Context-dependent effects of proline residues on the stability and folding pathway of ubiquitin Maria D. Crespo, Geoffrey W. Platt, Roger Bofill and Mark S. Searle School of Chemistry, Centre for Biomolecular Sciences, University Park, Nottingham, UK Substitution of trans-proline at three positions in ubiquitin (residues 19, 37 and 38) produces significant context- dependent effects on protein stability (both stabilizing and destabilizing) that reflect changes to a combination of parameters including backbone flexibility, hydrophobic interactions, solvent accessibility to polar groups and intrinsic backbone conformational preferences. Kinetic analysis of the wild-type yeast protein reveals a predominant fast-folding phase which c onforms to an apparent two- state f olding model. Temperature-dependent studies of the refolding rate reveal thermodynamic details of the nature of the transition s tate fo r f olding consistent with hydrophobic collapse providing the overall driving force. Brønsted analysis of the refolding and unfolding rates of a family of mutants w ith a variety o f side c hain substitutions for P 37 and P38 reveals that the two prolines, which are located in a surface l oop adjacent to the C terminus of the m ain a-helix (residues 24–33), are not significantly structured in the transition state for folding and appear to be consolidated into the native structure only late in the folding process. We draw a similar conclusion regarding position 19 in the loop connecting the N-terminal b-hairpin to t he main a-helix. T he proline residues of ubiquitin are passive spectators in the folding process, but influence protein stability in a variety of ways. Keywords: folding kinetics; NMR structural analysis; proline mutations; p rotein folding pathway; protein stability. Proline is unique amongst the natural amino a cid residues; the five-membered ring significantly reduces the flexibility of the polypeptide chain by restricting r otation around the N-Ca bond to a relatively small region of conformational space. This factor, coupled with the lack of an amide NH hydrogen bond donor means that proline i s not readily accommodated into r egular (a-helical or b-sheet) protein secondary structure. It is, however, more abundant in connecting loops playing a specific role in b-turn sequences [1,2], and as a helix capp ing residue or as a helix terminator [3–5]. Prolines confer pre-organization and rigidity in the context of small peptide protease inhibitors [6,7], a c oncept that has been widely used in biomolecular and supramole- cular design to overcome t he potential energetic cost of loss of conformational entropy when dynamic molecules asso- ciate, or when a fl exible polypeptide chain folds. In the context of protein folding, the observation that cis and trans forms of the Xaa-Pro peptide bond are n early isoenergetic [8], and separated by a significant activation barrier, can lead to slow-folding kinetic phases due to the population of the non-native cis-form in the unfolded state, where the rate limiting step is the isomerization of the Xaa-Pro peptide bond [9–12]. Heterogeneity in the unfolded state due to slow isomerization reactions potentially complicates the kinetic elucidation of folding pathways and t he ability to ide ntify partially folded intermediate states or parallel folding pathways [13–21]. However, the observation of a wide variation in the amplitude of slow folding phases associated with prolyl isomerizatio n (in many cases less than expected on the basis of frequency of occurrence in the primary amino acid sequence) suggests that not all non-native cis prolines result in slow folding p hases, and that cis–trans isomerization in some structural contexts need not be rate limiting [22–29]. More recent studies demonstrate that nonprolyl cis-peptide bonds also contribute to the hetero- geneous pool of unfolded molecules [18,30]. Although individual cis-peptide bonds contribute little to the popu- lation (% 0.15–0.5%) in the unfolded protein, their large number generates a significant proportion of slow folding molecules [18,30,31]. We report on the effects of proline on the stability and folding kinetics of ubiquitin, a small model system of 76 residues that is uncomplicated by disulphide bonds and bound cofactors [32]. Ubiquitin has been the subject of a number of investigatio ns regarding i ts folding m echanism. Early studies had suggested that the protein populates an intermediate state identified on the basis of deviations of kinetic data from linearity in the refolding arm of chevron plots at low denaturant concentrations [33]. M ore recent studies [13,14,34,35] report apparent two-state kinetics under similar conditions, suggesting that t he roll-over effect in the r efolding kinetics may b e a consequence o f either transient aggregation that is exacerbated by the stabilizing effects of inorganic salts [15,35], or due to data fitting at rates near the instrumental limits where interference from slower phases can decrease apparent folding rates resulting Correspondence to M. S. Searle, School of Chemistry, Centre for Biomolecular Sciences, University Park, Nottingham NG7 2RD, UK. Tel.: +44 115 9513567, E-mail: mark.searle@nottingham.ac.uk Abbreviations: TSE, transition state ensemble; GdmCl, guanidinium chloride. (Received 8 July 2004, accepted 30 September 2004) Eur. J. Biochem. 