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β protein: characterization of a truncation mutant of the N-terminal domain of the ribosomal protein L91
Article No. jmbi.1999.2742 available online at http://www.idealibrary.com on
J. Mol. Biol. (1999) 289, 167±174
Effects of Varying the Local Propensity to Form Secondary Structure on the Stability and Folding Kinetics of a Rapid Folding Mixed a /b b Protein: Characterization of a Truncation Mutant of the N-terminal Domain of the Ribosomal Protein L9 Donna L. Luisi1, Brian Kuhlman1, Kostandinos Sideras1, Philip A. Evans2 and Daniel P. Raleigh1,3* 1
Department of Chemistry State University of New York at Stony Brook, Stony Brook NY 11794-3400, USA 2
Department of Biochemistry University of Cambridge Tennis Court Rd., Cambridge CB2 1QW, UK 3
Graduate Program in Biophysics and Graduate Program in Molecular and Cellular Biology, State University of New York at Stony Brook, Stony Brook NY 11794-8661, USA *Corresponding author
The N-terminal domain of the ribosomal protein L9 forms a split bab structure with a long C-terminal helix. The folding transitions of a 56 residue version of this protein have previously been characterized, here we report the results of a study of a truncation mutant corresponding to residues 1-51. The 51 residue protein adopts the same fold as the 56 residue protein as judged by CD and two-dimensional NMR, but it is less stable as judged by chemical and thermal denaturation experiments. Studies with synthetic peptides demonstrate that the C-terminal helix of the 51 residue version has very little propensity to fold in isolation in contrast to the C-terminal helix of the 56 residue variant. The folding rates of the two proteins, as measured by stopped-¯ow ¯uorescence, are essentially identical, indicating that formation of local structure in the C-terminal helix is not involved in the rate-limiting step of folding. # 1999 Academic Press
Keywords: protein folding; ribosomal protein L9; two-state kinetics; a-helix
Introduction The effect of local interactions on the rate of protein folding is an issue of considerable interest Present address: B. Kuhlman, Department of Biochemistry, University of Washington School of Medicine, Seattle, WA 98195, USA. Abbreviations used: 2D, two-dimensional; CD, circular dichroism; DQF-COSY, double quantum ®ltered correlation spectroscopy; Fmoc, Na-9-¯uoroenylmethyloxycarbonyl; GuDCl, guanidinium deuterium chloride; HPLC, high pressure liquid chromatography; MALDI-TOF, matrix assisted laser desorption and ionization time of ¯ight; NOESY, nuclear Overhauser effect spectroscopy; PAL, polystyrene Fmoc support for peptide amides; TBTU, 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetra¯uoroborate; TFA, tri¯uoroacetic acid; TOCSY, total correlation spectroscopy; TSP, sodium 3-trimethylsilyl (2,2,3,3-d4) propionate. E-mail address of the corresponding author: [email protected] 0022-2836/99/210167±8 $30.00/0
and also of some controversy. Some theoretical work using simpli®ed models suggests that the optimal conditions for folding are when non-local interactions predominate (Abkevich et al., 1995; Govindarajan & Goldstein, 1995), while other computational studies have suggested that the initial formation of local substructure is important to the overall foldability of a molecule (Doyle et al., 1997; Unger & Moult, 1996). The N-terminal domain of the ribosomal protein L9 from Bacillus stearothermophilus offers an interesting system to examine the effects of varying the tendency to form local structure on folding kinetics and on protein stability. L9 contains two domains connected by a long partially solvent-exposed a-helix (Figure 1; Hoffman et al., 1994, 1996). The N-terminal domain adopts a mixed a/b structure and is connected to the C-terminal domain of L9 by the long helix. This helix contributes residues to the hydrophobic core of both domains and the start of the helix forms the C-terminal portion of the N-terminal domain. We have previously # 1999 Academic Press
168 characterized the folding transitions of a 56 residue construct, designated NTL91-56, showing that it folds rapidly in a two-state fashion (Kuhlman et al., 1988a). NTL91-56 is one of the fastest folding proteins characterized to date, making an analysis of the effect of varying the strength of local interactions on the rate of folding particularly interesting. We have also shown that a peptide corresponding to the last 17 residues of the domain, residues 40-56 in the intact protein, is more than 50 % helical in isolation (Luisi et al., 1999). There are very few examples of short peptides derived from proteins which are as structured as this fragment, making this region of NTL91-56 an attractive target for mutational studies. In some cases where peptides have been shown to adopt signi®cant structure in isolation it has been demonstrated that this structure plays a role in folding (An®nsen, 1972; Dyson et al., 1992; Freund et al., 1996; Itzhaki et al., 1995a; Wright et al., 1988). In order to test whether this is the case for NTL9 we have studied the effects upon folding of varying the stability of the C-terminal helix. NOE experiments show that the solution structure of NTL91-56 is very similar to the corresponding region of the intact protein. In the solution structure of NTL91-56 the C-terminal helix packs against and contributes residues to the hydrophobic core of the structure. Here we report the results of a study of a truncation mutant of NTL91-56 in which the propensity to form locally stabilized helical structure is considerably reduced. The truncated protein, designated NTL91-51, is a 51 residue variant (residues 1-51 of the intact protein) in which the ®ve C-terminal residues of NTL91-56 have been eliminated. Removal of these residues greatly reduces the tendency of the C-terminal helix to fold in isolation. These residues project away from the globular portion of NTL9 and do not contact the rest of the protein. The remainder of the helix (e.g. residues 41-51) interacts with the globular domain and further truncation may compromise the tertiary structure. Thus, removing this portion of the protein is expected to primarily modulate local structure in the helix and to have relatively
Figure 1. A ribbon diagram of the ribosomal protein L9 from Bacillus stearothermophilus constructed using the program MOLSCRIPT (Kraulis, 1991) with residues 1-51 shaded in black and residues 52 to 56 shaded in gray.
Folding of a Truncation Mutant of NTL9
little direct effect upon longer range tertiary interactions. This makes NTL9 a particularly attractive candidate for studies designed to test the role of local structure in folding. Here we examine the kinetic and thermodynamic consequences of this truncation on the folding process.
Results Control experiments: the tertiary fold of NTL91-51 and NTL91-56 are identical The near and far-UV CD spectra of NTL91-51 indicate that it is folded. The far-UV CD spectrum is typical of that expected for mixed a/b proteins and it is similar to the far-UV CD spectrum of the 56 residue protein (Figure 2). The protein lacks tryptophan and the near-UV CD signal is dominated by the single tyrosine residue located at position 25. The near-UV CD spectra of the two proteins are also very similar. NMR experiments provide more direct evidence that the two proteins adopt similar folds. There are a set of characteristic ring current shifted methyl resonances in the spectra of NTL91-56. These peaks are also present in the 1H spectrum of NTL91-51. The chemical shifts of the methyl resonances of residues Ile4, Ile18, Leu44 and Leu47 in NTL91-51, which make up part of the hydrophobic core, are within 0.03 ppm of their values in NTL91-56. There are a set of Ca proton resonances from residues in the b-sheet that are found down®eld of the water peak in the spectrum of NTL91-56. These peaks are also observed in the spectrum of NTL91-51, providing good evidence that the b-sheet structure is preserved. More detailed analysis using twodimensional (2D) NMR methods indicate that the Ca chemical shifts of NTL91-51 are virtually identical to those of NTL91-56. All of the Ca shifts are within 0.1 ppm of the Ca values for NTL91-56 (Figure 3(a)). The Ca proton resonances of NTL91-56 span a range of more than 2.3 ppm. Thus, the very close agreement between the observed Ca proton
Figure 2. Far-UV CD spectra of NTL91-51 (®lled circles) and NTL91-56 (open circles). Experiments were performed at 25 C in 2H2O, 20 mM sodium acetate and 100 mM NaCl at p2Hcorr 5.4.
169
Folding of a Truncation Mutant of NTL9
Figure 4. Plots of fraction folded versus temperature for NTL91-51 (®lled circles) and NTL91-56 (open circles). Experiments were performed in 2H2O, 20 mM sodium acetate and 100 mM NaCl at p2Hcorr 5.4.
