Folding kinetics of the SH3 domain of PI3 kinase by real-time NMR combined with optical spectroscopy1

J. Mol. Biol. (1998) 276, 657±667

Folding Kinetics of the SH3 Domain of PI3 Kinase by Real-time NMR Combined with Optical Spectroscopy J. InÄaki Guijarro1, Craig J. Morton2, Kevin W. Plaxco1, Iain D. Campbell2 and Christopher M. Dobson1* 1

New Chemistry Laboratory and 2Department of Biochemistry, Oxford Centre for Molecular Sciences, University of Oxford, South Parks Road, Oxford, OX1 3QT UK

The refolding kinetics of the chemically denatured SH3 domain of phosphatidylinositol 30 -kinase (PI3-SH3) have been monitored by real-time one-dimensional 1H NMR coupled with a variety of other biophysical techniques. These experiments indicate that the refolding kinetics of PI3SH3 are biphasic. The slow phase (27 (8)% amplitude) is due to a population of substantially unfolded molecules with an incorrectly con®gured cis proline residue. The fast phase (73 (8)% amplitude) corresponds to the folding of protein molecules with proline residues in a trans con®guration in the unfolded state. NMR experiments indicate that the ®rst species populated after the initiation of folding exhibit poor chemical shift dispersion and have spectra very similar to that of the denatured protein in 8 M guanidine hydrochloride. Linear combinations of the ®rst spectrum and of the spectrum of the native protein accurately reconstruct all of the spectra acquired during refolding. Consistent with this, native side-chain and backbone Ha atom packing (NMR), secondary structure (far-UV circular dichroism), burial of aromatic residues (near-UV circular dichroism), intrinsic ¯uorescence and peptide binding activity are all recovered with effectively identical kinetics. Equilibrium unfolding and folding/unfolding kinetics yield, within experimental error, identical values for the free energy of unfolding (
Gu ± H2O ˆ 3.38 kcal molÿ1) and for the slope of the free energy of unfolding versus denaturant concentration (meq ˆ 2.33 kcal molÿ1 Mÿ1). Together, these data provide strong evidence that PI3± SH3 folds without signi®cant population of kinetic well-structured intermediates. That PI3 ±SH3 folds slowly (time constant 2.8 seconds in H2O at 20 C) indicates that slow refolding is not always a consequence of kinetic traps but may be observed even when a protein appears to fold via a simple, two-state mechanism. # 1998 Academic Press Limited

*Corresponding author

Keywords: real-time NMR; protein folding; two-state transition; proline isomerization; SH3 domain

Introduction The mechanisms by which polypeptides fold to their native conformation remains an area of active Present addresses: C. J. Morton, Department of Chemistry, University of Melbourne, Parkville 3052, Australia; K. W. Plaxco, Department of Biochemistry, University of Washington, Seattle, Washington 98195, USA Abbreviations used: ANS, 1-anilino-naphthalene-8 sulfonic acid; CD, circular dichroism; GuHCl, guanidine hydrochloride; PI3, phosphatidylinositol 30 -kinase; PI3SH3, SH3 domain of the p85a subunit of bovine phosphatidylinositol 30 -kinase; 1D, one-dimensional; FUV, far-UV; NUV, near-UV. 0022±2836/98/080657±11 $25.00/0/mb971553

research in structural biology. Nuclear magnetic resonance (NMR) has proved to be an important technique for studying the complex phenomenon of protein folding because of the structural and dynamical data that it can provide at atomic resolution (for a recent review, see Dyson & Wright, 1996). NMR has been widely used to characterize models of folding intermediates at equilibrium and denatured or unfolded proteins (Dobson, 1992; WuÈthrich, 1994; Shortle, 1996; Smith et al., 1996). Amide-hydrogen exchange methods have played a major role in these studies and have furnished a wealth of information on partly folded and denatured states. Studies on small peptides (Dyson & Wright, 1996) have been helpful in clarifying the # 1998 Academic Press Limited