271, 4474–4484 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04392.x in chevron rollover effects [13,14]. HX labelling studies and stopped-flow CD similarly found no evidence for an early intermediate in the first 2 m s of folding [36]. We show that proline s ubstitutions in yeast ubiquitin at positions 19, 37 and 38 produce context-depend ent effects on stability with r emoval of proline at specific sites having the effect of either significantly increasing stability (P38A) or destabilizing the protein (P19S and P37A). A full kinetic analysis of the major fast folding phase of wild-type yeast ubiquitin (WT*) and of a number of nondisruptive single-point Ala mutants and several double mutants, using F-value analysis and Brønsted plots, s hows t hat t he transition state ensemble (TSE) is tolerant to proline substitutions at positions 19, 37 and 38, and that these residues are not well structured in the t ransition state for folding. Materials and methods Protein expression A pKK223-3 plasmid construct containing the yeast ubiquitin gene was used to express the wild-type protein in Escherichia coli strain BL21(DE3) under the control o f the isopropyl thio-b- D -galactoside (IPTG)-inducible tac pro- moter. The F45W m utant gene was cloned b y overlap PCR methodology using the wild-type y east ubiquitin g ene in pKK223-3 (Pharmacia Biotech) as a template. The mutated cassette was inserted between the Eco RI and HindIII restriction sites of pKK223-3, and the mutation confirmed by DNA sequencing. Competent E. coli cells were trans- formed with this construct. Expression and purification were as described for the wild-type yielding typically 10–15 mgÆL )1 of ubiquitin, as previously described [34]. NMR structural analysis All NMR experiments were performed on a Bruker Avance600 spectrometer. TOCSY and NOESY experi- ments were used as p reviously described [34] on 1 -m M protein samples at pH 5.5. Spectra were referenced to internal trimethylsilylpropionate. D ata were processed and assigned using Bruker XWINNMR and ANSIG software [37]. Structural models were visualized using MOLMOL [38]. Equilibrium stability measurements Protein stability was determined by fluorescence measure- ments on 1 .5 l M solutions of protein i n 2 5 m M acetate buffer at pH 5.0 and 298 K. The change in fluorescence at 358 nm was monitored as a function of guanidinium chloride (GdmCl) concentration. The linear extrapolation method was used [39–42] assuming that the stability v aries with the c oncentration of denaturant [D], according to the expression DG D ¼ DG eq + m [D], where DG D is the stability at a given [D], m is the constant of p roportionality, and DG eq is the stability in water alone. The fraction of folded protein F f is derived from fluorescence measurements according to F f ¼ (f D ) f U )/(f N ) f U ), where f D is the measured fluorescence at a g iven [D] a nd f U and f N are the limiting values for the unfolded and native states, respectively. The mid-point of the unfolding transition [D] 50% for each mutant was determined by nonlinear least squares fitting to the expression: F f ¼ exp½mð½DÀ½D 50% Þ=RT=ð1 þ exp½mð½D À½D 50% Þ=RTÞ ð1Þ The e quilibrium stability DG eq was d etermined from the expression DG eq ¼ –m[D] 50% ,wherem forasetofmutants is assumed constant (10.9 ± 0.23 kJÆmol )1 Æ M )1 ) [34,43]. This approach is justified by the NMR analysis which shows that all of the mutants fold to a native-like structure with only minor localized chemical shift pertu rbations. Thus, mutations are n ot producing s ignificant c hanges in the hydrophobic surface area buried, justifying the use of the same m-value for stability measurements. Additional cor- rections were used to allow for a small linear denaturant dependence o f t he fluorescence of both the folded and the unfolded state [39]. Kinetics experiments Fluorescence-detected kinetic unfolding and refolding measurements were performed using an Applied Photo- physics Pi-star 1 80 spectrophotometer. T emperature was regulated using a Neslab RTE-300 circulating program- mable water bath. All kinetics experiments were per- formed in 25 m M acetate buffer pH 5.0 at 298 K. Refolding experiments were performed by 1 : 10 dilution of unfolded protein (15 l M in 7 M GdmCl) into buffered solutions of different GdmCl concentrations yielding a final protein concentration of 1.36 l M . For unfolding experiments, a buffered solution of native protein was unfolded by a 1 : 10 dilution to yield final concentrations of GdmCl near or above the midpoint of the equilibrium unfolding transition (concentrations of GdmCl in the range 3.7–7.3 M ). Kinetic measurements for both unfold- ing and refolding reactions were averaged four to six times at each GdmCl concentration. In all cases, the GdmCl c oncentration w as determined using a refracto- meter [ 40]. Analysis of kinetic data The kinetic traces were analysed using a multiexponential fitting procedure (two o r three components). The kinetic data wer e analysed assuming an apparent two-state model using standard equations described in detail by others [41,43,44]. T he observed rate constant k obs is the sum of t he folding and unfolding rates, k obs ¼ k fold + k unfold where k obs is dependent on [D] a ccording to t he expression: lnk obs ¼ ln½k unfold expðm unfold ½D=RTÞ þ k fold expðm fold ½D=RTÞ ð2Þ The dependence of lnk obs on [D] gives extrapolated values for k unfold and k fold in water a lone, together w ith t he slopes of the f olding and unfolding components m unfold and m fold . The temperature dependence of the refolding rate w as examined at a denaturant concentration of 0.4 M GdmCl and 1.81 l M protein and the data fitted according to the following expressions [30]: Ó FEBS 2004 Proline residues in ubiquitin stability and folding (Eur. J. Biochem. 