 amino acid residues.

Evaluation of the free energy in theenhanced aaa-helices

The change in free energy for a-helix formationupon mutation of each a-helix cannot be calculatedprecisely using a standard two-state model. Amore precise estimation can be obtained by ®ttinga helix/coil transition algorithm to the changes inhelical content detected by far-UV CD (MunÄ ozet al., 1996). The difference in free energy for a-helix formation (�GHel) between the pseudo-wild-type and mutated peptides was calculated fromtheir differences in helical content as measured byfar-UV CD, after aromatic correction. The algor-ithm for the helix/coil transition AGADIR1s wasused to ®t the far-UV CD data of the peptides topolyalanine sequences with the same length as thepeptides analysed here. The ®tting was carried outby modifying the intrinsic propensity of alanine,maintaining the mean-residue enthalpic contri-bution (hydrogen bond) at a constant value ofÿ0.792 kcal/mol, to reproduce the helical contentshown by the wild-type and mutated peptides.This procedure intends to eliminate assumptionson the individual energy contributions and on thedistribution of helicity throughout the peptide(MunÄ oz et al., 1996). The net change in free energyfor a-helix formation is obtained ®nally from thedifference in Ala intrinsic propensity between thewild-type and mutated peptides as indicated inequation (1), where n is the peptide length:

��GHel �Xnÿ2

��Gintri MUT ÿ�Gintri WT� �1�Such a calculation indicates that we have providedÿ0.2 kcal/mol to mutant S, ÿ0.4 kcal/mol to mu-tant AE, ÿ1.2 kcal/mol to mutant AE-S andÿ1.9 kcal/mol to mutant AE-K (Table 2). An inde-pendent way in which to estimate the free energyprovided to the helical conformation upon mu-tation is to consider that TFE is having an effectopposite to that of urea or guanidium hydrochlor-ide (Sancho et al., 1992). If the increase in helicalcontent follows a two-state transition, which seems

to be the case in our peptides as indicated by anisodicroic point at 202.4 nm (data not shown), it ispossible to estimate the equilibrium constant forthe formation of helix using equation (2):

KTFE � KH2O exp��m=RT��TFE�=�water�� 2�where KTFE is the equilibrium constant for the for-mation of helix in TFE/water and m is character-istic for each peptide but appears to beproportional to the lengths of related helices(Jasanoff & Fersht, 1994; Kippen et al., 1994). It ispossible to obtain the free energy required to ob-tain an a-helix in the different peptides by ®ttingthe TFE titration curves (Figure 3D) to equation(3):

E � fEN ÿ EU exp��m�TFE� ÿ�GH2O�=RT�g=� f1� exp��m�TFE� ÿ�GH2O�=RT�g �3�

where E, EN and EU are the observed mean residueellipticity at a particular TFE concentration, themean residue ellipticity of the native state and themean residue ellipticity of the unfolded state, re-spectively. �GH2O is the free energy of unfolding inthe absence of TFE. Fitting the data of Figure 2D tothis equation shows that the S mutation has stabil-ised the helical conformation by �0.1 kcal/mol, theAE by �0.6 kcal/mol, the AE-S mutations by �1.4and the AE-K by �1.9 kcal/mol (Table 2). Thesenumbers are in very good agreement with those de-termined with the helix/coil algorithm method.

NMR analysis

Figure 4 shows the NOE summary for the differ-ent peptides. In the WT and S peptides there areno daN(i, i�3) and dab(i, i�3) NOEs, indicative ofthe presence of a helical population. In the WTpeptide there are two NOEs involving the side-chains of residues 11 and 13, and 12 and 15, show-ing the existence of some local conformation inthat region. Substituting Thr4 and Gly5 with Alaand Glu, respectively, in the AE peptide, results inthe appearance of a signi®cant number of i, i�3NOEs in the region Glu3 to Ala11, characteristic ofhelical conformation. Introducing the Asp14Sermutation into the EA peptide (EA-S), results in anextension of the peptide region presenting NOEscharacteristic of a-helices (residues 3 to 14). TheAsp14Lys mutation in the AE peptide (AE-K)further extends the helical region up to Gln16.Table 1 shows the length of the helical segments,which is in very good agreement with the predic-tion by AGADIR1s.

Small deviations from random coil values couldbe due to the chemical environment resulting fromthe surrounding residues. One way to determine ifthey really correspond to a conformational changeis to use the conformational shifts of the wild-typepeptide as reference values. In this way, except forthe mutated residues, the chemical environmentshould be the same. Figure 5 shows the differencein the conformational shifts of Ca protons with re-

Figure 4. NOE summary of the ®ve peptides in aqueous solution. The labelling at the top of each summary indicatesthe peptide analysed. Asterisks (*) represent NOEs that could not be detected because of signal overlapping. Theintensity of the bars for the sequential NOEs are proportional to the intensity of the NOE crosspeaks (weak, mediumand strong). dsch, Side-chain to side-chain NOEs.


spect to the WT values for the four mutant pep-tides. In the case of peptide S, we can identify asmall up®eld shift of the CaH protons in the centreof the peptide, with respect to the WT protein,

Figure 5. Conformational shifts of the Ca protons. Thedifference between the conformational shifts of the wild-type and mutant CaH protons are shown with respectto the amino acid sequence: open triangles, S; ®lled cir-cles, AE; ®lled triangles; AE-S; open squares, AE-K. TheXs represent the mutated positions. On top we show theextension of the a-helix conformation in the AE-K pep-tide as determined from the i, i�3 and i, i�4 NOEs(Figure 4) and the conformational shifts.