658 role of local structures in early folding events. The rate of interconversion between native and unfolded states at speci®c residues has been obtained by magnetization transfer and line shape analysis techniques for proteins under equilibrium conditions (Dobson & Evans, 1984; Evans et al., 1989; Roder, 1989; Huang & Oas, 1995a; Zhang & Forman-Kay, 1995). An NMR technique that has been widely used to follow indirectly the kinetics of protein folding is hydrogen-exchange pulse labeling (Roder et al., 1988; Udgaonkar & Baldwin, 1988; Roder, 1989; Englander & Mayne, 1992; Baldwin, 1993; Radford & Dobson, 1995). Undoubtedly, pulse labeling and equilibrium NMR experiments have made major contributions to our current understanding of the folding processes. A powerful complementary approach to these studies of protein folding is to monitor folding kinetics directly by real-time NMR. Time-resolved NMR is the only available technique that can directly probe the packing of side-chains and backbone atoms at individual sites. Real-time NMR provides a means of directly monitoring folding kinetics at multiple speci®c sites, thus allowing the detection of intermediates and the determination of the cooperativity of the folding process. In spite of the potential of the technique, its application has so far largely been limited to a few examples of slowly folding (or unfolding) proteins, due to its inherent relatively poor time-resolution. Real-time NMR has been used to monitor the folding of thermally denatured ribonuclease A (Blum et al., 1978; Adler & Scheraga, 1988; Akasaka et al., 1991), staphylococcal nuclease (Kautz & Fox, 1993), and a collagen-like, triple-helical peptide (Liu et al., 1996). Studies of the refolding of chemically denatured apoplastocyanin have indicated an intermediate trapped by an incorrect trans proline conformation (Koide et al., 1993). When applied to the unfolding of ribonuclease A, real-time NMR suggested the existence of a novel unfolding intermediate (Kiefhaber et al., 1995). Stopped-¯ow technology can signi®cantly reduce the dead time of real-time NMR experiments and has been used to characterize the folding (Hoeltzli & Frieden, 1996) and unfolding (Hoeltzli & Frieden, 1995) of 19F-labeled dihydrofolate reductase and the unfolding of 19F labeled intestinal fatty acid binding protein (Frieden et al., 1993). A transient intermediate with a 1H NMR spectrum which resembles that of the well-characterized equilibrium molten globule of a-lactalbumin at acid pH has been detected by onedimensional (1D) NMR stopped-¯ow experiments (Balbach et al., 1995), and a single two-dimensional (2D) 1H ± 15N experiment acquired during refolding has been used to show that the protein backbone forms cooperatively (Balbach et al., 1996). More recently, photo-CIDNP (chemically induced dynamic nuclear polarization) experiments coupled to a rapid mixing device have been developed and applied to study the refolding of hen lysozyme (Hore et al., 1997). Here, we report stopped-¯ow

Folding Kinetics of PI3-SH3 by Real-time NMR

1D NMR experiments in conjunction with optical spectroscopy to study the renaturation of the SH3 domain of the p85a subunit of the bovine phosphatidylinositol 30 -kinase (PI3 ±SH3). The SH3 domain (approximately 60 to 85 residues) is a small, modular domain that often occurs in proteins involved in intra-cellular signal transduction (Morton & Campbell, 1994; Musacchio et al., 1994). Despite the low sequence homology across the SH3 domain family, all SH3 domains exhibit a common fold which has been well characterized both by NMR spectroscopy and X-ray crystallography. In particular, the structure of PI3-SH3 (84 residues) is composed of two perpendicular antiparallel b-sheets of three and two strands, respectively, and two helix-like turns (Booker et al., 1993). SH3 domains provide simple model systems for the study of protein folding, as they are small, monomeric, structurally independent domains and lack complicating factors such as disul®de bonds, prosthetic groups or native cis-proline residues.

Results Equilibrium unfolding The equilibrium unfolding of PI3-SH3 was monitored by the change of intrinsic ¯uorescence upon unfolding (Figure 1). A single transition is observed between the native and denatured states. The ¯uorescence of the denatured state shows a linear dependence on denaturant concentration as has been reported for other proteins (for example Koide et al., 1993; Viguera et al., 1994). Data are well described by a two-state model that assumes that the free energy of unfolding (
Gu) is a linear function of denaturant concentration:
Gu ˆ
GuÿH2 O ÿ meq ‰GuHClŠ

…1†

where
Gu ± H2O is the free energy of unfolding in the absence of denaturant, and meq is the slope of
Gu versus [GuHCl]. The variable meq, which re¯ects the cooperativity of the transition, includes a term that is proportional to the increase in surface exposure on unfolding. From the ®tting, the apparent value of
Gu ± H2O is 3.23 (0.19) kcal molÿ1 (13.52 (0.80) kJ molÿ1), meq ˆ 2.33 (0.14) kcal molÿ1 Mÿ1, and the apparent [GuHCl] at the midpoint of the transition is 1.39 M ([GuHCl]50% ˆ
Gu ± H2O/meq). The value of meq obtained for PI3± SH3 is consistent with the values determined for other proteins of similar size (Myers et al., 1995). Refolding kinetics by real-time NMR A series of 1D spectra was acquired during the refolding of chemically denatured PI3± SH3 at 6 C (Figure 2). The intensities of a number of resolved signals which are absent in the ®rst spectrum increase with time. Concomitantly, some of the sig-

Folding Kinetics of PI3-SH3 by Real-time NMR

Figure 1. Equilibrium unfolding of PI3 ±SH3 at 20 C in GuHCl dissolved in buffer A followed by intrinsic ¯uorescence (excitation at 268 nm, emission at 303 nm). Filled circles represent experimental data. The best ®t to a two-state transition equation is shown as a continuous line. The equation used (Koide et al., 1993) takes into account the linear dependence of the denatured state ¯uorescence on GuHCl concentration.