271) 4475 lnk obs ¼ lnk o À DGz =RT ð3Þ where k o is the t emperature independent pre-exponential factor (% 10 8 ), and the temperature dependence of the activation free energy DGà is given by: DGz¼DHzþDC p zðT À 298Þ À T½DSzþDC p z lnðT=298Þ ð4Þ with DHà, DC p à and DSà representing the change in activation enthalpy, heat capacity and entropy of formation of the TSE for folding (U-à). Reported errors reflect the quality of th e nonlinear l east squares fi t to t he experimental data. Results Context-dependent effects of proline substitutions on protein stability We have used the F 45W mutant of yeast ubiquitin as our Ôwild-typeÕ protein (WT*) for mutational and biophysical studies. The partially buried indole side chain (Fig. 1) undergoes a significant (fourfold) quenching of fluorescence on folding but has previously been shown to have only a relatively small effect on the stability (DDG % 1kJÆmol )1 ) and structure of human ubiquitin [45]. Our own structural analysis of F45W mutants of the yeast protein confirms this. We have explored the context-dependent effects of proline on ubiquitin stability by introducing a number of substitu- tions at positions P37 a nd P38. The equilibrium stability of the mutants was determined from the change in fluorescence at 358 nm as a function of GdmCl concentration. The data show that in each case the fraction unfolded fits well to a two-state t ransition with t he observation of a r ange of mid- point denaturant concentrations, [D] 50% values, indicating significant context-dependent effects of the mutations on protein stability (Fig. 2; Table 1). The P37A mutation produces a large shift in the transition mid-point for denaturation from 2.62 M GdmCl (WT*) to 2.18 M GdmCl. This equates to a reduction in stability o f 4 .5 ± 0.6 kJÆ mol )1 . In contrast, the P38A mutation re sults in a significant increase in stability of )4.6±0.6kJÆmol )1 .TheA37A38 double mutant is slightly less stable than WT* ( 1 . 1 ± 0. 6 k J Æ mol )1 ), showing that the contributions from P37A and P38A are approximately additive. We also examined the effects of substituting a proline residue at position 19 in the loop region connecting the N-terminal b-hairpin to t he main a-helix (Fig. 1). Proline is highly conserved at t his site in many s pecies; however, in yeast ubiquitin residue 19 is serine. The mutation S19P produces a significant increase in stability o f )5.3 ± 0.7 kJÆmol )1 . Thus, the P19S, P37A a nd P38A mutations produce contrasting effects that do not appear to simply relate to entropic factors concerning changes in backbone flexibility. Structural analysis of the proline mutants by NMR NMR structural analysis was used to establish whether the substitutions of P37 and P38 are substantially perturbing the conformation and dynamics in this region of the protein, or more specifically, whether st ructural effects are transmit- ted t o the C terminus of the a djacent main a-helix (residues 24–33). We have completed an NMR backbone assignment of WT* for comparison with P37A, P38A and the A37A38 double m utant a nd have examined chemical shift p erturba- tions and the pattern of NOEs in the vicinity of the mutation sites. Deviations of Ha signals from random coil chemical shifts pro vide a sensitive probe of local perturba- tions to secondary structure [ 46,47]. We find that perturba- tions are largely confined to the residues immediately adjacent to the m utation s ite, in particular Ile36 (Fig. 3). In the case of the P37A mutant, some small (< 0.1 p.p.m.) longer range effects are observed involving residues on the Pro37 f ace of the main a-helix (namely, Asp24, Ser28 and Gln31). The characteristic pattern of NH–NH sequential NOEs enables us to map the e xtent of structure formation within the main a-helix (residues 24–33), and examine the integrity o f the helix C-capping motif and of the s hort helix (residues 3 8–40). In ubiquitin, the C-capping motif involves a hydrogen bond between Gly35 NH and the backbone carbonyl of G ln31. This i nteraction positions Ile36 to form hydrophobic contacts to Ile30 and results in strong NH-NH sequential NOEs between Gln34 « Gly35 « Ile36. Fur- ther, the NH signal of Ile36 is > 1 p.p.m. upfield shifted b y these interactions. These NOEs are clearly evident in the NOESY data for WT*, P37A, P38A and A37A38. Further, Ile36 NH has the characteristic upfield shift that confirms that the C terminus of the helix and the C-capping motif are not disrupted by the proline mutations. Extending the analysis to the short helix (residues 38–40), the strong sequential NH–NH NOEs