which is indicative of a nascent helix, and moresigni®cant changes in the region around Tyr13 andTyr15. These changes are probably not signi®cant,since aromatic residues can produce a large changein the chemical shifts of the neighbour protons(Merutka et al., 1995) and mutation of Asp14 couldproduce a large change in the surrounding pro-tons, due to small conformational rearrangementsof the aromatic rings in the random coil state. Forpeptides AE, AE-S and AE-K, the up®eld-shiftedCaH resonances with respect to the WT protein(Figure 5), are indicative of the presence of a helicalpopulation in approximately the same regionsdetermined by the NOE analysis (Table 1).Calculation of the helical content from the confor-mational shift of the CaH protons (JimeÂnez et al.,1993; Table 1 and Materials and Methods), is invery good agreement with the values obtained byCD and predicted by AGADIR1s. An independentway to determine structured populations of pep-tides in aqueous solution is to use the 3JHNa coup-ling constants (Ramirez-Alvarado et al., 1996). Thismethod works well when there is a large spreadingof the signals in the monodimensional NMR spec-trum of a peptide. In the case of helical peptidesthe spread is small and therefore the coupling con-stant values must be obtained from the 2D spectra,which is more prone to experimental errors (an es-timated error of � � 1 Hz in our case, due to the1.6 Hz resolution in the TOCSY spectra). This largeerror precludes obtaining a reliable estimate of thepopulation in all the peptides. However, in thecase of AE-S and AE-K, the 3JHNa values are clearlylower than in WT, for the whole peptide region


considered helical in the chemical shift and NOEanalysis (data not shown). We have also calculatedthe temperature coef®cients of the WT, AE-S andAE-K peptides. Although the protection factors areclose to random-coil values, they are much largerin the AE-S and AE-K (specially in the AE-K) pep-tides than in the WT protein, in the region corre-sponding to the helical conformation estimatedfrom the NOEs and chemical shifts (data notshown).

Figure 6. X-ray structure of the region around the mutatedsional structure of the region around Asp14 in the WT (vioshow the H-bond made by Ser14 and Asp14 in the AE-S aSer14 and Lys14 in the three proteins, respectively, are showBottom: Density map of residues Tyr13, Ser14 and Tyr15. Tbetween the WT and the AE-S mutant.

Three-dimensional structure of the mutants

The AE-S and AE-K mutants crystallised underthe same conditions as WT-SH3 (Musacchio et al.,1992) and have isomorphous cell dimensions. The3D structures of the AE-S and AE-K mutants ofSH3 have been solved by difference Fouriermethods and re®ned at 2.05 AÊ resolution (Figure 6and Table 3). The structures are virtually identicalwith that of the WT SH3 structure (Musacchio et al.,

positions of proteins AE-S and AE-K. Top: Three-dimen-let), AE-S (red) and AE-K (green) mutants. In yellow wend WT mutants, respectively. The side-chains of Asp14,n, as well as the main chain of the surrounding residues.he green density corresponds to the difference in density

Table 3. Crystallographic statistics

AE-S AE-K

A. Crystallographic data:No. collected data 15,343 14,234No. unique data 4736 3726Maximum resolution (AÊ ) 2.05 2.05Completeness (%) 94.5 73.8Rmerge (%)a 3.8 4.0

B. Re®ned modelsb

No. residues 57 56No. protein atoms 470 464No. water molecules 27 25hBi main-chain (AÊ 2) 28.1 28.7hBi side-chain (AÊ 2) 35.1 35.5hBi water molecules (AÊ 2) 39.4 39.1Rcrystal (%)c 21.3 22.4Rfree (%)c 29.2 28.3rms (bond length) 0.010 0.010rms (bond angles) (deg.) 1.44 1.40

a Rmerge��hkl �jjIhkl,j ÿ hIhklij/�hkl,jhIhkli, using Friedel mates asindependent observations.

b All observed data between 6.0 AÊ and 2.05 AÊ with intensities51 s were used for re®nement.

c R��hkl IFobs ÿ Fcalcj/�hkl Fobs, where 90% of the data wereused for re®nement (Rcrystal) and the remaining 10% of the datareserved for the calculation of Rfree.

Figure 7. Urea denaturation of the wild-type andmutant proteins. Each curve represents the normaliseddata of two experiments, performed on two differentdays: open circles, WT protein; open triangles, S protein;®lled circles, AE protein; ®lled triangles, AE-S protein;open squares, AE-K protein.


1992). The rms deviation of each mutant with theWT structure is 0.17 AÊ . Residues Ala4 and Glu5are not visible in the electron density. The same re-sidues are delocalised in the WT-SH3 structure(Musacchio et al., 1992). In the AE-K mutant theside-chain of Lys14 can be well traced in the elec-tron density, but it has rather high B-values,suggesting that it is rather ¯exible. Its terminalamino group is not involved in hydrogen bondswith the rest of the molecule and this should lar-gely destabilise the protein. In the AE-S mutant theg-hydroxyl group of Ser14 functions as a hydrogenbond donor to the main-chain nitrogen atom ofLys27, thereby replacing the hydrogen bond ofAsp14 (Od1) in the WT structure (Figure 6). There-fore, in this mutant the only changes introduced bymutating Asp14 to Ser are the substitution of acharged hydrogen-bond for a neutral one and theelectrostatic interactions made by Asp14 with therest of the protein.

Urea denaturation

The urea denaturation experiments of the ®veproteins are shown in Figure 7. Fitting the datawith equation (3) (see Materials and Methods), al-lows us to calculate the thermodynamic par-ameters in equilibrium for the different proteins.Mutation of Thr4 and Gly5 to Ala and Glu, re-spectively, results in only a small destabilisation ofthe protein. On the other hand, mutating Asp14along (S mutant), or in the AE mutant (AE-S andAE-K mutants), produces a large destabilisation. Inthe case of the AE-K mutant the protein is partly

denatured at 25�C, as expected from the fact thatthe side-chain to main-chain hydrogen bond ofAsp14 with Lys27 is lost. This is con®rmed by pre-liminary NMR analysis, which shows two sets ofsignals, not only in the region close to the mu-tations, but also in regions far away (i.e. Ala55packs against one of the Trp residues, has itscharacteristic signal corresponding to the foldedstate and another one corresponding to random-coil values; data not shown). The NMR signals ofthe folded state are identical with that of the WTprotein (Blanco et al., 1997), in agreement with thecomparison of the crystallographic structures ofthe two proteins. To ®t the AE-K mutant we as-sume that the ¯uorescence of the folded state andits dependence on urea concentration is similar tothat of the WT. This is a reasonable assumption,since the 3D structure of this mutant is identicalwith that of the WT protein (see above). Compari-son of the free energies for these proteins showsthat the effect of the mutations at positions Thr4/Gly5 and Asp14 are additive, within experimentalerror (Table 4). The slope m, which is related to thechange in solvent-accessible area when going fromthe folded to the unfolded state (Pace, 1986), seemsto be related to the stability of the protein. The lessstable the protein is, the smaller the m value,although some of the changes are within exper-imental error (Table 4). In the case of the AE-K mu-tant, the slope m is clearly smaller; however, wemust regard this value with caution, since we aremissing part of the denaturation curve.