Figure 3. (a) Plot of the CaH chemical shift differences between NTL91-56 and NTL91-51. (b) Plot of the CaH chemical shift differences between NTL91-51 and random coil values. (c) Plot of the CaH chemical shift differences between NTL91-56 and random coil values. Experiments were performed at 25 C in 90 % H2O and 10 % 2H2O, pH 5.4.
chemical shifts for NTL91-51 and NTL91-56 provides excellent evidence that the structures are identical. The differences are much smaller than the deviations from random coil values which range up to 1.25 ppm (Figure 3). NTL91-51 is less stable than NTL91-56 Thermal and chemical denaturation experiments were performed in order to compare the stability of NTL91-51 and NTL91-56. Thermal unfolding
curves are displayed in Figure 4, and it is clear that NTL91-51 is less thermostable. The apparent cooperativity of the unfolding transition is also reduced for NTL91-51 relative to NTL91-56. The smaller Ho, broader thermal transitions may re¯ect, in part, the Ho of helix formation since the missing ®ve residues are part of the C-terminal helix and largely do not directly interact with the globular portion of the domain. The Tm of NTL91-51 is 75 (ÿ2.8, 1.6) C, while the Tm of NTL91-56 is 81 (1.5) C. The numbers in brackets represent the 95 % con®dence limit. The thermal denaturations are reversible. NTL91-51 is also less resistant to chemical denaturation than NTL91-56. A plot of fraction folded versus [GuDCl] is shown in Figure 5. The midpoint, Cm, of the unfolding curve is 2.00 M for NTL91-51 and 2.98 M for NTL91-56. The value of Go extrapolated to 0 M GuDCl is 2.6 (ÿ0.46, 0.40) kcal molÿ1 for NTL91-51 and 4.6 (0.50) kcal molÿ1 for NTL91-56 at 25 C. The m values of the two proteins are similar, 1.28 (0.15) kcal molÿ1 Mÿ1 for NTL91-51 and 1.51 (0.16) kcal molÿ1 Mÿ1 for NTL91-56. This is entirely as expected since the m value is thought to be directly related to the change in solvent-exposed surface area upon unfolding. The removal of the C-terminal ®ve residues is expected to have a small effect on the surface area change on folding, since these residues form part of the helix which projects out from the globular portion of the protein. The thermodynamic parameters are summarized in Table 1. The propensity to form locally stabilized helical structure is considerably smaller in NTL91-51 than in NTL91-56 We have previously examined the conformational propensities of a set of peptides corresponding to the entire sequence of NTL91-56 (Luisi et al., 1999). Peptides corresponding to the three b-strands and to the ®rst helix were found to be
170
Folding of a Truncation Mutant of NTL9
Figure 5. Plots of fraction folded versus [GuDCl] for NTL91-51 (®lled circles) and for NTL91-56 (open circles). Experiments were performed at 25 C in 2H2O, 20 mM sodium acetate and 100 mM NaCl at p2Hcorr 5.4.
unstructured in isolation. In contrast, a 17 residue peptide corresponding to the C-terminal helix, residues 40-56, was remarkably structured. The peptide was found to be 53 % helical at low temperatures and 41 % helical at 25 C. NTL91-51 lacks the last ®ve residues of this helix and a peptide corresponding to residues 40-51 is noticeably less structured than the 40-56 peptide. The mean residue ellipticity at 222 nm of the shorter peptide is only ÿ7450 at 4 C corresponding to 23 % helix. The helicity decreases to 13 % at 25 C. CD spectra of the two peptides are shown in Figure 6. The decreased tendency to adopt helical structure clearly must contribute to the change in stability of NTL91-51. However, other effects may potentially play a role. In the structure of the full length protein residues Ile4 , Ile18, Leu44 and Leu47 form part of the hydrophobic core. Leu44 and Leu47 are part of the C-terminal helix and it is possible that destabilizing the helix might affect the packing of these residues. However, the chemical shifts of the methyl resonances of all four of these residues are not affected by the truncation, suggesting that there are not any signi®cant changes in the packing of the core. In the full length protein there are also potential interactions between the backbone carbonyl atoms of Lys14 and Lys15 located in the loop and the amide protons of the side-chain of Gln50. If these interactions Table 1. Summary of thermodynamic parameters for NTL91-51 and NTL91-56
Tm ( C) Gou (kcal molÿ1) m (kcal molÿ1 Mÿ1)
NTL91-51
NTL91-56
75 (ÿ2.8, 1.6) 2.6 (ÿ0.46, 0.40) 1.28 (0.15)
81 (1.5) 4.6 (0.50) 1.51 (0.16)
All experiments were performed in 2H2O, 20 mM sodium acetate, 100 mM NaCl, p2Hcorr 5.4 and 25 C. Gou refers to the Go value for unfolding. The m value is the slope of Gou versus [GuDCl]. The numbers in parentheses represent the 95 % con®dence limit.
Figure 6. (a) Far-UV CD spectra of the peptides 40-51 (open circles) and 40-56 (®lled circles). Experiments were performed at 4 C in H2O, 20 mM sodium acetate and 100 mM NaCl at pH 5.4. (b) Peptide fragments and their sequences used in these experiments are listed. NH2 denotes an amidated C terminus, and Ac denotes an acetylated N terminus.
are present in solution, then they might be affected by the truncation. Nevertheless, it is clear that destabilization of the helix reduces the free energy of folding. The kinetic consequences of destabilizing the C-terminal helix NTL91-51 offers an interesting opportunity to examine the consequences of decreasing the propensity to form secondary structure on folding kinetics. We have previously shown that NTL91-56 folds in a two-state fashion. The chevron plot shows no hint of rollover and the kinetic amplitudes are consistent with folding. Kinetic experiments provide excellent evidence that NTL91-51 also folds by a two-state mechanism. The kinetic curves are single exponential and the plot of ln (k) versus [GuDCl] shows the classic V-shape indicative of two-state folding. The experiments were conducted in 2H2O in order to allow a direct comparison with our earlier study of the folding kinetics of NTL91-56 which were carried out in 2H2O (Kuhlman et al., 1998b). Chevron plots of the observed rate versus [GuDCl] are shown in Figure 7, for both NTL91-56 and NTL91-51. Further evidence for two-state folding comes from comparing the stability of the protein deter-
171
Folding of a Truncation Mutant of NTL9
Table 3. A comparison of the folding and the unfolding rates measured at 25 C and at 4 C for NTL91-51 and NTL91-56 NTL91-51
NTL91-56
942 (327) 6.50 (ÿ2.7, 2.1)
1053 (ÿ277, 213) 0.55 (ÿ0.40, 0.24)
144 (ÿ61, 72) 0.57 (ÿ0.32, 0.22)
90a 0.018a
25 C kf (sÿ1) ku (sÿ1) 4 C kf (sÿ1) ku (sÿ1)
Figure 7. A plot of ln(k) versus [GuDCl] for NTL91-51 (®lled circles) and NTL91-56 (open circles). Experiments were performed at 25 C in 2H2O, 20 mM sodium acetate and 100 mM NaCl at p2Hcorr 5.4.
mined via equilibrium and kinetic measurements. The folding and the unfolding rate constants in the absence of denaturant were determined by extrapolation from the plot of ln (k) versus [GuDCl]. The curve was ®t assuming the standard linear dependence of ln (kf) and ln (ku) on [GuDCl]: ln k lnkf 0 M GuDCl exp mz f GuDCl=RT ku 0 M GuDCl exp mz u GuDCl=RT 1 where m{u is the slope of the unfolding branch and m{f is the slope of the folding branch. kf (0 M GuDCl) and ku (0 M GuDCl) are the folding and unfolding rate constants at 0 M GuDCl. For NTL91-51, the value of kf extrapolated to zero molar GuDCl is 942 (327) sÿ1, and the value for ku is 6.5 (ÿ2.7, 2.1) sÿ1. This corresponds to an apparent stability of 2.95 (0.19) kcal molÿ1, which is in good agreement with the value determined from the equilibrium measurements, 2.60 (ÿ0.46, 0.40) kcal molÿ1. The minimum in the ln (k) versus [GuDCl] plot occurs at 2.2 M GuDCl. This is in Table 2. A comparison of the kinetic and equilibrium parameters for the folding of NTL91-51 Equilibrium ÿ1
kf (s ) ku (sÿ1) m{f (kcal molÿ1 Mÿ1) m{u (kcal molÿ1 Mÿ1) ym m (kcal molÿ1 Mÿ1) Gou (kcal molÿ1)
Kinetic
Ð 942 (327) Ð 6.50 (ÿ2.7, 2.1) Ð ÿ1.06 (ÿ0.15, 0.11) Ð 0.42 (0.04) Ð 0.71 1.28 (0.15) 1.48 (0.08) 2.60 (ÿ0.46, 0.40) 2.95 (0.19)
All experiments were performed at 25 C in 2H2O, 20 mM sodium acetate, 100 mM NaCl, p2Hcorr 5.4. Gou refers to the Go value for unfolding. The m value is the slope of Gou versus [GuDCl]. The equilibrium values are derived from equilibrium unfolding experiments. The numbers in parentheses represent the 95 % con®dence limit.