from D39 through to Q41 are preserved in all mutants. The P38A mutation appears to extend the helical turn by one residue with Ala38 having a 3 J NH–Ha value < 6 Hz with evidence of i,i+3 NOEs to Gln41. NOE contacts from Ala38 protons to the side chains of Lys27 and Gln31 in the main a-helix are also evident and confirm t hat the Ala38 methyl g roup occupies the same hydrophobic pocket as the side chain of Pro38. Mod elling the structure with Ala substitutions imposed on the backbone conformation of WT* shows that the pattern of P37 P38 P19 W45 Fig. 1. Ribbon structure modelled on the X-ray structure of human ubiquitin [32]. The position and orientation of the side chains of Pro19, Pro37 and Pro38 are highlighted along with the F45W mutation (drawn using MOLMOL [38]). The sequences o f human and yeast ubiquitin differ at the f ollo wing positions: P19S, E24D and A28S. 4476 M. D. Crespo et al.(Eur. J. Biochem. 271) Ó FEBS 2004 NOEs is entire ly consistent with native-like /,w angles. Thus, w e conclude that the Pro to Ala substitutions are not significantly perturbing the backbone conformation and dynamics of the protein around the mutation sites and in the adjacent a-helix. Analogous NMR s tudies of the S19P mutant (data not shown) also establish that chemical shift perturbations are entirely l ocalized to the mutation site and immediately flanking residues. Kinetic analysis of ubiquitin folding The folding kinetics of WT* have been analysed from refolding and unfolding stopped-flow exper iments in GdmCl at 298 K a nd pH 5.0 in 2 5 m M acetate buff er. The refolding traces for WT* in the range 0–2.5 M GdmCl are best analysed in terms of a multiexponential fit reflecting at least three resolved folding phases. The fast phase, which accounts for % 87% of the amplitude of the fluorescence change, has an extrapolated folding rate in water of 303 s )1 , while seve ral minor slower folding phases are also evi- dent with extrapolated rate constan ts k 2 ¼ 34 s )1 and Fig. 2. Equilibrium denatura tion curves for yeast ubiquitin (WT*) a nd various mutan ts. Fraction unfolded is p lotted against c oncen- tration of GdmCl at pH 5.0 in 25 m M acetate buffer at 298 K and was mo nitored by tryptophan fluorescence. Stability d ata are shown in T able 1. Table 1. Equilibrium stability dat a for ubiquitin mutants (pH 5. 0, 25 m M acetate b uffer, 298 K) determined by GdmCl denaturation monitored b y changes i n tryptophan fluorescence. Mutant m eq a (kJÆmol )1 Æ M )1 ) [D] 50% b DG eq c (kJÆmol )1 ) WT* 11.3 2.62 )28.6 (± 0.6) P37A 11.9 2.21 )24.1 (± 0.5) P38A 10.2 3.05 )33.2 (± 0.7) SQ 10.8 2.21 )24.1 (± 0.5) QL 10.4 2.39 )26.0 (± 0.5) AA 11.2 2.52 )27.5 (± 0.5) VV 11.2 2.22 )24.2 (± 0.5) S19P 10.0 3.11 )33.9 (± 0.7) a Errors in m eq are less than ± 0.35. b Denaturant concentration at the mid-point of the folding/unfolding transition; fitting errors are less than ± 0.008. c Equilibrium stability determined from the [D] 50% value assuming a mean m-value (± SE) of 10.9 ± 0.23 kJÆmol )1 Æ M )1 . Fig. 3. Ha chemical shift analysis of residues 22–46 of the yeast ubiquitin mutants P37A, P38A and the double mutant A37A38. These residues span the main a-helical region (res i- dues 21–35) N terminal to the X37 and X38 mutation sites, and t he sequence of the short helix (residues 38–40) and fourth strand of b-sheet (re sidues 4 2–46) on the C-terminal side of the mutation sites (Fig. 1). Differences in chemical shifts with respect to r ando m coil values [46,47] are plotte d against sequence position. Ó FEBS 2004 Proline residues in ubiquitin stability and folding (Eur. J. Biochem. 271) 4477 k 3 ¼ 0.14 s )1 , and relative amplitudes o f 1 1% and 2 %, respectively. The k 2 and k 3 processes, also identified for human ubiquitin [13,33], have previously been attributed to slow rate-limiting cis–trans prolyl isomerization reactions. However, we have shown using double-jump (interrupted unfolding) experiments (data not shown) that k 2 is a direct refolding event whose amplitude is unaffected by the equilibration time of the dou ble-jump experiment. I n an isomerization-limited process, the pop ulation of t he non- native cis-isomer w ould be expected to build up only slowly in the unfolded state (rate constant < 2 s )1 [30]). While k 2 does not show these c haracteristics, the slowest phase (k 3 )is consistent with a cis–trans rate-limiting event, s howing a significant reduction in amplitude at short aging times. We concentrate h ere on t he major fast f olding p hase which yields a chevron plot with both the folding and unfolding arms varying linearly with the concentration of denaturant. Linearity is clearly observed when either GdmCl or urea are used as denaturants ( Fig. 4A). The kinetic stability calculated from the folding and unfolding rate constants are in good agreement with those e stimated from the equilibrium denaturation measurements. F urther, as can be seen in Fig. 4A, the linear refolding and unfolding arms of the chevron plots in GdmCl and urea extrapolate to v ery similar ln k obs values at [D] ¼ 0, and give closely similar stability estimates, consistent with two- state folding under these different conditions. A ddition- ally, we see no evidence for a burst-phase in fluorescence amplitude in