Temperature denaturation

Figure 8A shows the temperature denaturationpro®le of the wild-type and three mutants at pH7.0. As we found for the chemical denaturation ex-periments, introduction of the different mutations

Table 4. Thermodynamic parameters in equilibriumobtained from chemical denaturation of the wild-typeand mutant proteins

meqa �Geq

b ��Geqc

(kcal/(mol M)) (kcal/mol) (kcal/mol)

WT 0.72 � 0.01 3.58 � 0.03 ±AE 0.71 � 0.01 3.30 � 0.06 0.28S 0.70 � 0.01 2.52 � 0.05 1.06AE-S 0.69 � 0.01 2.24 � 0.05 1.34AE-Kd 0.64 � 0.03 0.30 � 0.14 3.38

The experimental conditions and analysis are described inMaterials and Methods. The data shown here were obtainedfrom ®tting the normalized data corresponding to two differentexperiments. The errors shown correspond to the ®tting errors.Independent ®tting of the two experiments produce differencesthat are of the same order as the errors shown here.

a Variation of the natural logarithm of the equilibrium con-stant with urea concentration.

b Free energy in kcal molÿ1 of unfolding in water, obtainedby ®tting the curves to a two-state transition, as indicated inMaterials and Methods.

c Differences in free energy of unfolding between WT and themutant proteins.

d The thermodynamic parameters for this mutant wereobtained by considering that the ¯uorescence emission of thefolded state and its dependence on urea, was the same as thatof the other four proteins. This is reasonable, since its three-dimensional structure is identical with that of the WT protein.


destabilises the protein. The AE-K mutant is partlyunfolded, even at 5�C (data not shown). Fitting thetemperature denaturation curves involves moreparameters than urea denaturation curves, andproduces larger errors. Therefore, we have not ana-lysed the AE-K mutant, since the thermodynamicvalues will be subjected to a large error. Analysisof the temperature denaturation curves of the WTprotein and of the other three mutants shows a sig-ni®cant change in the Tm value for the S and AE-Smutants (Table 5). The free energy values at 298 K(�G) are in good agreement with the values ob-tained from chemical denaturation.

Figure 8. Temperature denaturation of the wild-type andvalues: wild-type protein, AE mutant, S mutant and AE-S mtype protein, S mutant, AE mutant and AE-S mutant.

The temperature denaturation of the a-spectrinSH3 domain is fully reversible at pH 2.5 (Vigueraet al., 1994). In Figure 8B we show the temperaturedenaturation pro®les for the WT, S, AE and AE-Sproteins at pH 2.5, and Table 5 shows the corre-sponding thermodynamic values. In all our mu-tants we have modi®ed the net charge of the SH3domain. Since electrostatic interactions are long-range, it is quite possible that although we havenot disturbed local interactions, we have intro-duced new favourable or unfavourable tertiaryelectrostatic interactions. If this is the case, it couldbe that the destabilising effect produced by thedifferent mutations is not due to an increase in thenon-native helical propensity, but rather to electro-static interactions. Performing temperature dena-turation at low pH allows us to eliminate theelectrostatic interactions lost when replacing Asp14by Ser or introduced when mutating Gly5 to Glu.Surprisingly, the denaturation curves of the WTand S mutants, and those of the AE-S and AE pro-teins, can be superimposed. In fact, approximatelysimilar Tm values are found between the WT and Smutants, indicating that the main destabilizing ef-fect of the S mutation is electrostatic in nature. Thisis not the case for the Gly5 to Glu mutation, sincethe AE and AE-S mutants still have a signi®cantdifference in the Tm values with the WT. Far-UVCD analysis of the corresponding peptides at pH2.5 shows that the destabilising effect produced byAsp14 at pH 7.0 has disappeared (Table 5). Again,this is not the case for the helix stabilising effectproduced by mutating residues 4 and 5 (AE-mu-tant). The increase in the helical content of some ofthe peptides (AE and AE-S) is correctly predictedby AGADIR1s (data not shown) and re¯ects thecomplex electrostatic interactions present in thepeptides.

mutant proteins. A, pH 7.0. In order of decreasing Tm

utant. B, pH 2.5. In order of decreasing Tm values: wild-

Table 5. Thermodynamic parameters in equilibrium obtained from temperature denaturation of thewild-type and mutant proteins at pH 7 and pH 2.5

Tma �H(Tm)b �H(298)c �G(298)d Helixe

(K) (kcal/mol) (kcal/mol) (kcal/mol) (%)

A. pH 7.0WT 338.9 � 0.1 49.0 � 0.5 16.1 3.8 � 0.7 5.5AE 336.2 � 0.1 43.0 � 0.4 12.4 3.1 � 0.6 8.6S 330.9 � 0.2 39.3 � 0.5 13.3 2.5 � 0.5 6.2AE-S 325.0 � 0.3 32.5 � 0.2 10.6 1.8 � 0.4 14.3

B. pH 2.5WT 324.6 � 0.5 34.4 � 0.7 12.9 1.9 � 0.4 5.5AE 317.7 � 0.5 32.0 � 0.4 16.0 1.5 � 0.3 18.0S 322.4 � 0.3 34.7 � 0.5 14.9 1.9 � 0.4 4.7AE-S 317.0 � 0.4 31.5 � 0.6 16.1 1.4 � 0.3 18.0

The thermodynamic values for the different proteins were obtained after ®tting the curves shown in Figure 8Aand B with equation (4) shown in Materials and Methods.

a The temperature at which 50% of the molecules are denatured (Tm).b Enthalpic change at the temperature in which 50% of the molecules are denatured.c Enthalpic change at 298 K.d Free energy of denaturation at 298 K. The error is around 20% as described by Viguera et al. (1994).e The helical content of the peptides, at pH 7.0, is shown in Table 1 and here for comparative purposes. The

far-UV CD spectra and determination of the helical contents of the same peptides at pH 2.5 was done in thesame way as experiments at pH 7.0. The pH was adjusted with HCl and the ionic strength was the same as atpH 7.0.