All experiments were performed in 2H2O, 20 mM sodium acetate, 100 mM NaCl, p2Hcorr 5.4. The numbers in parentheses represent the 95 % con®dence limit. a These values are derived from our previously reported global analysis of folding rates of NTL91-56 in 2H2O as a function of temperature and GuDCl (Kuhlman et al., 1998b).
good agreement with the midpoint of the equilibrium unfolding curve, which is 2.0 M. If the folding is two-state then the m value calculated from the kinetic experiments, mu ÿ mf, should agree with the equilibrium value. m{u is found to be 0.42 (0.04) kcal molÿ1 Mÿ1 and m{f is ÿ1.06 (ÿ0.15, 0.11) kcal molÿ1 Mÿ1. The total m value calculated from m{u and m{f is 1.48 (0.08) kcal molÿ1 Mÿ1, which is in reasonable agreement with equilibrium unfolding experiments, 1.28 (0.15) kcal molÿ1 Mÿ1. The relative size of m{u and m{f provide an estimate of how much surface area is buried in the transition state ensemble for folding (Tanford, 1970). The ratio of m{f /(m{u ÿ m{f ), referred to as ym, is 0.71 indicating that approximately 70 % of the surface area buried upon folding is buried in the transition state ensemble for NTL91-51. The value of ym for NTL91-56 is 0.59, which corresponds to approximately 60 % of the surface area being buried. The kinetic parameters are summarized in Table 2. Comparison of the chevron plots of NTL91-51 and NTL91-56 shows that the folding rates of the two proteins are virtually identical while the unfolding rates differ by a factor of 10. The folding rate for NTL91-51 is 942 (327) sÿ1 and the folding rate for NTL91-56 is 1053 (ÿ277, 213) sÿ1. The unfolding rates are 6.50 (ÿ2.7, 2.1) and 0.55 (ÿ0.40, 0.24) sÿ1, respectively. These experiments were carried out at 25 C over a GuDCl concentration of approximately 0.5 to 6 M, similar behavior is observed if the experiments are repeated at low temperature (data not shown). Table 3 compares the folding and unfolding rates for the two proteins, at 25 C and 4 C.
Discussion We have shown that the tertiary folds of NTL91-51 and NTL91-56 are the same. The truncation does result in a signi®cant decrease in the propensity to form locally stabilized helical structure. NTL91-51 is less stable than NTL91-56 and the decrease in stability is due almost entirely to differences in the unfolding rates. The simplest interpretation of these results is that local structure formation in the
172 C-terminal helix is not involved in the rate-limiting step in folding. Consolidation of the helical structure must take place after the rate-limiting step. There may be a considerable population of helix in the transition state but because it is effectively decoupled from whatever other structure is present in the productive folding nucleus it does not affect the rate of folding. The work reported here provides a very dramatic illustration of the point that the ability of a local element of structure to fold autonomously, even where this is very pronounced, does not necessarily have any relevance for its role in the folding mechanism. Independent structure does not necessarily equate with participation in a folding nucleus. This is a particularly interesting observation for this protein since NTL9 folds extremely rapidly. It is interesting to compare our results with other studies that have examined the effects of varying local structural propensities upon folding rates. In most cases increasing local stability enhances the stability of the native state, but it is more dif®cult to predict how changes in local stability will affect folding rates. In some cases the rate increases while in others it is largely unperturbed or even reduced. Degrado, Englander and coworkers have studied the coiled coil region of GCN4 (Sosnick et al., 1996). Mutations that altered the helix propensity of the individual peptides did not alter the folding rates but did affect the unfolding rates. In contrast, work from the Oas laboratory has demonstrated that Gly to Ala mutations in monomeric l repressor greatly accelerated folding (Burton et al., 1997). Although no comparative studies of peptides corresponding to the mutated region have been reported, Gly to Ala substitutions should clearly lead to enhanced helical structure. Serrano and co-workers have demonstrated that increasing the stability of individual helices in the proteins Che Y and ADA2h stabilized the native state and increased the folding rate (Viguera et al., 1996). There is also at least one example where increasing the strength of local interactions in the unfolded state hindered folding (Lopez-Hernandez et al., 1997). One model for protein folding that may account for these results invokes a nucleation condensation mechanism. In this model the ratelimiting step for folding involves formation of a subset of native structure, a folding nucleus (Abkevich et al., 1995; Fersht, 1995; Itzhaki et al., 1995b). Mutations that stabilize the folding nucleus will enhance folding rates. Therefore, stabilizing secondary structure that is involved in the folding nucleus should speed up folding while there will be no affect on folding rates if the structure is not involved in the nucleus. In principle, the structure in the transition state ensemble for NTL9 could be quite extensive, so long as it speci®cally excludes the interactions between the C-terminal helix and the rest of the structure.
Folding of a Truncation Mutant of NTL9
Materials and Methods Peptide synthesis, purification and characterization The proteins and peptides were prepared by solidphase peptide synthesis using standard Fmoc chemistry on a Millipore 9050 Plus or a Rainin PS-3 automated peptide synthesizer. Polystyrene Fmoc support (PAL) for peptide amides were purchased from Perseptive Biosystems (Framingham, MA). 2-(1H-benzotriazole-1yl)-1,1,3,3-tetramethyluronium tetra¯uoroborate (TBTU) was purchased from Advanced Chemtech (Louisville, KT). Fmoc-protected amino acids were purchased from Advanced Chemtech and Perseptive Biosystems. The proteins were cleaved from the resin using a mixture of tri¯uoroacetic acid (TFA), anisole, thioanisole, and ethanedithiol (91 %: 3 %: 3 %: 3 %). The crude samples were puri®ed by high pressure liquid chromatography (HPLC) using a Vydac Reverse Phase C-4 column with an A-B gradient. Buffer A was H2O (0.1 % TFA) and buffer B was 90 % isopropanol and 10 % H2O (0.1 % TFA). All of the proteins and peptides were greater than 98 % pure as judged by analytical HPLC. Characterization of the samples were carried out using amino acid analysis, matrix assisted laser desorption ionization time of ¯ight mass spectroscopy (MALDI-TOF) and NMR. The calculated and the observed molecular weights are: peptide 40-51 calculated 1324 Da, observed 1326 Da; peptide 4056 calculated 1992 Da, observed 1992 Da; NTL91-51 calculated 5548 Da, observed 5544 Da; NTL91-56 calculated 6218 Da, observed 6217 Da. 1
H-NMR
NMR spectra were recorded on Varian INOVA 500 MHz and 600 MHz spectrometers. Samples were dissolved in 90 % H2O/10 % 2H2O, and the pH was adjusted to 5.4. The concentration of the NMR samples were between 3 mM and 6 mM. Sodium 3-trimethylsilyl (2,2,3,3-d4) propionate (TSP) was used as an internal reference (0.0 ppm). The spectra were recorded at 4 and 25 C. Two-dimensional (2D) total correlation spectroscopy (TOCSY; Bax & Davis, 1985) and 2D double quantum ®ltered correlated spectroscopy (DQF-COSY; Rance et al., 1993) were used for spin system assignments. A mixing time of 80 ms was used for the TOCSY experiments. Sequential assignment of the peptides was achieved using 2D nuclear Overhauser effect spectroscopy (NOESY; Kumar et al., 1980). The data matrices were 512 real by 4096 complex for the DQF-COSY, TOCSY, and NOESY experiments. Quadrature detection was achieved using TPPI (Marion & WuÈthrich, 1983). All the data were zero ®lled once in t1 and t2, and a 90 phase shifted sinebell window function was applied prior to Fourier transformation. The ®rst t1 point was multiplied by 0.5 to reduce t1 noise. Circular dichroism spectroscopy CD spectra were recorded on an Aviv model 62A DS circular dichroism spectrometer equipped with a Peltier temperature control system. Far-UV spectra were recorded over the range of 190 to 260 nm at 25 C and 4 C. Spectra were the average of three scans and were corrected by subtraction of the buffer signal. The buffer contained 20 mM sodium acetate and 100 mM sodium chloride at p2Hcorr 5.4. The reading on the pH meter was adjusted to 5.0 in order to compensate for the expected