the refolding exp eriment at low denaturant concentrations (Fig. 5A). Only when refolding experi- ments a re conducted in moderate concentrations of stabilizing salts, such as 0 .4 M Na 2 SO 4 ,doweseeany evidence for deviations from a two-state model. Under these conditions rollover effects are now apparent in the refolding data at low denaturant concentrations (Fig. 4B), together with burst-phase changes in the fluorescence intensity (Fig. 5B) [33,35]. We conclude that the data collected for yeast ubiquitin at protein concentrations <2 l M are adequately described in terms of a two-state folding model in concurrence with recent detailed studies of human ubiquitin [13,14,35]. Kinetic experiments on the Pro mutants reveal that the changes in protein stability associated with the P ro substi- tutions are largely manifested in effects on the unfolding rather than refolding kinetics (Table 2). The chevron plot analysis shown in Fig. 6 reveals little change in the m-values for either the refold ing or unfolding phas es, indicating that the TSE is not significantly perturbed by the mutations, nor do we see any evidence for deviation from the two-state folding model using the criteria described above. Tolerance to substitutions at the P37P38 site Kinetic studies with other systems, aimed at probing the nature of the TSE for folding, have focused primarily on nondisruptive Ala or Gly s ubstitutions, a rguing that more sterically demanding substitutions have the potential to shift the position of the TSE along the folding pathway or even stabilize intermediate s tates [48,49]. We have exam- ined the robustness of the TSE for folding in the current context by also introducing more polar or sterically more diverse mutations in place of P37 and P38. We have considered three double mutants with a combination of polar, nonpolar and b-branched side chains: SQ, QL and VV, in addition to the Ala substitutions already described. Equilibrium denaturation experiments monitored by fluorescence show that these double mutations have a modest destabilizing effect of < 5 kJÆmol )1 (Fig. 2; Table 1 ), suggesting that their loca tion close to the surface Fig. 4. Chevron plot analysis of the logarithm of the refolding and unfolding r ates vs. c oncentration of denaturant (GdmCl). (A) WT* in GdmCl and ure a (29 8 K in 25 m M acetate buffer, pH 5.0). D otted lines extend the unfolding arms to the y-axis to determine the unfolding rate constan ts i n b uffer a lon e, [d en aturant] ¼ 0. The estimated sta- bility constants from DG ¼ –RT ln(k fold /k unfold )are)25.5 kJÆmol )1 (GdmCl) and )25.8 kJÆmol )1 (urea); m-values are estimated as follows in urea, m fold ¼ 1604 ± 88 JÆmol )1 Æ M )1 and m unfold ¼ 2919 ± 4 3 J mol )1 Æ M )1 . (B) Refolding and unfolding data f or WT* as in (A) and in the presence of 0.4 M Na 2 SO 4 . The data for the latter were fitted to a three-state on-pathway model (U«I«N) in which the intermediate state is significantly populated with an equilib- rium constant K UI ¼ 204. Rate constants and m-values are as follows: m UI ¼6992 ± 250 JÆmol )1 Æ M )1 , k IN ¼ 468 ± 70 s )1 , m IN ¼ 1001 ± 3 78 J Æmo l )1 Æ M )1 , k NI ¼ 0.0034 ± 0.0011 s )1 and m NI ¼ 3103 ± 1 68 J Æmo l )1 Æ M )1 . 4478 M. D. Crespo et al.(Eur. J. Biochem. 271) Ó FEBS 2004 of the protein may allow some fl exibility in accommodating these side chains. NMR analysis o f H a chemical shifts for the SQ and QL mutants, in line with structural studies described above, confirms that only relatively small local perturbations to the structure have taken place. Detailed kinetic analysis shows that the reduction in stability of these mutants is largely manifested in perturbations to the unfolding rates with the degree of compactness of the TSE (a D ) and linearity of the chevron plots very similar to WT* (Fig. 6). The analysis o f m ultiple mutations at a common site (P37/P38) is conveniently expressed in terms of a Brønsted plot, allowing the r elationship to be e xamined between the logarithm o f the re folding and unfolding r ates and the effect on protein stability [50]. Such a relationship should enable us to assess the extent to which P37 and P38 are involved in native-like contacts in the TSE. Linear Brønsted p lots have been interpreted as indicating that the r esidues a t the mutation site give rise to the same degree of partial structure in the transition s tate as in WT*, and that the substitutions are not significantly perturbing the position o f the TS E along the folding pathway [51,52]. We have con sidered th e P37/P38 mutations simultaneously and constructed the Brønsted plot shown in Fig. 7 on the basis of the following: lnk fold ¼ lnk fold  À b f DDG=RT ð6Þ lnk unfold ¼ lnk unfold  þð1 À b f ÞDDG=RT ð7Þ where k fold ° and k unfold ° are the rate constants for folding and unfolding of WT*, k fold and k unfold are the folding and unfolding rates of the mutants derived from the chevron plot analysis, a nd b f is a constant describing the degree of native-like structure formation in the TSE at the P37/P38 site. The plots of k fold and k unfold vs. DDG/RT (both DDG eq /RT and DDG kin /RT; Fig. 7) are linear demonstra- ting that all mutants show the same degree of structure formation in the TSE, which appears to be tolerant to