Unfolding and refolding analysis

The variation of the refolding and unfolding rateconstants of the wild-type and the three mutantswith urea is shown in Figure 9. Fitting the datawith equation (8) allows us to obtain the corre-sponding kinetic and thermodynamic parameters(Table 6). As we found with the wild-type protein,there is very good agreement between the thermo-dynamic parameters (�G and m) obtained in equi-librium and kinetically, once we correct for thecontribution of the cis-trans Pro isomerization(Viguera et al., 1994). With respect to the kineticparameters, the unfolding slope mu, which is re-

Figure 9. Kinetic analysis of the folding and unfoldingreactions of the wild-type and mutant proteins. Eachcurve represents the normalized data of two experi-ments, performed on two different days: open circles,WT protein; open triangles, S protein; ®lled circles, AEprotein; ®lled triangles, AE-S protein; open squares AE-K protein.

lated to the difference in solvent accessibility be-tween the folded and transition states, is the samefor all the proteins (around 0.25), except for theAE-K protein (0.29). In refolding there are somedifferences in the mF values, which are clearly sig-ni®cant in the case of the AE-S and AE-K mutants(Table 6). The calculated mkin values correlate verywell with the equilibrium m values (R�0.93),although the former are larger. The fact that in thiscase the AE-K mutant still shows the smaller mvalue, argues in favour of the differences found inequilibrium to be real. The small discrepancy be-tween equilibrium and kinetic values in all the pro-teins is due to the fact that we are not consideringthe refolding slope of those denatured moleculesthat contain a cis proline bond. This SH3 domaincontains two trans proline bonds, which isomerizein the denatured state (Viguera et al., 1994). Con-sidering a population of around 10% moleculeswith one or more cis proline bond, the mkin valuesare identical with those in equilibrium, within ex-perimental error. More interestingly, although thedifferences in mF values are very small for theother two mutants, there is a very good correlationbetween the mF values and the changes in helicalcontent observed with the corresponding peptides(Figure 10).

Discussion

The mutants used in this work have been de-signed to increase the non-native helical propensityin a region of the spectrin SH3 domain that corre-sponds to the ®rst 19 residues and contains its ®rstb-strand. Opposite to what has been done in thechemotactic protein from E. coli, CheY (MunÄ ozet al., 1996), or in the activation domain of procar-boxypeptidase A, Ada2h (Villegas et al., 1996), here

Table 6. Kinetic and thermodynamic parameters for the wild-type and mutant proteins

mU mF kU kF mkin �Gkin

Protein (kcal/(mol M)) (kcal/(mol M)) (sÿ1) (sÿ1) kcal molÿ1 Mÿ1 (kcal/mol)

WT ÿ0.24 � 0.01 0.55 � 0.01 0.007 � 0.001 3.90 � 0.08 0.79(0.74) � 0.01 3.78(3.58) � 0.1AE ÿ0.25 � 0.01 0.52 � 0.01 0.007 � 0.001 3.01 � 0.05 0.77(0.72) � 0.01 3.57(3.37) � 0.1S ÿ0.25 � 0.01 0.53 � 0.01 0.019 � 0.001 2.90 � 0.05 0.78(0.73) � 0.01 2.98(2.78) � 0.1AE-S ÿ0.24 � 0.01 0.48 � 0.01 0.026 � 0.001 2.13 � 0.03 0.72(0.67) � 0.01 2.62(2.42) � 0.1AE-K ÿ0.29 � 0.02 0.39 � 0.05 0.184 � 0.020 0.66 � 0.03 0.68(0.64) � 0.04 0.76(0.56) � 0.4

The experimental conditions and analysis are described in Materials and Methods. The data shown here were obtained from the ®t-ting of the normalized data corresponding to two different experiments. The errors shown correspond to the ®tting errors. Indepen-dent ®tting of the two experiments produces differences of the same order as the errors shown here. mU, dependence of the naturallogarithm of unfolding with urea; mF, dependence of the natural logarithm of refolding with urea; kU, unfolding rate constant inwater; kF, refolding rate constant in water; mkin, dependence of the natural logarithm of the equilibrium constant with urea, obtainedfrom the kinetic parameters mU and mF, which should be equivalent to the m value obtained from equilibrium denaturation analysis.�Gkin, free energy of unfolding determined from the kinetic parameters. In parentheses we show the mkin and free energy of dena-turation values after correcting for the isomerization of the two trans Pro residues. In the refolding experiments of chicken spectrinSH3 domain there is a slow phase with a rate constant of 0.0176(�0.0023) sÿ1, independent of urea concentration, which correspondsto the cis-trans isomerization of Pro residues (Viguera et al., 1994). This isomerization affects the free energy of unfolding in equili-brium and needs to be considered in the kinetic experiments to compare both sets of data. Assuming that 10% of the molecules inthe denatured state contain one or both Pro residues in the cis conformation (which is a conservative estimate), we ®nd that weoverestimate �Gkin by around 0.2 kcal/mol and mkin by around 0.045.


we have introduced non-native local conformationsto stabilise an a-helical conformation not present inthe folded state. By mutating the solvent-exposedunstructured Thr4 and Gly5 residues to Ala andGlu, respectively, as well as Asp14 to Ser or Lys,we favoured a helical conformation spanning resi-dues 3 to 17 of this SH3 domain.

Conformational behaviour of themutant peptides

The far-UV CD and the NMR analysis of thedifferent peptides indicate that all the mutations, toa different extent, increase the helical tendency ofthe sequence. This is clearer in the AE, AE-S andAE-K mutant peptides than in the S peptide. Theexact quanti®cation of the increase in the helicalcontent, either from the far-UV CD spectra or theNMR conformational shifts of the Ca protons, is

Figure 10. Correlation analysis between the changes inthe refolding mF value for the wild-type and mutantproteins and the helical content at pH 7.0 of the corre-sponding peptides encompassing residues 1 to 19.

subject to some error due to the presence of twoTyr residues (Tyr13 and 15). The two Tyr residuesshould decrease the negative ellipticity at 222 nm(Chakrabartty et al., 1993) and therefore producean underestimation of the helical content. On theother hand, the two aromatic residues could up-®eld shift the Ca protons of the surrounding resi-dues and consequently produce an overestimationof the helical content by NMR (Wishart et al.,1995). In principle, it is possible to correct forthe aromatic contribution to these parameters(Chakrabartty et al., 1994; Viguera & Serrano, 1995;Viguera et al., 1995), but the presence of two aro-matic residues so close in the sequence can resultin speci®c interactions in the random-coil state, notfound in model peptides (Viguera et al., 1995).However, the fact that we obtain a good agreementbetween the helical content determined by far-UVCD and NMR, after correcting for the aromatic ef-fect, indicates that our estimations of the helicalcontent are approximately correct. However, wemust consider that small errors in the determi-nation of the helical content can result in largeerrors in the quanti®cation of the free energy pro-vided by the newly introduced local interactions.This is especially important in the peptides withvery low helical content, due to the co-operativityof the helix/coil transition (MunÄ oz & Serrano,1995a; Viguera & Serrano, 1995). Consequently,although we have obtained similar values by usingtwo different methods, they must be regarded withcaution and must be considered only qualitatively.