173
Folding of a Truncation Mutant of NTL9 glass electrode artifacts in 2H2O. The temperature-dependent experiments were performed over the range of 2 C to 98 C, with a two degree step size and a 45 second equilibration time. The ellipticity at 280 nm was monitored. Guanidine denaturations were performed at 25 C, as previously described (Kuhlman & Raleigh, 1998). The ellipticity at 222 nm was monitored. The protein concentrations for the temperature melts and the wavelength scans were 200-250 mM, the protein concentrations for the guanidine denaturations were 30-50 mM. Experiments were carried out in 2H2O in order to allow direct comparison to earlier studies of NTL91-56. The experimental data, plots of ellipticity versus temperature, were ®t with the program Kaleidagraph (Abelbeck Software) to equation (2): o
f T
aN bN T aD bD Teÿ GDÿN T=RT o 1 eÿ GDÿN T=RT
2
where T o GoDÿN T HDÿN Tm 1 ÿ Tm T o ÿ Cp Tm ÿ T T ln Tm
concentrations. The ®nal GuDCl concentrations were checked by measuring the refractive index of the solutions. The temperature of the stopped-¯ow cell compartment was maintained using a circulating water bath. A minimum of three traces were averaged for each ®nal concentration of GuDCl. The averaged kinetic traces were ®t to a single-exponential using a non-linear leastsquares algorithm. Error analysis Errors were determined by perturbing a parameter from its best ®t value and then observing how this change affects its chi-squared value for a new ®t. The parameters which are not being analyzed are allowed to change. An F test was performed to determine what chisquared value corresponds with the 95 % con®dence limit. The error for the analysis is determined by the range of parameter values that give chi-squared values lower than maximum allowed chi-squared determined from the F test. This statistical procedure is described in more detail by Shoemaker et al. (1989).
3
f is the ellipticity and T is temperature. aN, bN, aD and bD are parameters that de®ne the ellipticity of the native (N) and denatured states (D). a and b describe a line with a slope equal to b and a y intercept equal to a. Tm and Cm are the transition midpoints for the temperature and chemical denaturations, respectively. oHD-N(Tm) is the change in enthalpy at Tm. The value of CO p was set to 0.64 kcal molÿ1 Kÿ1. This value was experimentally determined for NTL91-56 in 2H2O and is a reasonable estimate for NTL91-51 (Kuhlman & Raleigh, 1998). Fraction folded at a given temperature was determined by using f (T), aN, bN, aD and bD. Thermal denaturation was 8590 % reversible. The fraction helix was determined using the equations given by Rohl & Baldwin (1997) for the ellipticity of a full helix. The ellipticity of the coil state was determined from measurements of the peptides in 6 M urea. The concentrations of the protein samples were determined by tyrosine absorbance (Gill & von Hippel, 1989). The concentrations of the peptide samples were determined by quantitative amino acid analysis. Stopped-flow kinetic measurements All measurements were made using an Applied Photophysics SX.18MV stopped-¯ow instrument. The refolding and unfolding of NTL91-56 and NTL91-51 in various GuDCl concentrations were followed by monitoring changes in the intrinsic tyrosine ¯uorescence emission. Experiments were performed in deuterated GuDCl in order to allow direct comparison to our previous studies of NTL91-56. The ¯uorescence of Tyr25 was excited at 279 (4) nm, and the change in ¯uorescence emission above 305 nm was collected using a 305 nm cutoff ®lter. The refolding reaction was measured by an 11-fold dilution of protein in 6.5 M GuDCl with 0 to 3.5 M GuDCl solutions to give the desired ®nal GuDCl concentrations. The ®nal protein concentrations were between 50 and 80 mM. The buffer and all the GuDCl solutions contained 20 mM sodium acetate and 100 mM sodium chloride at p2Hcorr 5.4. The unfolding reaction was measured after an 11-fold dilution of native protein in buffer with 3.5 to 6.5 M GuDCl solutions to give the desired ®nal GuDCl
Acknowledgments We thank Peter Leadlay for allowing us to use his stopped-¯ow ¯uorimeter for our initial experiments and we thank Nico ThomaÈ for instructing us in its use. We thank Daniel F. Moriarty for his assistance with the MALDI-TOF mass spectroscopy analysis. This work was supported by NSF grant MCB 9600866 to D.P.R. D.P.R. is a Pew Scholar in the Biomedical Sciences. P.A.E. is a Lister Institute Research Fellow. Travel to the UK was supported by a NATO grant to P.A.E. and D.P.R. D.L.L. was supported in part by a GAANN fellowship from the Department of Education. K.S. was supported in part by a ``RAIRE'' fellowship from SUNY Stony Brook. The RAIRE fellowship program is supported by a grant from the NSF. The NMR facility at SUNY Stony Brook is supported by a grant from the NSF, Che9413510.
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Folding of a Truncation Mutant of NTL9
its implications. Proc. Natl Acad. Sci. USA, 92, 10869-10873. Freund, S. M. V., Wong, K. & Fersht, A. R. (1996). Initiation sites of protein folding by NMR analysis. Proc. Natl Acad. Sci. USA, 93, 10600-10603. Gill, S. C. & von Hippel, P. H. (1989). Calculation of protein extinction coef®cients from amino acid sequence data. Anal. Biochem. 182, 319-326. Govindarajan, S. & Goldstein, R. A. (1995). Optimal local propensities of protein folding. Proteins: Struct. Funct. Genet. 22, 413-418. Hoffman, D. W., Davies, C., Gerchman, S. E., Porter, S. J., White, S. W. & Ramakrishnan, V. (1994). Crystal structure of prokaryotic ribosomal protein L9: a bi-lobed RNA-binding protein. EMBO J. 13, 205-212. Hoffman, D. W., Cameron, C. S., Davies, C., White, S. & Ramakrishnan, V. (1996). Ribosomal protein L9: a structure determination by the combined use of X-ray crystallography and NMR spectroscopy. J. Mol. Biol. 264, 1058-1071. Itzhaki, L. S., Neira, J. L., Ruiz-Sanz, J., de Prat, Gay G. & Fersht, A. R. (1995a). Search for nucleation sites in smaller fragments of chymotrypsin inhibitor 2. J. Mol. Biol. 254, 289-304. Itzhaki, L. S., Otzen, D. E. & Fersht, A. R. (1995b). The structure of the transition state for folding of chymotrypsin inhibitor 2 analysed by protein engineering methods: evidence for a nucleationcondensation mechanism for protein folding. J. Mol. Biol. 254, 260-288. Kraulis, P. J. (1991). MOLSCRIPT: a program to produce both detailed and schematic plots of protein structure. J. Appl. Crystallog. 24, 946-950. Kuhlman, B. & Raleigh, D. P. (1998). Global analysis of the thermal and chemical denaturation of the N-terminal domain of the ribosomal protein L9 in H2O and D2O. Determination of the thermodynamic parameters, Ho, So, and Cop, and evaluation of solvent isotope effects. Protein Sci. 7, 1-8. Kuhlman, B., Boice, J. A., Fairman, R. & Raleigh, D. P. (1998a). Structure and stability of the N-terminal domain of the ribosomal protein L9: evidence for the rapid two-state folding. Biochemistry, 37, 10251032. Kuhlman, B., Luisi, D. L., Evans, P. A. & Raleigh, D. P. (1998b). Global analysis of effects of temperature and denaturant on the folding and unfolding kinetics of the N-terminal domain of the protein L9. J. Mol. Biol. 284, 1661-1670. Kumar, A., Ernst, R. R. & WuÈtrich, K. (1980). A twodimensional nuclear Overhauser enhancement (2D