the variety o f changes introdu ced. Values of b f ¼ 1 have been interpreted as evidence that residues at the mutation site occupy a highly native-like environment in the TSE, whereas much smaller values (close to zero) suggest that these residues are largely unstructured in the rate-limiting step for folding. The linear plots in Fig. 7 indicate a b f -value of 0.09 supporting the latter model. We see that the proline mutations produce very small effects on the folding rate of ubiqutin with only a two-fold difference between the fastest and slowest folding mutants. In contrast, we see a 26-fold range in the rate of unfolding. This trend i s also r eflected i n t he effects o f t he S19P mutation on the kinetics. The significant stabilizing effect of this mutat ion ()5.3 kJÆmol )1 ) is also manifested largely in a deceleration of the unfolding rate. By a nalogy with the above analysis, F-values provide an estimate, on the scale of 0–1, of the extent to which a s ide chain interact ion formed in the native state, and which is deleted through mutation, is present (F ¼ 1) or absent (F ¼ 0) in the TSE for folding [53,54]. Formerly, the F-value was calculated as: U ¼ÀRT lnðk fold WTà =k fold mut Þ=DDG eq ð8Þ where k fold WT *andk fold mut are the folding r ates for t he WT* and mutant protein, and DDG eq is the difference in equilibrium stability between mutant and WT*. The single point S19P mutation leads to a F ¼ 0.37, which points to the stabilizing effect of this mutation not being realized in the folding TSE, indicative of the loop between the N-terminal b-hairpin and the main a-helix remaining flexible in the TSE, with native-like contacts and back- bone F,w angles becoming consolidated at a late stage in the folding process. Fig. 5. Amplitude of the raw fluorescence signal for the refolding of WT* ubiquitin. In the absence (A) and presence of 0.4 M Na 2 SO 4 (B) a t 298 K in 25 m M acetate buffer, pH 5 .0. The b lac k dots and solid line are the fit to the refolding data enabling a two-state equilibrium unfolding curve to be constructed. The dashed line (circles) is a linear fit in (A) t o the denaturant dependence of the fluorescence signal of the unfolded state. In (B), in the presence of stabilizing salt, the fluores- cence s ignal of th e unfolded state (dashed line, circles) shows deviations from a linear extrapolation, providing evidence for a burst phase around 1 M GdmCl w here the fluorescence intensity increases signifi- cantly as the collapsed state is destabilized by t he denaturant. This is consistent w ith the curvature observed in the corresponding chevron plot in Fig. 4B and formation of an intermediate co llap sed state at low denaturant concentrations. Ó FEBS 2004 Proline residues in ubiquitin stability and folding (Eur. J. Biochem. 271) 4479 Activation parameters for folding The temperature-dependence of the refolding kinetics were examined in detail for WT* and the A37A38 double mutant under fixed refolding conditions (0.4 M GdmCl) to deter- mine thermodynamic parameters for formation of the folding TSE. Because formation of the TSE buries a significant hydrophobic surface area (a D values 0.66–0.71), the temperature dependence of t he refolding rate should be associated with a nonzero change in heat capacity [8,30]. The experimental data show a pronounced curvature, consistent with the large a D values observed (Fig. 8) 1 .The data were fitted to Eqn (3) over the temperature range 283– 310 K to give DC p à values of )2.1 (± 0 .3) and )2.4 (± 0.5) kJÆK )1 Æmol )1 , respectively. The activation enthalpy and entropy t erms for folding are also very similar for the two proteins. The positive entropy change (25 ± 4 and 28 ± 6 JÆK )1 Æmol )1 , respectively) reflects a small f avour- able stabilization of the TS, however, t he enthalpy te rm (66 ± 2 and 67 ± 2 kJÆmol )1 , respectively) is highly unfa- vourable to folding and dominates the size of t he activation barrier, DGà [9]. Discussion Context-dependent effects of proline residues on protein stability Ubiquitin i s highly conserved across species with the y east and human forms differing in on ly three r esidues (S19P, E24D and A28S). The first of these is located in a loop region which connects the N-terminal b-hairpin sequence (residues 1–17) to the main a-helix (residues 24–33) (Fig. 1). The E24D and A28S substitutions lie within the main a-h elix. Both structures have conserved prolines (P37 and P38) in adjacent po sitions at the N terminus of a short a-h elix (residues 38–40) in an otherwise extended loop region connecting the C terminus of the main a-helix to subsequent strands of b-sheet (Fig. 1). We have investigated the context-dependent effects of mutations at these sites on Fig. 6. Chevron plot analysis of the logarithm of the r efolding and unfolding rates v s. concen- tration of denaturant (GdmCl). Da ta shown for WT* and all ubiquitin m utants studied ( 298 K in 25 m M acetate buffer, pH 5.0). Refolding and unfolding were monitored by changes in tryptophan fluorescence at 358 nm. Kinetic data were determined by fi tting to Eqn (2); results are shown in Table 2. Table 2. Kinetic data for the refolding (U fi N)/unfolding (N fi U) of ubiquitin mutants (298K, pH 5.0 in 25 m M acetate buffer) monitored by changes i n tryptophan fluorescence using GdmCl denaturant. a D -values determined from m UN /(m UN + m NU ). Mutant k NfiU (s )1 ) m N fi U (JÆmol )1 Æ M )1 )k U fi N (s )1 ) m U fi N (JÆmol )1 Æ M )1 ) a D WT* 0.0090 (± 0.0008) 2876 (± 44) 304 (± 11) 5934 (± 58) 0.67 P38A 0.0036 (± 0.0009) 2992 (± 113) 243 (± 15) 5236 (± 94) 0.64 P37A 0.042 (± 0.004) 2383 (± 51) 161 (± 11) 5881 (± 125) 0.71 AA 0.0204 (± 0.002) 2614 (± 54) 250 (± 13) 5725 (± 89) 0.69 SQ 0.066 (± 0.007) 2370 (± 61) 142 (± 16) 5904 (± 205) 0.71 QL 0.092 (± 0.008) 2555 (± 47) 271 (± 27) 6000 (± 184) 0.70 VV 0.060 (± 0.003) 2428 (± 31) 228 (± 11) 6133 (± 93) 0.71 S19P 0.0038 (± 0.0005) 2953 (± 67) 501 (± 22) 5761 (± 62) 0.66 4480 M. D. Crespo et al.