Analysis of the mutant peptides reveals that themost effective mutations are those occurring at theN terminus of the WT peptide (Thr4Ala, Gly5Glu;peptide AE). Mutation of these two residues intro-duces a capping box motif in the peptide (DXXE;Harper & Rose, 1993). NMR analysis of the AE-Sand AE-K peptides, shows the expected NOEs in acapping box: between the Cb and Cg protons ofGlu5 and the amide proton of Asp2 and between


the Cb protons of Asp2 and the amide proton ofAla4. The point mutation Asp14Ser has a very lim-ited increase in the helical propensity of the ®rst b-strand region, which is within experimental error.However, the fact that mutating Asp14 to Ser inthe AE peptide (peptide AE-S) produces a signi®-cant increase of its helical content as well as ex-tending the helix limits to the C terminus, showsthat the increase is real. Finally, substitution ofSer14 by Lys in the AE peptide (AE-K) produces amore dramatic increase in the helical content andin water, and further extends the helix confor-mation up to Glu17.

Comparison between the predicted peptidehelical contents and experimental results

The program we used to design the mutations isa helix/coil-based algorithm, AGADIR1s, tuned forpredicting the helical behaviour of monomeric pep-tides in aqueous solution. In previous work weused the same algorithm to rationally modify thehelical content of protein fragments correspondingto a-helices, and in all the cases the designed pep-tides acquired the predicted helical content withina small error (MunÄ oz & Serrano, 1995a; Villegaset al., 1996; MunÄ oz et al., 1996). When comparingthe predicted helical content for the ®ve peptidesin this work with the values obtained by far-UVCD or NMR in aqueous solution, we ®nd a verygood agreement for the peptides having less than15% helix, and overprediction above this number.Helix/coil transition algorithms assume that thereare only two conformations, helix or random coil,and that in the latter there is no energy couplingbetween residues. It is probable than in a non-helical protein fragment there are favourable side-chain to side-chain interactions, not compatiblewith the helical conformation. Therefore, sincenon-helical conformations in the helix/coil theoryis part of the random-coil state, the rule that thereshould not be energy coupling between residues inthis state could not apply. This could result in anoverprediction of the helical content of the de-signed peptides, which should be worst when thedesigned peptide is expected to have between �20to �70% helix (the increase in helical content withfree energy is not linear and is most sensitive inthat region; MunÄ oz & Serrano, 1995a).

Another important aspect when designing thehelical propensities in a peptide sequence is thelength of the helical segment. As we have seen inResults, AGADIR1s predicted a small helical ten-dency for a central region of the WT peptide thatcorresponds to the ®rst b-strand in the protein. Thedifferent mutations we have introduced were pre-dicted to raise the helical content and to enlargethe helical region of the peptides. The NMRanalysis of the different peptides in water indicatesthat AGADIR1s, in general, correctly estimatesthe length of the helical segments in the differentpeptides.

Local versus non-local interactions indetermining the secondary structure of thefolded state

How important is the role of local interactions indriving the formation of secondary structure el-ements is still an open question (for a review, seeMunÄ oz & Serrano, 1996). On one hand, the struc-tural characterisation by far-UV CD and NMR ofprotein fragments spanning the whole sequence ofseveral different proteins (lysozyme, Segawa et al.(1991); myohemerytrin, Dyson et al. (1992a); plasto-cyanin, Dyson et al. (1992b); thermolysin C-term-inal domain, Jimenez et al. (1993); BPTI, Kemmink& Creighton (1993); B1 domain of the IgG bindingprotein, Blanco & Serrano (1995); barley chymo-trypsin inhibitor, CI-2, Itzhaki et al. (1995); andspectrin SH3 domain, Viguera et al. (1995)), hasshown that, in general, the majority of the frag-ments have a tendency either to populate native-like conformations, although never to a largeextent, or random-coil conformations, thussuggesting that there is a selective pressure to keepa minimum local native-secondary structure ten-dency in proteins (MunÄ oz et al., 1996; MunÄ oz &Serrano, 1996). Recently, Minor & Kim (1996), indi-cated that context effects are the main determinantsof secondary structure formation, based on an ex-periment in which they placed the same aminoacid sequence in two different environments of aprotein. However, this result must be interpretedwith caution, since the chosen sequence did not ex-hibit any particular conformational tendency inaqueous solution. Our experiment therefore is theopposite, we have engineered a non-native helicalpropensity in a region of the a-spectrin SH3 do-main without disturbing the packing against therest of the protein. Determination of the structuresof the AE-S and the AE-K mutants by X-ray crys-tallography shows that although, as determinedfrom the peptide analysis, we have provided a sig-ni®cant amount of free energy to the non-nativeconformation (up to 1.9 kcal/mol), in the nativestate the modi®ed region of the protein adopts thenative conformation. It seems that tertiary contactsare responsible for the ®nal structure of this regionof the SH3 protein. However, it is dangerous togeneralise to other proteins or even other segmentsin this protein, since a large part of the helix stabi-lising local interactions are located in the ®rst ®veresidues of the protein, which are non-structuredin the folded state. Therefore, it is possible thatpart of the local helix stabilising interactions arecompatible with the native conformation.

Non-native interactions and protein stability

Introduction of native local interactions in pro-teins results in an increase in their resistance tochemical and temperature denaturation. However,in the two cases analysed so far the increase instability is smaller than expected from the free en-ergy introduced by the new local interactions


(MunÄ oz et al., 1996; Villegas et al., 1996). Thereason for this has been a compaction and in somecases a stabilisation of the denatured state, inducedby native local interactions, which results in lessco-operative transitions. Introduction of non-nativelocal interactions should, in principle, stabilise non-native conformations only in the denatured stateand therefore destabilise the protein. Equilibriumdenaturation by temperature or urea at pH 7.0, aswell as kinetic analysis of the mutant proteins withnon-native helical tendencies, shows that they havebeen destabilised to different extents and that thedestabilising effect of the mutations is additive.However, the free energy changes obtained fromthe peptides and from the proteins are not thesame. This could be explained partly by the factthat we have measured the helical content of thepeptides at 278 K (the helical content is higher atthis temperature, allowing us to improve the resol-ution) and by errors in the estimation of the freeenergy change in the peptides. However, it wouldnot explain the larger discrepancy in the case ofthe Asp14Ser mutation (0.2 kcal/mol in the peptideand 1.0 kcal/mol in the protein). In fact, the exper-iments done at pH 2.5 indicate that favourableelectrostatic interactions made by the side-chain ofAsp14 (i.e. a charged side-chain to main-chain hy-drogen bond; Serrano & Fersht, 1993), are the maincontributors to the free energy change observedand not the change in helical propensity.