nals present in the ®rst spectrum lose intensity over time. After 1000 seconds, the protein has achieved its ®nal conformation as judged by a lack of further change in the spectrum. Figure 3 shows the ®rst spectrum acquired approximately four seconds after refolding was initiated (Figure 3(b)) and a spectrum of the protein 1500 seconds later (Figure 3(d)). The spectrum of the latter state of the protein displays the characteristic chemical shift dispersion of a structured protein and is identical with the spectrum of the native protein recorded under the same conditions. This indicates that the protein folds to its native state during the course of the experiment; we will thus refer to the spectrum of this ®nal state as the native spectrum. The ®rst spectrum, however, is characteristic of a largely unfolded protein lacking a single preferred conformation. Indeed, this spectrum exhibits limited chemical shift dispersion and resembles the spectrum of the fully denatured protein in 8 M guanidine hydrochloride (Figure 3(a)); the differences in resonant frequencies between these two spectra can be attributed to small, solvent-induced shifts (Plaxco et al., 1997) and to differing linewidths. The lines in the ®rst spectrum are signi®cantly broader than the corresponding resonances from the protein in 8 M GuHCl. Line broadening in the ®rst spectrum could arise from an equilibrium between different conformations at an intermediate rate on the chemical shift time scale (as observed for folding intermediates at equilibrium (Alexandrescu et al., 1993; Guijarro et al., 1995) or during refolding (Balbach et al., 1995)), or from magnetic ®eld inhomogeneities due to incomplete mixing. In order to discriminate between these possibilities, Tris-HCl (a molecule too small to undergo signi®cant slow conformational changes) was added to the protein

659

Figure 2. Stacked plot of the high-®eld region of a series of one-dimensional 1H NMR spectra acquired during the refolding of PI3 ± SH3 (6 C). Refolding was initiated by injecting 50 ml of PI3 ± SH3 in 8 M Gu2HCl into the NMR tube containing 500 ml of deuterated buffer A. The acquisition of the ®rst spectrum shown was initiated 3.12 seconds after mixing. The time interval between spectra is 2.08 seconds. Two independent kinetic experiments were averaged. Only the ®rst 200 seconds of the kinetics are shown.

solution in 8 M GuHCl and a control kinetic experiment was performed. The Tris-HCl signal was also broad in the ®rst spectrum and its linewidth decreased with time during the ®rst 12 seconds. This strongly argues that the line broadening observed for PI3 ±SH3 signals in the initial spectra is caused, at least in part, by residual magnetic ®eld inhomogeneity. Thus, the spectrum recorded approximately four seconds after the initiation of refolding is consistent with a substantially unfolded protein in which the backbone and sidechain atoms are not close packed. The ®nal spectrum corresponds to the native protein. The kinetics of the appearance and disappearance of several peaks in the NMR spectra were determined. In each case, the kinetic traces were well described by a sum of two exponential functions. The kinetics of appearance of the wellresolved signals of the native protein were, within experimental error, identical with the kinetics of disappearance of signals arising from the substantially unfolded ®rst state. This suggests that no partially folded intermediates with signi®cant structure accumulate during refolding. The average and standard deviation values for the kinetic parameters calculated for ten signals distributed throughout the spectrum were: 78.5 (2.6)% amplitude and 0.0575 (0.0053) secondÿ1 rate for the fast phase, and 21.5 (2.6)% amplitude and 0.00446 (0.00104) secondÿ1 rate for the slow

660

Folding Kinetics of PI3-SH3 by Real-time NMR

Figure 3. High-®eld region of the 1D 1H NMR spectrum of PI3-SH3 in 8 M Gu2HCl in deuterated buffer A, p2H 7.2 (a) and of spectra acquired at 4.2 seconds (b), and 1500 seconds (d) of refolding of the protein at 6 C. (c) Difference between the spectrum of the native protein and the kinetic spectrum after 104 seconds of refolding. The population fraction of native SH3 after 104 seconds of refolding was determined from the kinetics prior to calculating the difference spectrum in (c). After appropriate referencing (Plaxco et al., 1997), the spectrum of the protein in 8 M Gu2HCl (a) was right-shifted 0.1 ppm in order to align the major aliphatic peak with that of the spectrum of the protein after 4.2 seconds of initiating refolding (b).

Figure 4. High-®eld region of the experimental ((a), (c) and (e)) and simulated ((b), (d) and (f)) 1D 1H NMR spectra of PI3± SH3 after 12.5 seconds ((a) and (b)), 29 seconds ((c) and (d)) and 106 seconds ((e) and (f)) of refolding at 6 C. Simulated spectra were calculated by a weighted combination of the spectra at 4.2 seconds (®rst-state spectrum) and at 1500 seconds (native-state spectrum) when refolding is complete. The parameters used to calculate the population of the ®rst and native state at each time were: 78.5% amplitude and 0.0575 secondÿ1 rate for the fast phase and 21.5% amplitude and 0.00446 secondÿ1 rate for the slow phase. These values were derived from the mean of the respective values calculated from the ®tting of the kinetic traces of the signals indicated by an asterisk (*).

phase. To test further if any partially folded intermediate is populated, experimental spectra were simulated as linear combinations of the ®rst and the native spectra using the above rates and amplitudes to calculate the relative populations at each time point. These simulations describe all of the intermediate spectra recorded during the refolding of the protein to within experimental error (Figure 4). Any small difference between the experimental and simulated spectra can be ascribed to the line broadening observed in the ®rst spectrum. It should be mentioned that the aromatic region and the region down-®eld of the solvent signal where the Ha protons involved in b-sheets resonate are also well simulated as linear combinations of the ®rst and native spectra (data not shown). As described above, at 6 C and 0.727 M ®nal concentration of GuHCl, the kinetics of refolding of PI3-SH3 are biphasic. The fast phase has a time constant of 17.5 (1.8) seconds while the slow phase has a time constant of 238 (60) seconds.