(Eur. J. Biochem. 271) Ó FEBS 2004 protein stability, and their involvement in the folding pathway from studies of refolding/unfolding kinetics. While the single point mutation P37A is destabilizing b y 4.5 kJÆmol )1 , in contrast the P38A mutation produces an equal and opposite en hancement of stability of )4.6 kJÆmol )1 . T he reduction in stability of t he A37A38 double mutant approximates to the additive effects of the single point mutations (1.1 kJÆmol )1 ). Thus, the observed changes in stability cannot be inte rpreted purely in terms of entropic effects on the flexibility of the polypeptide back- bone si nce i n one case removal of proline leads to an enhancement of stability. The side chain solvent accessibility of P37 and P38 is quite similar in t he native protein (53% and 46%, respectively). Truncation of the P37 side chain removes van der Waals c ontacts with the side c hain of Q40, and these may account for some loss of stability. In contrast, structural analysis suggests that removal of the P38 side chain, which substantially enhances stability b y )4.6 kJÆmol )1 , favours greater solvent accessibility o f the partially buried Q41 side chain and this may be a contributing factor to the stability changes. Further, proline is a g ood helix capping residue and P38 is found to N-cap the short three-residue helix spanning residues 38–40. The S19P mutation produces a substantial increase in stability ()5.3 kJÆmol )1 ) which we can also attempt to rationalize on the basis of the X-ray structure of human ubiquitin which already has Pro at this position. The structure shows t hat the Pro19 side chain forms significant hydrophobic contacts with the side chain of Met1, which becomes more solvent accessible when P ro is replaced with Ser. There may also be solvation implications for the Ser hydroxyl group, which may also contribute a small destabilizing effect. The contrasting effects of the S19P, P37A and P38A mutations on stability appear to reflect a c omplex balance between entropic factors relating to changes in backbone flexibility, changes in hydrophobic surface burial, effects on solvent accessibility t o other polar group s and changes in intrinsic backbone conformational preferences. These observations are consistent with those of others that proline residues play a variety of context-dependent roles in modulating protein stability [10–12,16,19]. Apparent two-state model for folding of ubiquitin There have been conflicting reports as to whether ubiquitin folds via an apparent two-state model o r via a m ore complex process involving a significantly populated inter- mediate, which forms rapidly in t he dead-time of the stopped-flow experiment [13,14,33]. In the case of the yeast protein d escribed here, the linear dependence o f the folding and unfolding rates on denaturant concentration ( both GdmCl and urea), and the lack of a burst phase change in fluorescence intensity at low denaturant concentrations, is indicative of an apparent two-state model in which any intermediate state is too high in energy to be significantly populated [34,35]. However, k inetic experiments a t low temperature, using multiple probes including CD and SAXS, suggest rapid formation of a c ompact ensemble which is invisible by fluorescence [55]. All of the mutants studied here by fluorescence conform to the t wo-state model. Only in the presence of stabilizing inorganic salts (0.4 M Na 2 SO 4 ) do we see any evidence for nonlinear effects consistent with rapid collapse t o a compact intermediate [15,33,35]. Recent results describing folding studies of human ubiquitin have established that transient aggregation effects are an important factor in accoun ting for nonlinear effects on refolding rates [35]. Possible errors in determining rate constants near the limit of detection, further compli- cated by slow isomerization-limited phases, have also been proposed to result in roll-over effects in chevron-plot analysis [13,14]. Fig. 8. Temperature dependence of the refolding rate for WT* yeast ubiquitin and the proline-free A37A38 m utant. Data collected in 0 .4 M GdmCl at pH 5.0 in 25 m M acetate buffer. The logarithm of the observed rate constant vs. 1/T sh ow s distinc t curvatu re refl ecting a significant change in heat ca pacity associa ted with TS formation. Solid lines represent the b est fit t o E qn (3) f rom w hich activation p arameters (DHà, DSà and DC p à) have been determined. Fig. 7. Brønsted plot showing logarithm of the observed r ate (refolding and unfolding) v s. change in stability (DDG/RT) for th e family of P 37 / P38 mutants. DDG values were estimated from both equilibrium (cir- cles) and kinetic data (squares). D ata were fitted t o the lin ear corre- lations represented by equations 6 an d 7. A b f value of 0.09 indicates that the loop r egion containing the two adjacent proline residues is largely u nstructured in the rate-limiting s tep for folding. Ó FEBS 2004 Proline residues in ubiquitin stability and folding (Eur. J. Biochem. 