NOE) experiment for elucidation of complete proton-proton cross relaxation networks in biological macromolecules. Biochem. Biophys. Res. Commun. 95, 1-6. Lopez-Hernandez, E., Cronet, P., Serrano, L. & Munoz, V. (1997). Folding kinetics of Che Y mutants with enhanced native a-helix propensities. J. Mol. Biol. 266, 610-620. Luisi, D. L., Wu, W-J. & Raleigh, D. P. (1999). Conformational analysis of a set of peptides corresponding to the entire primary sequence of the N-terminal domain of the ribosomal protein L9: evidence for stable native-like secondary structure in the unfolded state. J. Mol. Biol. 287, 395-407. Marion, D. & WuÈthrich, K. (1983). Application for phase sensitive two-dimensional correlated spectroscopy (COSY) for measurements of 1H-1H spin-spin coupling constants in proteins. Biochem. Biophys. Res. Commun. 113, 967-974. Rance, M., Sorenson, O. W., Bodenhausen, G., Wagner, G., Ernst, R. R. & WuÈtrich, K. (1993). Improved spectral resolution in COSY 1H NMR spectra of proteins via double quantum ®ltering. Biochem. Biophys. Res. Commun. 117, 479-485. Rohl, C. A. & Baldwin, R. L. (1997). Comparison of NH exchange and circular dichroism as techniques for measuring the parameters of the helix-coil transition in peptides. Biochemistry, 36, 8435-8442. Shoemaker, D. P., Garland, C. W. & Nibler, J. W. (1989). Experiments in Physical Chemistry, 5th edit., pp. 819820, McGraw-Hill Inc., New York. Sosnick, T. R., Jackson, S., Wilk, R. R., Englander, S. W. & DeGrado, W. F. (1996). The role of helix formation in the folding of a fully a-helical coiled coil. Proteins: Struct. Funct. Genet. 24, 427-432. Tanford, C. (1970). Protein denaturation. Part C. Theoretical models for the mechanism of denaturation. Advan. Protein Chem. 24, 1-95. Unger, R. & Moult, J. (1996). Local interactions dominate folding in a simple model of protein folding kinetics. J. Mol. Biol. 259, 988-994. Viguera, A. R., Villegas, V., Aviles, F. X. & Serrano, L. (1996). Favourable native-like helical local interactions can accelerate protein folding. Fold. Des. 2, 23-33. Wright, P. E., Dyson, H. J. & Lerner, R. A. (1988). Conformation of peptide fragments of proteins in aqueous solution: implications for initiation of protein folding. Biochemistry, 27, 7167-7175.
Edited by P. E. Wright (Received 15 January 1999; received in revised form 22 March 1999; accepted 22 March 1999)
J. Mol. Biol. (1999) 289, 167±174
Effects of Varying the Local Propensity to Form Secondary Structure on the Stability and Folding Kinetics of a Rapid Folding Mixed a /b b Protein: Characterization of a Truncation Mutant of the N-terminal Domain of the Ribosomal Protein L9 Donna L. Luisi1, Brian Kuhlman1, Kostandinos Sideras1, Philip A. Evans2 and Daniel P. Raleigh1,3* 1
Department of Chemistry State University of New York at Stony Brook, Stony Brook NY 11794-3400, USA 2
Department of Biochemistry University of Cambridge Tennis Court Rd., Cambridge CB2 1QW, UK 3
Graduate Program in Biophysics and Graduate Program in Molecular and Cellular Biology, State University of New York at Stony Brook, Stony Brook NY 11794-8661, USA *Corresponding author
The N-terminal domain of the ribosomal protein L9 forms a split bab structure with a long C-terminal helix. The folding transitions of a 56 residue version of this protein have previously been characterized, here we report the results of a study of a truncation mutant corresponding to residues 1-51. The 51 residue protein adopts the same fold as the 56 residue protein as judged by CD and two-dimensional NMR, but it is less stable as judged by chemical and thermal denaturation experiments. Studies with synthetic peptides demonstrate that the C-terminal helix of the 51 residue version has very little propensity to fold in isolation in contrast to the C-terminal helix of the 56 residue variant. The folding rates of the two proteins, as measured by stopped-¯ow ¯uorescence, are essentially identical, indicating that formation of local structure in the C-terminal helix is not involved in the rate-limiting step of folding. # 1999 Academic Press
Keywords: protein folding; ribosomal protein L9; two-state kinetics; a-helix
Introduction The effect of local interactions on the rate of protein folding is an issue of considerable interest Present address: B. Kuhlman, Department of Biochemistry, University of Washington School of Medicine, Seattle, WA 98195, USA. Abbreviations used: 2D, two-dimensional; CD, circular dichroism; DQF-COSY, double quantum ®ltered correlation spectroscopy; Fmoc, Na-9-¯uoroenylmethyloxycarbonyl; GuDCl, guanidinium deuterium chloride; HPLC, high pressure liquid chromatography; MALDI-TOF, matrix assisted laser desorption and ionization time of ¯ight; NOESY, nuclear Overhauser effect spectroscopy; PAL, polystyrene Fmoc support for peptide amides; TBTU, 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetra¯uoroborate; TFA, tri¯uoroacetic acid; TOCSY, total correlation spectroscopy; TSP, sodium 3-trimethylsilyl (2,2,3,3-d4) propionate. E-mail address of the corresponding author: [email protected] 0022-2836/99/210167±8 $30.00/0
and also of some controversy. Some theoretical work using simpli®ed models suggests that the optimal conditions for folding are when non-local interactions predominate (Abkevich et al., 1995; Govindarajan & Goldstein, 1995), while other computational studies have suggested that the initial formation of local substructure is important to the overall foldability of a molecule (Doyle et al., 1997; Unger & Moult, 1996). The N-terminal domain of the ribosomal protein L9 from Bacillus stearothermophilus offers an interesting system to examine the effects of varying the tendency to form local structure on folding kinetics and on protein stability. L9 contains two domains connected by a long partially solvent-exposed a-helix (Figure 1; Hoffman et al., 1994, 1996). The N-terminal domain adopts a mixed a/b structure and is connected to the C-terminal domain of L9 by the long helix. This helix contributes residues to the hydrophobic core of both domains and the start of the helix forms the C-terminal portion of the N-terminal domain. We have previously # 1999 Academic Press
168 characterized the folding transitions of a 56 residue construct, designated NTL91-56, showing that it folds rapidly in a two-state fashion (Kuhlman et al., 1988a). NTL91-56 is one of the fastest folding proteins characterized to date, making an analysis of the effect of varying the strength of local interactions on the rate of folding particularly interesting. We have also shown that a peptide corresponding to the last 17 residues of the domain, residues 40-56 in the intact protein, is more than 50 % helical in isolation (Luisi et al., 1999). There are very few examples of short peptides derived from proteins which are as structured as this fragment, making this region of NTL91-56 an attractive target for mutational studies. In some cases where peptides have been shown to adopt signi®cant structure in isolation it has been demonstrated that this structure plays a role in folding (An®nsen, 1972; Dyson et al., 1992; Freund et al., 1996; Itzhaki et al., 1995a; Wright et al., 1988). In order to test whether this is the case for NTL9 we have studied the effects upon folding of varying the stability of the C-terminal helix. NOE experiments show that the solution structure of NTL91-56 is very similar to the corresponding region of the intact protein. In the solution structure of NTL91-56 the C-terminal helix packs against and contributes residues to the hydrophobic core of the structure. Here we report the results of a study of a truncation mutant of NTL91-56 in which the propensity to form locally stabilized helical structure is considerably reduced. The truncated protein, designated NTL91-51, is a 51 residue variant (residues 1-51 of the intact protein) in which the ®ve C-terminal residues of NTL91-56 have been eliminated. Removal of these residues greatly reduces the tendency of the C-terminal helix to fold in isolation. These residues project away from the globular portion of NTL9 and do not contact the rest of the protein. The remainder of the helix (e.g. residues 41-51) interacts with the globular domain and further truncation may compromise the tertiary structure. Thus, removing this portion of the protein is expected to primarily modulate local structure in the helix and to have relatively
Figure 1. A ribbon diagram of the ribosomal protein L9 from Bacillus stearothermophilus constructed using the program MOLSCRIPT (Kraulis, 1991) with residues 1-51 shaded in black and residues 52 to 56 shaded in gray.