After 100 seconds of folding, the fast phase is complete to 99.6% while the slow phase is only 36% complete. Therefore, the difference between the native spectrum (weighted by the population fraction of native molecules expected from the kinetics) and the kinetic spectrum at 100 seconds yields the spectrum of the slowly refolding species (Figure 3(c)). The spectrum of the slowly refolding species is very similar to the ®rst spectrum acquired during refolding and to the spectrum of the protein in 8 M GuHCl. Furthermore, no change is observed in the shape of the difference spectrum during the refolding of the protein, and the kinetics of disappearance of the difference spectrum signals parallel the kinetics observed for the experimental spectra. Thus, it can be concluded that the slowly refolding species are not highly structured and that the biphasic behavior displayed by PI3 ±SH3 arises from a heterogeneity in a largely unfolded state of the protein.

661

Folding Kinetics of PI3-SH3 by Real-time NMR Table 1. Kinetic parameters for the refolding of chemically denatured PI3± SH3 at 20 C Amplitude (%) NRa CD, FUVb CD, NUVb Intrinsic fluorescenceb,c Peptide binding activityb,d Meane

80.4  1.8 70.4  8.7 73.5  3.0 60.5  0.9 81.7  2.3 73.3  7.7

Fast phase

Rate (secondÿ1)

0.1006  0.0278 0.0542  0.0104 0.0495  0.0036 0.0585  0.0016 0.0639  0.0037 0.0565  0.0053

Amplitude (%) 19.6  1.8 29.6  5.4 26.5  4.0 39.5  1.2 18.3  2.5 26.7  7.7

Slow phase Rate (secondÿ1) 0.0138  0.0043 0.0115  0.0013 0.0149  0.0011 0.0169  0.0003 0.0127  0.0018 0.0140  0.0021

a

Data obtained from refolding in deuterated buffer A at ®nal [Gu2HCl] of 0.727 M. Data obtained from refolding in aqueous buffer A. Final [GuHCl] was 0.727 M. Refolding was initiated by manual mixing. Errors shown are ®tting errors. Errors from averaging independent experiments are expected to be larger. c The ®t of the intrinsic ¯uorescence kinetic data extrapolates at time zero seconds to the ¯uorescence value expected for the unfolded protein in the refolding buffer, suggesting that no burst phase is occurring. d Refolding experiments at lower concentrations of GuHCl, performed with a stopped-¯ow ¯uorimeter with a dead time of 1.7 ms, are also well ®tted as a sum of two exponentials. This suggests that there is no fast phase occurring within the dead time of the manual mixing experiments reported in this Table. e Mean and standard deviation of the rates (omitting NMR data due to a kinetic isotopic effect) and amplitudes (all data). b

The refolding of PI3 ± SH3 was also monitored in real time by NMR at 20 C. Under these conditions, the ®rst spectrum has contributions from native signals because the time constant of the fast phase (10.6 seconds) is relatively close to the dead time of the experiment (4.2 seconds). Nevertheless, under these conditions the kinetics are also biphasic, the appearance of the native state is simultaneous with the disappearance of the ®rst state, the kinetic spectra are well described by linear combinations of the ®rst and the native spectra and the spectrum of the slowly refolding species is similar to the ®rst spectrum and to the spectrum of the denatured protein in 8 M GuHCl. The parameters obtained from ®tting the data to double exponential functions are reported in Table 1. Proline isomerization in the denatured state has long been recognized as a rate limiting process in the folding of many proteins. PI3± SH3 contains three proline residues at positions 50, 70 and 84 that are in a trans conformation in the native protein (Booker et al., 1993). Pro84 is unlikely to affect the folding kinetics of the protein because it is located in the unstructured C-terminal region of the molecule. However, cis proline residues at positions 50 and/or 70 in the denatured protein could well be the cause of the slow phase observed in the renaturation experiments. Indeed, the amplitude and the rate of the slow phase are consistent with that expected for a proline-isomerization-limited phase and the real-time NMR experiments show that the backbone and side-chains of the slowly folding species are not closely packed. We therefore synthesized two pentapeptides encompassing the sequence around Pro50 (AKPEE) and Pro70 (DFPGT) and determined the relative population of cis-trans proline isomers in 8 M GuHCl by 1H NMR at 20 C. Peptide AKPEE contains 7% of cis isomers and peptide DFPGT contains 23 (2)%. The latter is of the same order of magnitude as the amplitude of the slow phase (26.7 (7.7)%) as determined by real-time NMR and other spectroscopic techniques (see below and Table 1) and suggests that the slow phase is due to a population