271) 4481 A description of the TSE for f olding of ubiquitin, at the level of a detailed F-value analysis to map out interactions present in the TSE, has not yet been reported. However, human ubiquitin has been studied by Krantz et al .[56]using a combination of w-value analysis and protein engineering methods to introduce bis-His metal coordination sites to identify native noncovalent interactions involved in the folding TSE [57]. This approach, through metal complex- ation, enables the degree of partial structure formation at specific sites to be continuously varied over a wide range o f relative populations such that the effects on the rate-limiting step can be determined. The conclusions of this novel approach are that ubiquitin folds through a native-like TSE with a common nucleus but with heterogeneous structural features populated according to their relative stability. A broad TSE, a nd pathway diversity, reflects the variable degrees of structure formation which appears to b e formed around a common folding nucleus consisting of part of the major helix docked against native-like b-strand structure. Previously, HX exchange studies have suggested that the formation of hydrogen bonded structure (and hence pro- tection against NH/ND exchange) occurs in a sin gle co- operative event from which all of the major secondary structure emerges [36], suggesting a loose TSE driven by hydrophobic c ollapse in which secondary structure is y et to be consolidated. Analysis of the kinetic data for t he single and double P37P38 mutants using the Brønsted analysis [48,50] dem- onstrates that all mutants show the same degree of structure formation in the transition state, with a b-value close to zero (%0.09). The data indicate that these residues are largely unstructured in the rate-limiting step for folding, forming native like contacts at a late s tage along the folding co-ordinate. We draw a similar conclusion from the S19P single point mutation where we obtain an estimated F-value of 0.37 [53,54]. Although the w-value analysis described by Krantz et al. has implicated the N-terminal b-hairpin sequence (residues 1–17) and part of the main a-helix (Fig. 1) in the folding nucleus, t he loop connecting the two elements of secondary structure does not appear to be significantly ordered. Similarly, P37 a nd P38 i n adjacent positions at the N terminus of a s hort a-helix (residues 38–40) in an otherwise extended loop region connecting the C terminus of the main a-helix to subsequent strands of b-sheet (Fig. 1 ), also appears to play a passive role in the rate-limiting step for folding. Activation parameters for folding and formation of a compact transition state The temperature-dependence of the refolding rate provides thermodynamic insights into the nature of the TSE for folding. Curvature in the plot of 1/T vs. ln k fold is characteristic of a change in heat capacity associated with burial of hydrophobic surface area. The a D values d erived from the denaturant dependence of k fold and k unfold , namely from the m fold and m unfold values, a re consistent with a compact TSE (a D in the range 0.66–0.71). T he temperature dependence of the refolding r ate enables us to estimate a DC p à of )2.1 (± 0 .3) to )2.4 (± 0.5) k JÆK )1 Æ mol )1 for W T and the A37A38 double mutant. Despite the small fitting errors, the estimated DC p à values are subject to the uncertainties of having measured the refolding rates over a relatively narrow range (283– 310 K) w here the total curvature of the plot is small. Literature estima tes o f DC p UN for the full U–N folding transition from DSC and van’t Hoff analysis are close to %5000 JÆK )1 Æmol )1 [58,59]. It is not entirely clear whether burial of 66–71% of the hydrophobic surface area of the native state should account for all of the observed DC p à for folding, and how other factors relating to desolvation of polar groups, conformational dynamics and hydrogen bonding also contribute [60]. T he observation of a positive entropy o f a ctivation (DSà) s uggests that the favourable entropic contribution from r elease of ordered water associated with the hydrophobic effect is able to overcome the conformational entropy term associated with ordering the flexible polypeptide chain in TSE formation. The large positive enthalpy of activation also attributed to the thermodynamic consequences of the hydrophobic effect [9], dominates DGà for TSE formation. 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Context-dependent effects of proline residues on the stability and folding pathway of ubiquitin Maria D. Crespo, Geoffrey W. Platt, Roger Bofill and. proportion of slow folding molecules [18,30,31]. We report on the effects of proline on the stability and folding kinetics of ubiquitin, a small model system of