Folding of a Truncation Mutant of NTL9
little direct effect upon longer range tertiary interactions. This makes NTL9 a particularly attractive candidate for studies designed to test the role of local structure in folding. Here we examine the kinetic and thermodynamic consequences of this truncation on the folding process.
Results Control experiments: the tertiary fold of NTL91-51 and NTL91-56 are identical The near and far-UV CD spectra of NTL91-51 indicate that it is folded. The far-UV CD spectrum is typical of that expected for mixed a/b proteins and it is similar to the far-UV CD spectrum of the 56 residue protein (Figure 2). The protein lacks tryptophan and the near-UV CD signal is dominated by the single tyrosine residue located at position 25. The near-UV CD spectra of the two proteins are also very similar. NMR experiments provide more direct evidence that the two proteins adopt similar folds. There are a set of characteristic ring current shifted methyl resonances in the spectra of NTL91-56. These peaks are also present in the 1H spectrum of NTL91-51. The chemical shifts of the methyl resonances of residues Ile4, Ile18, Leu44 and Leu47 in NTL91-51, which make up part of the hydrophobic core, are within 0.03 ppm of their values in NTL91-56. There are a set of Ca proton resonances from residues in the b-sheet that are found down®eld of the water peak in the spectrum of NTL91-56. These peaks are also observed in the spectrum of NTL91-51, providing good evidence that the b-sheet structure is preserved. More detailed analysis using twodimensional (2D) NMR methods indicate that the Ca chemical shifts of NTL91-51 are virtually identical to those of NTL91-56. All of the Ca shifts are within 0.1 ppm of the Ca values for NTL91-56 (Figure 3(a)). The Ca proton resonances of NTL91-56 span a range of more than 2.3 ppm. Thus, the very close agreement between the observed Ca proton
Figure 2. Far-UV CD spectra of NTL91-51 (®lled circles) and NTL91-56 (open circles). Experiments were performed at 25 C in 2H2O, 20 mM sodium acetate and 100 mM NaCl at p2Hcorr 5.4.
169
Folding of a Truncation Mutant of NTL9
Figure 4. Plots of fraction folded versus temperature for NTL91-51 (®lled circles) and NTL91-56 (open circles). Experiments were performed in 2H2O, 20 mM sodium acetate and 100 mM NaCl at p2Hcorr 5.4.
Figure 3. (a) Plot of the CaH chemical shift differences between NTL91-56 and NTL91-51. (b) Plot of the CaH chemical shift differences between NTL91-51 and random coil values. (c) Plot of the CaH chemical shift differences between NTL91-56 and random coil values. Experiments were performed at 25 C in 90 % H2O and 10 % 2H2O, pH 5.4.
chemical shifts for NTL91-51 and NTL91-56 provides excellent evidence that the structures are identical. The differences are much smaller than the deviations from random coil values which range up to 1.25 ppm (Figure 3). NTL91-51 is less stable than NTL91-56 Thermal and chemical denaturation experiments were performed in order to compare the stability of NTL91-51 and NTL91-56. Thermal unfolding
curves are displayed in Figure 4, and it is clear that NTL91-51 is less thermostable. The apparent cooperativity of the unfolding transition is also reduced for NTL91-51 relative to NTL91-56. The smaller Ho, broader thermal transitions may re¯ect, in part, the Ho of helix formation since the missing ®ve residues are part of the C-terminal helix and largely do not directly interact with the globular portion of the domain. The Tm of NTL91-51 is 75 (ÿ2.8, 1.6) C, while the Tm of NTL91-56 is 81 (1.5) C. The numbers in brackets represent the 95 % con®dence limit. The thermal denaturations are reversible. NTL91-51 is also less resistant to chemical denaturation than NTL91-56. A plot of fraction folded versus [GuDCl] is shown in Figure 5. The midpoint, Cm, of the unfolding curve is 2.00 M for NTL91-51 and 2.98 M for NTL91-56. The value of Go extrapolated to 0 M GuDCl is 2.6 (ÿ0.46, 0.40) kcal molÿ1 for NTL91-51 and 4.6 (0.50) kcal molÿ1 for NTL91-56 at 25 C. The m values of the two proteins are similar, 1.28 (0.15) kcal molÿ1 Mÿ1 for NTL91-51 and 1.51 (0.16) kcal molÿ1 Mÿ1 for NTL91-56. This is entirely as expected since the m value is thought to be directly related to the change in solvent-exposed surface area upon unfolding. The removal of the C-terminal ®ve residues is expected to have a small effect on the surface area change on folding, since these residues form part of the helix which projects out from the globular portion of the protein. The thermodynamic parameters are summarized in Table 1. The propensity to form locally stabilized helical structure is considerably smaller in NTL91-51 than in NTL91-56 We have previously examined the conformational propensities of a set of peptides corresponding to the entire sequence of NTL91-56 (Luisi et al., 1999). Peptides corresponding to the three b-strands and to the ®rst helix were found to be
170
Folding of a Truncation Mutant of NTL9
Figure 5. Plots of fraction folded versus [GuDCl] for NTL91-51 (®lled circles) and for NTL91-56 (open circles). Experiments were performed at 25 C in 2H2O, 20 mM sodium acetate and 100 mM NaCl at p2Hcorr 5.4.
unstructured in isolation. In contrast, a 17 residue peptide corresponding to the C-terminal helix, residues 40-56, was remarkably structured. The peptide was found to be 53 % helical at low temperatures and 41 % helical at 25 C. NTL91-51 lacks the last ®ve residues of this helix and a peptide corresponding to residues 40-51 is noticeably less structured than the 40-56 peptide. The mean residue ellipticity at 222 nm of the shorter peptide is only ÿ7450 at 4 C corresponding to 23 % helix. The helicity decreases to 13 % at 25 C. CD spectra of the two peptides are shown in Figure 6. The decreased tendency to adopt helical structure clearly must contribute to the change in stability of NTL91-51. However, other effects may potentially play a role. In the structure of the full length protein residues Ile4 , Ile18, Leu44 and Leu47 form part of the hydrophobic core. Leu44 and Leu47 are part of the C-terminal helix and it is possible that destabilizing the helix might affect the packing of these residues. However, the chemical shifts of the methyl resonances of all four of these residues are not affected by the truncation, suggesting that there are not any signi®cant changes in the packing of the core. In the full length protein there are also potential interactions between the backbone carbonyl atoms of Lys14 and Lys15 located in the loop and the amide protons of the side-chain of Gln50. If these interactions Table 1. Summary of thermodynamic parameters for NTL91-51 and NTL91-56
Tm ( C) Gou (kcal molÿ1) m (kcal molÿ1 Mÿ1)
NTL91-51
NTL91-56
75 (ÿ2.8, 1.6) 2.6 (ÿ0.46, 0.40) 1.28 (0.15)
81 (1.5) 4.6 (0.50) 1.51 (0.16)
All experiments were performed in 2H2O, 20 mM sodium acetate, 100 mM NaCl, p2Hcorr 5.4 and 25 C. Gou refers to the Go value for unfolding. The m value is the slope of Gou versus [GuDCl]. The numbers in parentheses represent the 95 % con®dence limit.