with cis proline at position 70 in the denatured state that is not able to fold until the proline is in the trans conformation. Several experimental results further support this hypothesis. First, the enzyme cyclophilin A, known to catalyze proline isomerization, accelerates the slow phase of refolding of PI3-SH3 (J.I.G., unpublished results). Second, the rate constant of the slow phase is an exponential function of the inverse of the temperature between 279 and 323 K (i.e. Arrhenius behavior). This result indicates that the activation heat capacity (
Cp{) for the transition that de®nes the rate of this phase is near zero, as expected for a proline isomerization reaction in an unfolded state but not for a folding transition involving a hydrophobic collapse (Oliveberg et al., 1995; Plaxco et al., 1998). Furthermore, the activation enthalpy (9.6 (0.9) kcal molÿ1) is similar to that observed for a proline-isomerization-limited phase for chymotrypsin inhibitor 2 (Jackson & Fersht, 1991b). Third, the rate and amplitude of this reaction are independent of GuHCl concentration (see below and Figure 6; Schmid & Baldwin, 1979). Finally, that the kinetics of the slow phase are the same in H2O and in 2 H2O is also in agreement with this hypothesis (see below) because the proline-isomerization rate in model peptides is not in¯uenced by the isotopic composition of the solvent (Stein, 1993). The results shown here indicate that Pro70 cis to trans isomerization limits the rate of the slow phase. Whether or not Pro50 isomerization is also rate limiting cannot be determined as the estimated population of cis proline conformers at position 50 in the unfolded state is very small (7%). Refolding kinetics by circular dichroism and fluorescence The recovery of the native circular dichroism (CD) signal in the far-UV (FUV) and near-UV (NUV) regions and of native tyrosine ¯uorescence show, within experimental error, the same kinetics (Figure 5(c), (d) and (e) and Table 1). The recovery of the binding activity to a proline-rich peptide,

662 which monitors the formation of the peptide binding site, also displays the same kinetics (Figure 5(f) and Table 1). All four probes indicate biphasic kinetics with similar amplitudes and rates for the fast (A1 and k1) and slow (A2 and k2) phases. The average values for these parameters are: A1 ˆ 73.3 (7.7)%, k1 ˆ 0.0565 (0.0053) secondÿ1, A2 ˆ 26.7 (7.7)%, k2 ˆ 0.0140 (0.0021) secondÿ1. The amplitude of both phases and the rate of the slow phase are in close agreement with those obtained by NMR. However, the rate constant of the fast phase determined from the NMR experiment is larger than that obtained using optical probes. The

Folding Kinetics of PI3-SH3 by Real-time NMR

origin of this discrepancy is that the kinetic NMR experiments were performed in buffer prepared with 2H2O, while buffer prepared with H2O was used in all other experiments. To con®rm this, control experiments monitoring intrinsic tyrosine ¯uorescence in 2H2O buffer were carried out; these indicated fast-phase refolding kinetics identical (within experimental error) with those obtained by NMR. While the relative amplitude of both phases and the rate of the slow phase are hardly affected by 2H2O, the fast phase rate constant was increased by a factor of 1.6  0.1 relative to the rate constant in H2O. An isotopic effect of similar magnitude has

Figure 5. Refolding kinetics of PI3-SH3 at 20 C monitored by (a) NMR, disappearance of the ®rst state (signal at 0.91 ppm); (b) NMR, appearance of the native state (signal at 0.57 ppm); (c) CD in the far-UV region; (d) CD in the near-UV region; (e) intrinsic tyrosine ¯uorescence; and (f) recovery of the peptide binding activity. Continuous lines correspond to the best ®t to a double-exponential function. Data are normalized relative to the best ®t. The experimental direction of variation of the signal was maintained to produce this Figure. Final GuHCl concentration was 0.727 M for all experiments. In (a) and (b), two representative kinetic traces were chosen. Refolding kinetics followed by NMR were performed using deuterated buffer. An isotopic effect which accelerates the fast phase can be observed.

663

Folding Kinetics of PI3-SH3 by Real-time NMR

The slope (m{f ) of the plot of ln(kf) versus [GuHCl] is ÿ1.42 (0.05) kcal molÿ1 Mÿ1 after appropriate unit conversion. The rate of the slow renaturation phase shows a rather small dependence on the ®nal concentration of denaturant, as expected for a process in which the rate is limited by proline isomerization in an unfolded state (Jackson & Fersht, 1991b). The unfolding kinetics of PI3 ±SH3 are monophasic. At GuHCl concentrations above the transition region, the unfolding rate is a linear function of GuHCl concentration (Figure 6(b)). The unfolding rate in H2O (ku ± H2O) calculated from Figure 6(b) is 0.00067 (0.000046) secondÿ1 and the slope (m{u) is 0.93 (0.02) kcal molÿ1 Mÿ1. For a two-state transition, the equilibrium free energy of unfolding in the absence of denaturant (
Gu ± H2O) must be equal to the sum of the free energy between the native and transition state (
G{u ± H2O) and of the free energy between the transition state and the unfolded state (ÿ
G{f ± H2O; see Jackson & Fersht, 1991a). Similarly, the equilibrium m value (meq) must be equal to ÿm{f ‡ m{u. In order to calculate the equilibrium value of
Gu ± H2O, consider the following mechanism: Kiso

kfÿH2 O

Uc „ Ut „ N Figure 6. Dependence of the logarithm of the (a) refolding and (b) unfolding rates of PI3-SH3 on the ®nal concentration of GuHCl (20 C). *, Fast-phase folding rate; ~, slow-phase folding rate; & , unfolding rate. The rate of folding (fast phase) and unfolding in H2O calculated from the extrapolation of the respective linear ®t (continuous lines) are 0.3528 (0.0164) secondÿ1 and 0.00067 (0.000046) secondÿ1. The average rate for the slow folding phase is 0.0164 (0.0022) secondÿ1.

been reported for the slow refolding phase of lysozyme, though for this protein, the rate in H2O is larger than in 2H2O at neutral pH (Itzhaki & Evans, 1996). At low pH, this rate becomes larger in 2H2O as observed here for PI3 ± SH3. Hence, the acceleration of the fast phase is due to an isotope effect; when corrected for this, the kinetics obtained by NMR are essentially the same as those obtained by the other techniques.