In the case of the AE-mutant there is still a sig-ni®cant difference in the Tm value at pH 2.5 withthe wild-type, thus arguing in favour of a non-elec-trostatic destabilising effect of these mutations.Since this region of the protein is not structuredand does not contact the rest of the protein itseems that local helical interactions are responsiblefor the destabilising effect. In fact, kinetic analysisof this mutant shows that the main free energychange with respect to the wild-type protein occurswhen going from the denatured to the transitionstate, as expected if local interactions stabilise anon-native conformation in the denatured state. In-terestingly, the kinetic analysis of the S mutantshows that �0.2 kcal/mol of the free energy differ-ence with the wild-type protein is lost when goingfrom the denatured to the native state. This valueis the same as we found in the peptide analysisand points out that a minor part of the destabilis-ing effect of the Asp14Ser mutation at pH 7.0could be due to the increase in non-native helicalpropensity. All together, these results suggestthat non-native local interactions could destabiliseproteins.

Non-native interactions and the compaction ofthe denatured state

As mentioned above, stabilisation of native heli-cal conformations in CheY and Ada2h results in acompaction of the denatured state, as shown by achange in the equilibrium m value (MunÄ oz et al.,

1996; Villegas et al., 1996). These results suggestthat in the denatured ensemble under native con-ditions, compact structures with native-like sec-ondary structure are signi®cantly populated(MunÄ oz et al., 1996; MunÄ oz & Serrano, 1996). How-ever, little information is available about the exist-ence of compact denatured conformations havingnon-native secondary structure. If these confor-mations are abundant in the denatured ensemble,stabilisation of the non-native secondary structureshould result in an overall compaction of the de-natured ensemble. As a result we should observe adecrease in the equilibrium m value and in the re-folding mF value (as long as the refolding transitionstate of the protein is compact).

Equilibrium analysis of the four mutant proteinswith non-native helical propensities, analysed here,shows some changes in the equilibrium althoughthe differences are within experimental error, ex-cept for the AE-S protein (the data on the AE-Kprotein are not reliable). However, a very goodcorrelation is found between the kinetic mF values(which are reproducible in separate experiments)and the helical tendency (Figure 10). The kineticanalysis shows that there is no change in the un-folding slope mU, with the exception of the AE-Kmutant. The fact that the structure of the triple mu-tants, AE-S and AE-K, is identical with that of theWT protein, together with the above result,suggests that we have not signi®cantly altered thestructure of the transition state in the S, AE andAE-S mutants. In the AE-K mutant the decrease inmU could indicate a displacement of the transitionstate towards a more unfolded conformation. Inthe refolding process, the slope mF changes and thechanges correlate with the increase in helical ten-dency of the mutated region. Consequently, sincemU does not change (except for the AE-K protein),the WT (Viguera et al., 1994) and the mutants exhi-bit a two-state transition with no folding inter-mediate, the decrease in mF can be explained onlyby a decrease in the solvent accessibility of the de-natured state of the mutant proteins. In the AE-Kmutant, the decrease in mF cannot be explainedsolely by a decrease in the solvent accessibility ofits denatured state, since we know that there is adisplacement of the transition state towards a moreunfolded conformation. However, addition of mF

and mU results in a smaller equilibrium value thanin the other proteins, thus indicating that a changein the denatured state is also taking place.

These results suggest that in the denatured stateof the SH3 domain of a-spectrin, compact confor-mations with non-native helical secondary struc-ture are signi®cantly populated.

Conclusions

Introducing non-native helical propensities atthe N-terminal region of the SH3 domain, throughlocal interactions, is not enough to drive the for-


mation of an a-helix in the native state, thus indi-cating that non-local interactions are the mainstructure determinants in this region. Non-nativeinteractions destabilise proteins through a prefer-ential stabilisation of the denatured state. More in-teresting, we ®nd that non-native local interactionscan also produce a compaction of non-native con-formations in the denatured state. These resultssuggest that optimisation of protein folding andstability seems to require a certain ratio of localversus non-local interactions, which seems to belower in larger proteins, and there must be a selec-tion to prevent a large contribution of non-nativelocal interactions. Proteins seem to be very specialpolymers in which many different opposing forcesare neatly balanced.

Materials and Methods

Chemicals

Urea was purchased from BRL (Gaithersburg, MD),sodium phosphate from MERK (Darmstadt, Germany),ammonium sulphate and Tris were obtained from Sigma(St Louis, MO). Taq polymerase, phage T4 DNA ligase,and NcoI and BamHI restriction enzymes were fromBoehringer (Mannheim, Germany). Reaction bufferswere provided by the same company.

Peptide synthesis

The peptides were synthesised at the EMBL peptidesynthesis service by solid-phase synthesis methods. Pep-tide homogeneity, composition and molecular mass werechecked by analytical HPLC, amino acid analysis andmatrix-assisted laser desorption time-or-¯ight mass spec-trometry. The N and C termini were free in all cases.

Far-UV CD analysis

Far-UV CD spectra were recorded on a Jasco-710 di-chrograph previously calibrated with d-10-camphorsul-phonic acid. The spectra were acquired in the continuousmode with 1 nm bandwidth, one second response and ascan speed of 50 nm/minute. Thirty scans were accumu-lated to obtain the ®nal spectra. The samples were pre-pared either in water or in 30% (v/v) tri¯uoroethanol(TFE). The buffer used was 20 mM sodium phosphate(pH 7.0) and the temperature 278 K. Peptide concen-tration was determined by measuring the absorbance at280 nm using the method of Gill & Hippel (1989). Thesamples were prepared at 15 mM peptide concentrationand measured in a 0.5 cm pathlength cuvette.