Figure 6. (a) Far-UV CD spectra of the peptides 40-51 (open circles) and 40-56 (®lled circles). Experiments were performed at 4 C in H2O, 20 mM sodium acetate and 100 mM NaCl at pH 5.4. (b) Peptide fragments and their sequences used in these experiments are listed. NH2 denotes an amidated C terminus, and Ac denotes an acetylated N terminus.
are present in solution, then they might be affected by the truncation. Nevertheless, it is clear that destabilization of the helix reduces the free energy of folding. The kinetic consequences of destabilizing the C-terminal helix NTL91-51 offers an interesting opportunity to examine the consequences of decreasing the propensity to form secondary structure on folding kinetics. We have previously shown that NTL91-56 folds in a two-state fashion. The chevron plot shows no hint of rollover and the kinetic amplitudes are consistent with folding. Kinetic experiments provide excellent evidence that NTL91-51 also folds by a two-state mechanism. The kinetic curves are single exponential and the plot of ln (k) versus [GuDCl] shows the classic V-shape indicative of two-state folding. The experiments were conducted in 2H2O in order to allow a direct comparison with our earlier study of the folding kinetics of NTL91-56 which were carried out in 2H2O (Kuhlman et al., 1998b). Chevron plots of the observed rate versus [GuDCl] are shown in Figure 7, for both NTL91-56 and NTL91-51. Further evidence for two-state folding comes from comparing the stability of the protein deter-
171
Folding of a Truncation Mutant of NTL9
Table 3. A comparison of the folding and the unfolding rates measured at 25 C and at 4 C for NTL91-51 and NTL91-56 NTL91-51
NTL91-56
942 (327) 6.50 (ÿ2.7, 2.1)
1053 (ÿ277, 213) 0.55 (ÿ0.40, 0.24)
144 (ÿ61, 72) 0.57 (ÿ0.32, 0.22)
90a 0.018a
25 C kf (sÿ1) ku (sÿ1) 4 C kf (sÿ1) ku (sÿ1)
Figure 7. A plot of ln(k) versus [GuDCl] for NTL91-51 (®lled circles) and NTL91-56 (open circles). Experiments were performed at 25 C in 2H2O, 20 mM sodium acetate and 100 mM NaCl at p2Hcorr 5.4.
mined via equilibrium and kinetic measurements. The folding and the unfolding rate constants in the absence of denaturant were determined by extrapolation from the plot of ln (k) versus [GuDCl]. The curve was ®t assuming the standard linear dependence of ln (kf) and ln (ku) on [GuDCl]: ln k lnkf 0 M GuDCl exp mz f GuDCl=RT ku 0 M GuDCl exp mz u GuDCl=RT 1 where m{u is the slope of the unfolding branch and m{f is the slope of the folding branch. kf (0 M GuDCl) and ku (0 M GuDCl) are the folding and unfolding rate constants at 0 M GuDCl. For NTL91-51, the value of kf extrapolated to zero molar GuDCl is 942 (327) sÿ1, and the value for ku is 6.5 (ÿ2.7, 2.1) sÿ1. This corresponds to an apparent stability of 2.95 (0.19) kcal molÿ1, which is in good agreement with the value determined from the equilibrium measurements, 2.60 (ÿ0.46, 0.40) kcal molÿ1. The minimum in the ln (k) versus [GuDCl] plot occurs at 2.2 M GuDCl. This is in Table 2. A comparison of the kinetic and equilibrium parameters for the folding of NTL91-51 Equilibrium ÿ1
kf (s ) ku (sÿ1) m{f (kcal molÿ1 Mÿ1) m{u (kcal molÿ1 Mÿ1) ym m (kcal molÿ1 Mÿ1) Gou (kcal molÿ1)
Kinetic
Ð 942 (327) Ð 6.50 (ÿ2.7, 2.1) Ð ÿ1.06 (ÿ0.15, 0.11) Ð 0.42 (0.04) Ð 0.71 1.28 (0.15) 1.48 (0.08) 2.60 (ÿ0.46, 0.40) 2.95 (0.19)
All experiments were performed at 25 C in 2H2O, 20 mM sodium acetate, 100 mM NaCl, p2Hcorr 5.4. Gou refers to the Go value for unfolding. The m value is the slope of Gou versus [GuDCl]. The equilibrium values are derived from equilibrium unfolding experiments. The numbers in parentheses represent the 95 % con®dence limit.
All experiments were performed in 2H2O, 20 mM sodium acetate, 100 mM NaCl, p2Hcorr 5.4. The numbers in parentheses represent the 95 % con®dence limit. a These values are derived from our previously reported global analysis of folding rates of NTL91-56 in 2H2O as a function of temperature and GuDCl (Kuhlman et al., 1998b).
good agreement with the midpoint of the equilibrium unfolding curve, which is 2.0 M. If the folding is two-state then the m value calculated from the kinetic experiments, mu ÿ mf, should agree with the equilibrium value. m{u is found to be 0.42 (0.04) kcal molÿ1 Mÿ1 and m{f is ÿ1.06 (ÿ0.15, 0.11) kcal molÿ1 Mÿ1. The total m value calculated from m{u and m{f is 1.48 (0.08) kcal molÿ1 Mÿ1, which is in reasonable agreement with equilibrium unfolding experiments, 1.28 (0.15) kcal molÿ1 Mÿ1. The relative size of m{u and m{f provide an estimate of how much surface area is buried in the transition state ensemble for folding (Tanford, 1970). The ratio of m{f /(m{u ÿ m{f ), referred to as ym, is 0.71 indicating that approximately 70 % of the surface area buried upon folding is buried in the transition state ensemble for NTL91-51. The value of ym for NTL91-56 is 0.59, which corresponds to approximately 60 % of the surface area being buried. The kinetic parameters are summarized in Table 2. Comparison of the chevron plots of NTL91-51 and NTL91-56 shows that the folding rates of the two proteins are virtually identical while the unfolding rates differ by a factor of 10. The folding rate for NTL91-51 is 942 (327) sÿ1 and the folding rate for NTL91-56 is 1053 (ÿ277, 213) sÿ1. The unfolding rates are 6.50 (ÿ2.7, 2.1) and 0.55 (ÿ0.40, 0.24) sÿ1, respectively. These experiments were carried out at 25 C over a GuDCl concentration of approximately 0.5 to 6 M, similar behavior is observed if the experiments are repeated at low temperature (data not shown). Table 3 compares the folding and unfolding rates for the two proteins, at 25 C and 4 C.
Discussion We have shown that the tertiary folds of NTL91-51 and NTL91-56 are the same. The truncation does result in a signi®cant decrease in the propensity to form locally stabilized helical structure. NTL91-51 is less stable than NTL91-56 and the decrease in stability is due almost entirely to differences in the unfolding rates. The simplest interpretation of these results is that local structure formation in the
172 C-terminal helix is not involved in the rate-limiting step in folding. Consolidation of the helical structure must take place after the rate-limiting step. There may be a considerable population of helix in the transition state but because it is effectively decoupled from whatever other structure is present in the productive folding nucleus it does not affect the rate of folding. The work reported here provides a very dramatic illustration of the point that the ability of a local element of structure to fold autonomously, even where this is very pronounced, does not necessarily have any relevance for its role in the folding mechanism. Independent structure does not necessarily equate with participation in a folding nucleus. This is a particularly interesting observation for this protein since NTL9 folds extremely rapidly. It is interesting to compare our results with other studies that have examined the effects of varying local structural propensities upon folding rates. In most cases increasing local stability enhances the stability of the native state, but it is more dif®cult to predict how changes in local stability will affect folding rates. In some cases the rate increases while in others it is largely unperturbed or even reduced. Degrado, Englander and coworkers have studied the coiled coil region of GCN4 (Sosnick et al., 1996). Mutations that altered the helix propensity of the individual peptides did not alter the folding rates but did affect the unfolding rates. In contrast, work from the Oas laboratory has demonstrated that Gly to Ala mutations in monomeric l repressor greatly accelerated folding (Burton et al., 1997). Although no comparative studies of peptides corresponding to the mutated region have been reported, Gly to Ala substitutions should clearly lead to enhanced helical structure. Serrano and co-workers have demonstrated that increasing the stability of individual helices in the proteins Che Y and ADA2h stabilized the native state and increased the folding rate (Viguera et al., 1996). There is also at least one example where increasing the strength of local interactions in the unfolded state hindered folding (Lopez-Hernandez et al., 1997). One model for protein folding that may account for these results invokes a nucleation condensation mechanism. In this model the ratelimiting step for folding involves formation of a subset of native structure, a folding nucleus (Abkevich et al., 1995; Fersht, 1995; Itzhaki et al., 1995b). Mutations that stabilize the folding nucleus will enhance folding rates. Therefore, stabilizing secondary structure that is involved in the folding nucleus should speed up folding while there will be no affect on folding rates if the structure is not involved in the nucleus. In principle, the structure in the transition state ensemble for NTL9 could be quite extensive, so long as it speci®cally excludes the interactions between the C-terminal helix and the rest of the structure.