kuÿH2 O

…2†

where Kiso (Uc/Ut) is the equilibrium constant between the unfolded molecules with cis (Uc) and trans (Ut) proline residues and N represents the native state. The apparent equilibrium constant (Ku0 ) derived from the equilibrium unfolding experiment must be corrected according to: Ku ˆ Ku0 =…1 ‡ Kiso †

…3†

to obtain the true equilibrium constant Ku (ku ± H2O/ kf ± H2O) and thereby the true
Gu ± H2O. Using the relative amplitudes of the slow (27%) and fast (73%) phase to calculate Kiso (0.299), the corrected value of
Gu ± H2O is 3.38 (0.19) kcal molÿ1. This value is similar to the equilibrium free energy of unfolding calculated from the kinetics of folding and unfolding: 3.65 (0.05) kcal molÿ1. Similarly, the m values from equilibrium (2.33 (0.14) kcal molÿ1 Mÿ1) and from kinetics (2.35 (0.05) kcal molÿ1 Mÿ1) are in close agreement.

GuHCl concentration dependence of the folding and unfolding kinetics

Discussion

The kinetics of folding and unfolding at different concentrations of denaturant were studied by ¯uorescence spectroscopy. At GuHCl concentrations below the transition region, the logarithm of the rate of folding for the fast phase is a linear function of GuHCl concentration (Figure 6(a)). This suggests that no intermediate is formed, as deviations from a straight line are observed when an intermediate state is populated (Jackson & Fersht, 1991a; Khorasanizadeh et al., 1993). Extrapolation of the rate (kf) to zero denaturant concentration yields a rate constant (kf ± H2O) of 0.3528 (0.0163) secondÿ1.

Real-time NMR has been a particularly useful tool to probe the refolding of PI3-SH3. Indeed, this technique showed that (i) four seconds after initiating the refolding, the side-chains and backbone of the protein are not packed and that no burst-phase collapse leading to a highly structured intermediate occurs; (ii) the transition between the ®rst state (or more accurately, ensemble of states) and the native state is cooperative and no highly structured intermediate accumulates during refolding; (iii) the refolding kinetics are biphasic; and (iv) the slow refolding phase is due to molecules that are

664 trapped in a substantially unfolded state. Of particular importance is the fact that NMR can directly monitor the formation of the fully native state and distinguish between native-like and native conformations. The ®rst spectrum shows poor chemical shift dispersion and looks very much like the spectrum of the denatured protein in 8 M GuHCl. This indicates that the protein remains substantially unfolded after approximately four seconds of refolding. Indeed, if highly structured intermediate species were formed in the dead time of the experiment, the ®rst spectrum should display signi®cant chemical shift dispersion, as has been observed for staphylococcal nuclease (Kautz & Fox, 1993), for which the spectrum after eight seconds of refolding indicates the presence of a native-like intermediate. The ®rst spectrum of PI3-SH3 shows line broadening relative to the spectrum in 8 M GuHCl. The line broadening is, however, substantially less than that shown by the partly folded state of a-lactalbumin at the early stages of refolding (Balbach et al., 1995). Furthermore, control experiments indicate that this line broadening is caused, at least in part, by inhomogeneities in the magnetic ®eld following mixing. Nevertheless, the NMR results alone cannot rule out the possibility that the protein is partially folded and interconverts between different conformations on a millisecond time scale. Moreover, the real-time NMR experiments performed do not provide information about the compactness of the refolding protein. Other lines of evidence, however, demonstrate that the protein is substantially unfolded at this stage: PI3-SH3 does not bind the hydrophobic dye ANS during its refolding (J.I.G., unpublished results) as molten globule-like conformations with accessible hydrophobic cores do (Chaffotte et al., 1992; Engelhard & Evans, 1995); the refolding as probed by intrinsic ¯uorescence does not show any burst phase; and the recovery of peptide binding activity in which the dead time of measurement was only 1.7 ms does not show a fast phase that could lead to a folding intermediate within four seconds of refolding (see Table 1). The spectrum of the species trapped by proline isomerization also shows very little chemical shift dispersion and is very similar to the ®rst-state and to the denaturedprotein spectra. No broadening is observed in this spectrum. These results indicate that these species are substantially unfolded, in contrast to the proline-isomerization-trapped species detected for plastocyanin which have a spectrum signi®cantly different from that of the unfolded state (Koide et al., 1993). The best evidence that kinetic NMR provides on the cooperativity of the refolding of PI3-SH3 is that linear combinations of the ®rst and the native spectra weighted by their relative populations can accurately reconstruct the kinetic spectra. No other available spectroscopic technique can so convincingly and directly demonstrate the absence of intermediates by means of a single experiment.