Peptide aggregation

To determine if the peptides aggregated, we recordedfar-UV CD spectra in the 10 mM to 1 mM concentrationrange at pH 7 and pH 2.5. At pH 7.0 we did not ®ndany concentration dependence of the mean residue ellip-ticity. However, at pH 2.5 between 20 and 400 mM pep-tide concentration there was a clear difference in the

shape of the CD spectra. Between 5 and 20 mM the spec-tra remained the same (data not shown). Therefore, atpH 2.5 we used the CD data obtained at 20 mM. Com-parison of NMR monodimensional spectra obtained atpH 7 and peptide concentrations of 0.2 and 2.2 mM didnot show any difference in the chemical shift values andonly very small changes in the linewidth.

Quantification of the helical population

The helical content of the peptides was determinedfrom the ellipticity at 222 nm following the method ofChen et al. (1974). Since all the peptides contain two Tyrresidues a correction for the aromatic contribution at222 nm was done as indicated by Chakrabartty et al.(1993).

NMR spectroscopy

NMR samples were prepared by dissolving the lyo-philised peptides in 0.5 ml H2O/2H2O (9:1, v/v) at a con-centration of around 2.2 mM. The pH was adjusted to7.0. The pH measurements were not corrected for isotopeeffects. Sodium 3-trimethylsilyl(2,2,3,3-2H4)propionate(TSP) was used as an internal reference.

NMR experiments were performed on a Bruker AMX-600 spectrometer at 278 K. All the two-dimensional spec-tra were acquired in the phase-sensitive mode using thetime-proportional phase incrementation (TPPI) technique(Marion & WuÈ thrich, 1983) with presaturation of thewater signal. Conventional pulse sequences and phase-cycling procedures were used for COSY (Aue et al., 1976)and NOESY (Kumar et al., 1980) spectra. Mixing times of200 ms were used in the NOESY spectra. TOCSY (Rance,1987) spectra were acquired using the standard MLEV-16spinlock sequence and z-®lter with 80 ms mixing time.The size of the acquisition data matrix was 2048 � 512words in f2 and f1, respectively, and before. Eight scanswere acquired for the COSY and TOCSY experimentsand 64 for the NOESY. A presaturation of one secondwas done. The spectral width was 6666.7 Hz. Fouriertransformation of the two-dimensional data matrix wasmultiplied by a phase-shifted sine-bell or square-sine-bellwindow function in both dimensions. The correspondingshift was optimised in every experiment.

1H NMR assignment

Complete assignment of the 1H NMR spectra ofeach peptide in aqueous solution was made using thestandard two-dimensional sequence-speci®c methods(WuÈ thrich, 1986). Firstly, spin systems were identi®ed byjoint analysis of phase-sensitive COSY and TOCSY spec-tra. The second stage involves the assignment of thesespin systems to speci®c residues in the peptide sequenceon the basis of sequential bN, aN or/and NN spatial con-nectivities observed in the NOESY spectra. The Tableswith the assignment of the peptides are available upon re-quest. The 3JHNa values were measured in proton one-di-mensional spectra with high digital resolution and signal-to-noise ratio. Resolution enhancement functions wereused prior to Fourier transformation, thus minimising thedistorting effect of the linewidth in some of the amide sig-nals. Also the 3JHNa values were measured from 2D spec-


tra using the program MEDEA (Stonehouse & Keeler,1995) implemented by J. Santoro (unpublished results).

Estimation of helix population by 1H NMR

Based on the well-established fact that the Ha chemicalshift values are up®eld-shifted upon helix formation,quanti®cation of helix population was carried out as de-scribed (Jimenez et al., 1993). Thus, average helical popu-lations per residue were found by dividing the sum ofthe conformational shifts (�di � gobs

i ÿ dRCi ) of the pro-

tons involved in the helix by the helical length (n) andby �, the shift corresponding to 100% helix formation.The helical lengths were determined from the NMR dataaccording to the NOE and chemical shift criteria. Thus, aresidue is considered to form part of the helix when its�d conformational shift is negative and there is not morethan one residue with a null or positive shift intercalatedbetween it and the other residues with negative values,or when it participates in aN(i, i�3) and/or ab(i, i�3)NOEs. Only those residues considered to be part of thehelix were used for the calculation. Random coil, dRC, va-lues at 278 K were those given by Merutka et al. (1995).An average value of ÿ0.39 ppm was accepted for 100%helix (Wishart et al., 1991). Alternatively, the averageconformational shift changes for every amino acid typewhen going from the random to the helical conformationwere used as 100% a-helix references (Wishart et al.,1991). No signi®cant difference was found between theresults obtained from the two approaches. A correctionfor the aromatic contribution in the random coil, usingthe values found by Merutka et al. (1995), was done onthe peptides analysed in water (Viguera & Serrano, 1995;Viguera et al., 1995). This correction was not done in 30%TFE, since the effect of the aromatic ring on the i � 1,i � 2, i ÿ 1 and i ÿ 2 residues is due to the proximity ofthe ring to their Ca protons in the random coil confor-mation. In an a-helix the aromatic ring will be closer toresidues i � 4, i � 3, i ÿ 3 and/or i ÿ 4, depending onthe nature of the side-chain at these positions. In waterthe helical conformation of the peptides analysed here islow and therefore the random coil contribution will belarge, this is not the case in 30% TFE.

Mutagenesis and cloning

The pET3d plasmid coding for the wild-type SH3 do-main was a generous gift from Dr Saraste. Mutants wereobtained by mutagenesis using the polymerase chain re-action (PCR) method (Higuchi et al., 1988), NcoI andBamHI were the 50 and 30 restriction sites, respectively, ofthe inserts that were cloned in the plasmid pBAT-4(ParaÈnen et al., 1996). A methionine codon present in theNcoI site was used as a translation initiator. Chemical se-quencing of DNA was performed by the EMBL DNAservice of the puri®ed plasmids after cloning.

Amino acid sequences of all the constructions are asfollows:

The amino acid residues that were mutated are shown

in bold.

Expression and purification of the SH3 mutants

The wild-type domain was expressed and puri®ed asdescribed (Viguera et al., 1994). Protein concentrationwas determined by absorbance at 280 nm using the ex-tinction coef®cient calculated previously (16,147 Mÿ1

cmÿ1; Viguera et al., 1994).