Folding of a Truncation Mutant of NTL9
Materials and Methods Peptide synthesis, purification and characterization The proteins and peptides were prepared by solidphase peptide synthesis using standard Fmoc chemistry on a Millipore 9050 Plus or a Rainin PS-3 automated peptide synthesizer. Polystyrene Fmoc support (PAL) for peptide amides were purchased from Perseptive Biosystems (Framingham, MA). 2-(1H-benzotriazole-1yl)-1,1,3,3-tetramethyluronium tetra¯uoroborate (TBTU) was purchased from Advanced Chemtech (Louisville, KT). Fmoc-protected amino acids were purchased from Advanced Chemtech and Perseptive Biosystems. The proteins were cleaved from the resin using a mixture of tri¯uoroacetic acid (TFA), anisole, thioanisole, and ethanedithiol (91 %: 3 %: 3 %: 3 %). The crude samples were puri®ed by high pressure liquid chromatography (HPLC) using a Vydac Reverse Phase C-4 column with an A-B gradient. Buffer A was H2O (0.1 % TFA) and buffer B was 90 % isopropanol and 10 % H2O (0.1 % TFA). All of the proteins and peptides were greater than 98 % pure as judged by analytical HPLC. Characterization of the samples were carried out using amino acid analysis, matrix assisted laser desorption ionization time of ¯ight mass spectroscopy (MALDI-TOF) and NMR. The calculated and the observed molecular weights are: peptide 40-51 calculated 1324 Da, observed 1326 Da; peptide 4056 calculated 1992 Da, observed 1992 Da; NTL91-51 calculated 5548 Da, observed 5544 Da; NTL91-56 calculated 6218 Da, observed 6217 Da. 1
H-NMR
NMR spectra were recorded on Varian INOVA 500 MHz and 600 MHz spectrometers. Samples were dissolved in 90 % H2O/10 % 2H2O, and the pH was adjusted to 5.4. The concentration of the NMR samples were between 3 mM and 6 mM. Sodium 3-trimethylsilyl (2,2,3,3-d4) propionate (TSP) was used as an internal reference (0.0 ppm). The spectra were recorded at 4 and 25 C. Two-dimensional (2D) total correlation spectroscopy (TOCSY; Bax & Davis, 1985) and 2D double quantum ®ltered correlated spectroscopy (DQF-COSY; Rance et al., 1993) were used for spin system assignments. A mixing time of 80 ms was used for the TOCSY experiments. Sequential assignment of the peptides was achieved using 2D nuclear Overhauser effect spectroscopy (NOESY; Kumar et al., 1980). The data matrices were 512 real by 4096 complex for the DQF-COSY, TOCSY, and NOESY experiments. Quadrature detection was achieved using TPPI (Marion & WuÈthrich, 1983). All the data were zero ®lled once in t1 and t2, and a 90 phase shifted sinebell window function was applied prior to Fourier transformation. The ®rst t1 point was multiplied by 0.5 to reduce t1 noise. Circular dichroism spectroscopy CD spectra were recorded on an Aviv model 62A DS circular dichroism spectrometer equipped with a Peltier temperature control system. Far-UV spectra were recorded over the range of 190 to 260 nm at 25 C and 4 C. Spectra were the average of three scans and were corrected by subtraction of the buffer signal. The buffer contained 20 mM sodium acetate and 100 mM sodium chloride at p2Hcorr 5.4. The reading on the pH meter was adjusted to 5.0 in order to compensate for the expected
173
Folding of a Truncation Mutant of NTL9 glass electrode artifacts in 2H2O. The temperature-dependent experiments were performed over the range of 2 C to 98 C, with a two degree step size and a 45 second equilibration time. The ellipticity at 280 nm was monitored. Guanidine denaturations were performed at 25 C, as previously described (Kuhlman & Raleigh, 1998). The ellipticity at 222 nm was monitored. The protein concentrations for the temperature melts and the wavelength scans were 200-250 mM, the protein concentrations for the guanidine denaturations were 30-50 mM. Experiments were carried out in 2H2O in order to allow direct comparison to earlier studies of NTL91-56. The experimental data, plots of ellipticity versus temperature, were ®t with the program Kaleidagraph (Abelbeck Software) to equation (2): o
f T
aN bN T aD bD Teÿ GDÿN T=RT o 1 eÿ GDÿN T=RT
2
where T o GoDÿN T HDÿN Tm 1 ÿ Tm T o ÿ Cp Tm ÿ T T ln Tm
concentrations. The ®nal GuDCl concentrations were checked by measuring the refractive index of the solutions. The temperature of the stopped-¯ow cell compartment was maintained using a circulating water bath. A minimum of three traces were averaged for each ®nal concentration of GuDCl. The averaged kinetic traces were ®t to a single-exponential using a non-linear leastsquares algorithm. Error analysis Errors were determined by perturbing a parameter from its best ®t value and then observing how this change affects its chi-squared value for a new ®t. The parameters which are not being analyzed are allowed to change. An F test was performed to determine what chisquared value corresponds with the 95 % con®dence limit. The error for the analysis is determined by the range of parameter values that give chi-squared values lower than maximum allowed chi-squared determined from the F test. This statistical procedure is described in more detail by Shoemaker et al. (1989).
3
f is the ellipticity and T is temperature. aN, bN, aD and bD are parameters that de®ne the ellipticity of the native (N) and denatured states (D). a and b describe a line with a slope equal to b and a y intercept equal to a. Tm and Cm are the transition midpoints for the temperature and chemical denaturations, respectively. oHD-N(Tm) is the change in enthalpy at Tm. The value of CO p was set to 0.64 kcal molÿ1 Kÿ1. This value was experimentally determined for NTL91-56 in 2H2O and is a reasonable estimate for NTL91-51 (Kuhlman & Raleigh, 1998). Fraction folded at a given temperature was determined by using f (T), aN, bN, aD and bD. Thermal denaturation was 8590 % reversible. The fraction helix was determined using the equations given by Rohl & Baldwin (1997) for the ellipticity of a full helix. The ellipticity of the coil state was determined from measurements of the peptides in 6 M urea. The concentrations of the protein samples were determined by tyrosine absorbance (Gill & von Hippel, 1989). The concentrations of the peptide samples were determined by quantitative amino acid analysis. Stopped-flow kinetic measurements All measurements were made using an Applied Photophysics SX.18MV stopped-¯ow instrument. The refolding and unfolding of NTL91-56 and NTL91-51 in various GuDCl concentrations were followed by monitoring changes in the intrinsic tyrosine ¯uorescence emission. Experiments were performed in deuterated GuDCl in order to allow direct comparison to our previous studies of NTL91-56. The ¯uorescence of Tyr25 was excited at 279 (4) nm, and the change in ¯uorescence emission above 305 nm was collected using a 305 nm cutoff ®lter. The refolding reaction was measured by an 11-fold dilution of protein in 6.5 M GuDCl with 0 to 3.5 M GuDCl solutions to give the desired ®nal GuDCl concentrations. The ®nal protein concentrations were between 50 and 80 mM. The buffer and all the GuDCl solutions contained 20 mM sodium acetate and 100 mM sodium chloride at p2Hcorr 5.4. The unfolding reaction was measured after an 11-fold dilution of native protein in buffer with 3.5 to 6.5 M GuDCl solutions to give the desired ®nal GuDCl
Acknowledgments We thank Peter Leadlay for allowing us to use his stopped-¯ow ¯uorimeter for our initial experiments and we thank Nico ThomaÈ for instructing us in its use. We thank Daniel F. Moriarty for his assistance with the MALDI-TOF mass spectroscopy analysis. This work was supported by NSF grant MCB 9600866 to D.P.R. D.P.R. is a Pew Scholar in the Biomedical Sciences. P.A.E. is a Lister Institute Research Fellow. Travel to the UK was supported by a NATO grant to P.A.E. and D.P.R. D.L.L. was supported in part by a GAANN fellowship from the Department of Education. K.S. was supported in part by a ``RAIRE'' fellowship from SUNY Stony Brook. The RAIRE fellowship program is supported by a grant from the NSF. The NMR facility at SUNY Stony Brook is supported by a grant from the NSF, Che9413510.
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Edited by P. E. Wright (Received 15 January 1999; received in revised form 22 March 1999; accepted 22 March 1999)