Folding Kinetics of PI3-SH3 by Real-time NMR

The fact that PI3± SH3 folds slowly has allowed us to use real-time NMR to monitor the folding of a protein which folds via a two-state mechanism while in general these proteins fold very rapidly. The recovery of native secondary structure (circular dichroism in the far-UV region), aromatic residue side-chain packing (circular dichroism in the near-UV region and ¯uorescence), side-chain and backbone packing (NMR) and peptide binding activity are all concerted processes. Moreover, a single transition is observed in the equilibrium unfolding of PI3± SH3 which is also well described by a two-state model. Finally, the rate constants for folding and unfolding determined over a wide range of denaturant concentrations can be ®tted to a two-state model and yield thermodynamic parameters consistent with the values obtained from equilibrium unfolding. Taken together, these data provide compelling evidence that PI3± SH3 refolds through a two-state mechanism without accumulation of intermediates. To date, in addition to PI3 ± SH3, it has been demonstrated that several small, single-domain proteins fold in a process well described by a twostate model. Proteins for which this has been shown include chymotrypsin inhibitor 2 (Jackson & Fersht, 1991a), two IgG binding domains of streptococcal protein G (Alexander et al., 1992), the spectrin SH3 domain (Viguera et al., 1994), the l repressor (Huang & Oas, 1995b), acyl-coenzyme A binding protein (Kragelund et al., 1995), the Bacillus cold shock protein (Schindler et al., 1995), the activation domain of human procarboxypeptidase A2 (Villegas et al., 1995), the IgG binding domain of peptostreptococcal protein L (Scalley et al., 1997), the tenascin ®bronectin type III domain (Clarke et al., 1997), tendamistad (SchoÈnbrunner et al., 1997) and the Fyn SH3 domain (Plaxco et al., 1998). Except for PI3-SH3, all these proteins fold extremely rapidly with time constants in the absence of denaturant ranging from less than 1 ms (Bacillus cold shock protein, l repressor) to 340 ms (tenascin ®bronectin type III domain, 25 C). That all of these proteins fold with relatively rapid kinetics has contributed to a common view that the slow folding of larger, non-two-state proteins is a consequence of kinetic traps. The relatively slow folding of PI3± SH3 (time constant of 2.8 seconds at 20 C in the absence of denaturant), however, demonstrates that there is not a simple relationship between the absence of intermediates and rapid folding as has been suggested previously (Schindler et al., 1995) and shows that slow folding need not be a consequence of kinetic traps.

Materials and Methods Protein and synthetic peptides PI3 ± SH3 was expressed in Escherichia coli as a fusion protein with the enzyme glutathione S-transferase and puri®ed as described (Booker et al., 1993). The recombinant protein used in this study consists of 84 residues

665

Folding Kinetics of PI3-SH3 by Real-time NMR from the SH3 domain of PI3, plus a two-residue N-terminal extension (GS) and a four-residue C-terminal extension (WNSS). After puri®cation, PI3± SH3 was buffer exchanged to 20 mM NH4CO3 (pH 8.0) by dialysis at 4 C, lyophilized and stored at 4 C until further use. The protein was pure as assessed by SDS-PAGE, mass spectrometry and N-terminal sequencing. Peptides RKLPPRPSK, DFPGT and AKPEE were synthesized at the Oxford Centre for Molecular Sciences facilities on an Applied Biosystems automated synthesizer using Fmoc chemistry. The three peptides were produced with a free N terminus and a blocking amide group at the C terminus. Peptides were puri®ed by reverse-phase, high-performance liquid chromatography (RP-HPLC) and their purity checked by analytical RPHPLC and mass spectrometry. Guanidine hydrochloride denaturation Equilibrium denaturation of PI3 ± SH3 was followed by ¯uorescence spectroscopy on a Perkin Elmer LS 50B spectrometer thermostated to 20 (0.5) C. PI3± SH3 dissolved in buffer A (20 mM NaH2PO4 (pH 7.2)) was equilibrated at 20 C with different amounts of guanidine hydrochloride (GuHCl) also in buffer A for at least one hour. In all experiments, ®nal denaturant concentration was determined by refractrometry according to Nozaki (1972). The ®nal protein concentration was 0.25 mg/ml. Fluorescence excitation was at 268 (1.25) nm and the emitted signal was recorded at 303 (2.5) nm. Thermodynamic parameters were obtained using the linear extrapolation method. Data were ®tted to a two-state equation including a term for the dependence of the ¯uorescence of the unfolded state on denaturant concentration (Koide et al., 1993). All ®ttings for equilibrium and kinetic experiments were done using the non-linear least-squares algorithm provided with KaleidaGraph (Albeck Software, Reading, PA). The errors shown correspond to ®tting errors unless otherwise stated. NMR NMR experiments were performed at a 1H resonant frequency of 600.2 MHz on a home-built spectrometer based on an Oxford Instruments magnet and a GE-1280 computer. The sodium salt of 4,4-dimethyl-silapentane sulfonate (DSS) was used as an external reference taking into account the effect of GuHCl on the chemical shift of the water signal as reported (Plaxco et al., 1997). Data were analyzed on Sun Sparc stations with the software Felix 2.3 (Biosym). Folding kinetics were followed at 6 C and 20 C in real time using a pneumatic mixer as described by Balbach et al. (1995). Deuterated PI3± SH3 was unfolded in 8 M GuHCl in buffer A prepared in 2H2O with previously deuterated guanidine hydrochloride. Folding was triggered by mixing one part of the denatured PI3± SH3 solution with ten parts of the refolding buffer (buffer A prepared in 2H2O). The ®nal concentration of the protein was 10 mg/ml. A series of one-dimensional (1D) spectra was acquired with a relaxation delay of 1.04 seconds between each scan. Two accumulations were recorded for each spectrum to improve the signal-to-noise ratio. The time point for an experiment was taken to be half way through the acquisition time. The ®rst spectrum was recorded at 2.08 seconds of refolding but was discarded as mixing of the highly viscous GuHCl solution remained incomplete. The second spectrum was identical