Crystal growing and X-ray analysis

Crystals of the AE-S and AE-K mutants were obtainedunder the same conditions as for the WT SH3 domain(Musacchio et al., 1992). All crystallographic data werecollected under cryo-cooling conditions (100 K) using anOxford CryoSystem cryo-stream instrument with in-house CuKa X-ray generators. Prior to shock-freezing thecrystals were transferred into a solution that contained90% (v/v) reservoir solution and 10% (v/v) glycerol. TheX-ray data were recorded on a Small Mar imaging plate(radius � 90 mm) mounted on a Siemens/MacScienceMX18 generator. All data sets were reduced and pro-cessed with the program XDS (Kabsch, 1988). The differ-ences between the WT-SH3 structure (Musacchio et al.,1992) and each mutant was identi®ed by difference Four-ier methods. The structures were modi®ed with the pro-gram O (Jones et al., 1991) and re®ned with the X-PLORpackage (BruÈ nger, 1993). The crystallographic statisticsare summarised in Table 3.

Chemical denaturation experiments

Urea denaturation experiments were performed as de-scribed (Viguera et al., 1994), the main difference beingthat the buffer used here was 50 mM sodium phosphate(pH 7.0). The ®ve proteins were analysed one day andthe experiment was repeated on a different day underthe same conditions. The equilibrium constant for dena-turation and the thermodynamic parameters in equili-brium, at 25�C, were also calculated by ®tting thechanges in ¯uorescence to the following equation:

F � fFN � ��FU � B�urea�� exp ��GH2O � �m�urea�ÿ0:008295�urea�2��=RT��g=f1� exp��ÿ�GH2O

��m�urea� ÿ 0:008295�urea�2��=RT��g�4�

where B is the slope of the ¯uorescence dependence ofthe unfolded state with urea. F, FN and FU, are the ob-served ¯uorescence at a particular urea concentration,the ¯uorescence of the native state and the ¯uorescenceof the unfolded state, respectively. �GH20 is the free en-ergy of unfolding in the absence of urea. The term0.008395 [urea]2 has been introduced to take into accountthe non-linear dependence of the natural logarithm ofthe unfolding rate constant with urea (see below).

Temperature denaturation experiments

Thermally induced unfolding was monitored by CDspectroscopy at pH 7 and pH 2.5 in the temperaturerange 298 to 365 K. The temperature was increased atone deg. C intervals per minute. At both pH values andwith a protein concentration of 20 mM SH3, denaturationwas found to be 100% reversible (Viguera et al., 1994).The curves corresponding to the wild-type protein at pH7 and pH 2.5, together with the calorimetric data(Viguera et al., 1994), were used to ®nd the linear depen-dence of the mean residue ellipticity at 315 nm of the


native (EN) and denatured states (EU). The curves were®tted to the following equation:

E � �EN � AT � �EU � BT� exp��HTm�1ÿ T=Tm�ÿ�Cp;U�Tm ÿ T � T ln �T=Tm��=RT��=� �1� exp��H�Tm�1ÿ T=Tm� ÿ�C�p;U

� �Tm ÿ T � T� ln �T=Tm��=RT��

�5�

where:

�Cp;U � 6:58� 0:02392��T ÿ Tm� ÿ 0:0000195��T ÿ Tm�2�6�

The non-linear dependence of �Cp,U with temperaturewas found in a calorimetric study of the wild-type pro-tein (Viguera et al., 1994). The value used here is slightlydifferent from that previously published and has beenfound after several more experiments on this domain.

Kinetic analysis

Folding and unfolding kinetics were followed in a Bio-logic stopped-¯ow machine by ¯uorescence emission se-lected with a 305 nm cut-off ®lter upon excitation at290 nm. The experiments were repeated for each proteinon two different days. The unfolding reaction was per-formed by dilution of the native SH3 domain, in 50 mMsodium phosphate (pH 7.0), with the appropriate ratio ofdenaturing buffer containing different concentrations ofurea in 50 mM phosphate buffer (pH 7.0). For the refold-ing reaction, the unfolded domain in 50 mM phosphate(pH 7.0), containing 8.55 M urea, was mixed with an ex-cess of the same buffer without urea to give various ®nalconcentrations of urea. The cell chamber and the syr-inges were kept at 298 K. Apparent kinetic constantswere calculated by ®tting the experimental traces to amono-exponential transition by means of algorithms pro-vided by Biologic Software.

Originally when analysing the kinetic data of thewild-type SH3 spectrin the whole set of unfolding andrefolding kinetic constants versus urea concentrationwere ®tted to the following equation, after deleting thepoints in the transition region:

ln k � ln�kf;H2O eÿmkf �urea� � ku;H2O eÿmku�urea�� 7�where k is the rate constant at a given concentration ofdenaturant, kf,H2O is the rate constant of refolding inwater, ku,H2O is the rate constant of unfolding in water,and mkf and mku are the slopes of the refolding and un-folding reactions, respectively.

More recently, when studying the unfolding and re-folding reactions of very unstable SH3 mutants it wasfound that the changes in the natural logarithm of theunfolding rate constant with urea were not linear andcurve at high urea concentrations (Viguera et al., 1996).A similar phenomenon has been described for barnase(Johnson & Fersht, 1995). Fitting several of these unstablemutants has shown that the curvature of the unfoldingdata follows equation (8):

ln ku � ln ku;H2O � B �urea� ÿ 0:014 �urea�2 �8�Accordingly, the kinetic data were ®tted to equation (9):

ln k � ln�kf;H2O eÿmkf �urea� � ku;H2O eÿmkf �urea�ÿ0:014�urea�2 � �9��GH2O and m can then be calculated either by equili-brium measurements, by equation (4), or from kinetic

parameters by the following equations:

�GH2O � ÿRT ln

�kf;H2O

ku;H2O

��10�

m � mkf �mku �11�WT spectrin SH3 showed a good agreement in these par-ameters, calculated either by equilibrium or kinetics ex-periments, considering the difference due to prolineisomerization (Viguera et al., 1994).

Acknowledgements

We are very grateful to Victor MunÄ oz for helpful dis-cussions and for his assistance in some of the kinetic andequilibrium experiments. We are very grateful to JorgeSantoro, for providing the software used to determine3JHNa coupling constants for 2D spectra. J. P. is a recipi-ent of a Human Capital and Mobility Fellowship. The al-gorithm AGADIR1s is available on the web (http://www.embl-heidelberg.de/ExternalInfo/serrano/). Theassignment of the peptides as well as the 3JHNa couplingconstants and the temperature coef®cients of the amideprotons are available upon request. The structures of themutants are available upon request.

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Edited by A. R. Fersht

(Received 20 August 1996; received in revised form 9 December 1996; accepted 12 February 1997)

non-native local interactions in protein folding and stability: introducing a helical tendency in...

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