with the ®rst spectrum except for line narrowing. This spectrum was taken as the ®rst spectrum for the simulations (see Results). All spectra were corrected to have the same integral between 3.5 and ÿ0.1 ppm. Either the integral or the height of a peak were used to obtain kinetic traces that were ®tted to exponential decays with the KaleidaGraph software. The equilibrium relative population of proline cis-trans isomers was determined for the pentapeptides DFPGT and AKPEE dissolved in 8 M GuHCl prepared in deuterated buffer A by measuring the relative area of the peaks assigned to cis and trans conformers in 1D spectra. The errors for the relative population of cis and trans conformers represent one standard deviation from the mean of the integral values measured for different peaks. Spectra were acquired with a relaxation delay of 3.6 seconds on concentrated peptide samples (20 mM). Folding kinetics by circular dichroism Refolding of PI3± SH3 was monitored at 20 (0.1) C by circular dichroism in the near-UV (NUV) and far-UV (FUV) regions by means of a Jasco J-720 spectropolarimeter equipped with a water bath to control the temperature. The protein (16.3 mg/ml for NUV experiments and 7.5 mg/ml for FUV experiments) in 8 M GuHCl prepared in buffer A (GuHCl 8 M) was diluted 11-fold in buffer A to initiate refolding. Mixing was done manually. Excitation wavelength and light path length were respectively, 270 nm and 0.1 cm for kinetics followed in the NUV experiments, and 215 nm and 0.5 cm for FUV experiments. Averages of eight (NUV) or 16 (FUV) shots are reported. Folding and unfolding kinetics by fluorescence Kinetic experiments involving slowly folding conditions were conducted on the LS 50B ¯uorimeter using manual mixing with a typical dead time of approximately ten seconds. An Applied Photophysics SX-17MV stopped-¯ow spectrophotometer was used to follow fastrate kinetics (1.7 ms dead time). All experiments were conducted at 20 C using a Grant LTD6 water bath to maintain the temperature to 0.5 deg.C (LS 50B) or 0.1 deg.C (SX-17MV). PI3 ± SH3 contains two solvent-exposed tryptophan residues at positions 55 and 85 as well as seven tyrosine residues. Trp55 is located in the binding site and directly participates in the interaction with proline-rich peptides (Booker et al., 1993; Yu et al., 1994) while Trp85 is in the unstructured C-terminal region of the protein. Initial experiments showed that tryptophan ¯uorescence (excitation at 280 nm, emission at 340 nm) does not change upon folding. Therefore, tyrosine ¯uorescence was used to monitor the refolding of PI3 ±SH3. The protein (2 to 3 mg/ml) in GuHCl 8 M was refolded by dilution (1 : 10) with buffer A. Intrinsic tyrosine ¯uorescence was recorded at 303 nm with an excitation wavelength of 268 nm. The recovery of the binding activity of PI3-SH3 towards the peptide RKLPPRPSK was followed during refolding by means of the ¯uorescence enhancement of Trp55 upon binding. A ®vefold excess of peptide (1100 mM) was dissolved in the GuHCl 8 M solution containing the protein (220 mM). Excitation and emission wavelengths were, respectively, 280 and 340 nm. Under the conditions used, peptide binding is much faster than the folding reaction and no change in ¯uorescence is observed when the native protein is mixed with the pep-

666 tide in the refolding buffer. Control experiments indicated that the peptide does not exhibit a change in ¯uorescence when diluted from GuHCl to the refolding buffer. The dependence of the folding kinetics on GuHCl concentration was monitored as the recovery of peptide binding activity on the SX-17MV ¯uorimeter, with an excitation wavelength of 280 nm and a high-pass ®lter with a cut-off at 320 nm. The protein and the peptide in 4.3 M GuHCl were diluted into buffer A containing the appropriate concentration of GuHCl. All other conditions were as described above. Control experiments showed that the folding kinetics followed by intrinsic ¯uorescence and the peptide binding activity of PI3± SH3 were, within experimental error, the same between 0.7 and 1.3 M GuHCl ®nal concentration. Unfolding kinetics as a function of the ®nal GuHCl concentration were followed by the change of tyrosine ¯uorescence as PI3 ± SH3 in buffer A was diluted 11-fold into buffer A containing different concentrations of GuHCl. All other conditions were as described above.

Acknowledgements We thank Maureen Pitkeathly for peptide synthesis, Dennis Benjamin for his help with mass spectrometry and Sophie Jackson for kindly providing the cyclophilin A used in this study. This is a contribution from the Oxford Centre for Molecular Sciences which is supported by the UK Biotechnology and Biological Sciences Research Council, the Engineering and Physical Sciences Research Council and the Medical Research Council. This investigation was also supported in part by the Wellcome Trust (I.D.C.), an International Research Scholars award from the Howard Hughes Medical Institute (C.M.D.) and the European Community (C.M.D. and J.I.G.).

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Edited by P. E. Wright (Received 20 August 1997; received in revised form 18 November 1997; accepted 21 November 1997)