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Acceleration of the folding of hen lysozyme by trifluoroethanol1
J. Mol. Biol. (1997) 265, 112–117
COMMUNICATION
Acceleration of the Folding of Hen Lysozyme by Trifluoroethanol Hui Lu, Matthias Buck, Sheena E. Radford and Christopher M. Dobson* Oxford Centre for Molecular Sciences, New Chemistry Laboratory, University of Oxford, South Parks Road Oxford, OX1 3QT, UK
The refolding of a partially structured state of hen lysozyme formed in 60% (v/v) 2,2,2-trifluoroethanol (TFE) has been studied using hydrogen exchange pulse labelling monitored by 2D 1H NMR, and by stopped flow fluorescence and CD measurements. The results are compared with similar studies of the refolding of the protein denatured in 6 M guanidine hydrochloride (GuHCl). Two conclusions have emerged from these studies. First, provided that the refolding conditions are identical, the two denatured states fold with very similar kinetics, despite the fact the extensive secondary structure is present in the TFE-denatured state but not in the protein denatured in 6 M GuHCl. This arises because of the rapid equilibration of structure in the species formed in the initial stage of folding. Second, whilst addition of GuHCl to the refolding buffer decreases the rate of folding, low concentrations of TFE increase the rate of folding. The result is consistent with slow steps in the refolding of lysozyme being associated primarily with the reorganisation of hydrophobic interactions rather than of hydrogen bonded structure. 7 1997 Academic Press Limited
*Corresponding author
Keywords: rate enhancement; hydrophobic interaction; hydrogen bonding; partially folded states; kinetic traps
Investigation of the mechanism by which a protein attains its native three-dimensional structure represents a considerable challenge to experimentalists, not least because of the extreme rapidity of many of the steps involved (Kim & Baldwin, 1990; Matthews, 1993; Plaxco & Dobson, 1996). Although the refolding of some small proteins appears highly cooperative and effectively two state (Jackson & Fersht, 1991; Prat Gay et al., 1995; Kragelund et al., 1995; Schindler et al., 1995), refolding of other proteins involves the transient population of partially folded states (Matthews, 1993; Miranker & Dobson, 1996). One example of the latter class of proteins is lysozyme, studies Present addresses: M. Buck, Department of Chemistry, Harvard University, 12 Oxford Street, Cambridge, MA 02138, USA; S. E. Radford, Department of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, UK. Abbreviations used: TFE, 2,2,2-trifluroethanol; MeUdiNAG, 4-methylumbelliferyl-N,N'-diacetyl-bchitobiose; ANS, 1-anilino napthalene sulphonate; GuHCl, guanidine hydrochloride. 0022–2836/97/020112–06 $25.00/0/mb960715
of which (with the native disulphide bonds intact) have shown at least two distinct types of intermediate states (Radford et al., 1992; Dobson et al., 1994; Radford & Dobson, 1995). Firstly, within the first 4 ms of folding a nativelike component of secondary structure is formed, along with clustering of at least some aromatic residues, a high affinity for 1-anilino napthalene sulphonate (ANS) binding, but little protection against hydrogen exchange (Radford et al., 1992; Chaffotte et al., 1992; Dobson et al., 1994; Itzhaki et al., 1994). More persistently structured states with extensive protection against amide exchange emerge later. These do not yet, however, have persistent native-like tertiary structure. An additional feature of the lysozyme folding pathway is the existence of heterogeneity in folding, which has been attributed to the presence of kinetic traps generated in the initial stages of folding (Radford et al., 1992; Kiefhaber, 1995; Rothwarf & Scheraga, 1996). One of the most interesting issues in the folding of a protein such as lysozyme is the manner in which structure can form in the absence of many of 7 1997 Academic Press Limited
Folding of Hen Lysozyme in TFE
the interactions that stabilise the native state, and the significance of such structure for the process of folding. One approach to this has been the study of peptide fragments of a protein (Shin et al., 1993; Waltho et al., 1993; Peng & Kim, 1994; Prat Gay et al., 1995; Ittah & Haas, 1995; Itzhaki et al., 1995). A common feature of such peptides is that secondary structure, particularly helical structure, can be stabilised by the addition of solvents, such as trifluoroethanol (TFE) that enhance intramolecular hydrogen bonding (Nelson & Kallenbach, 1989; Lehrman et al., 1990; Segawa et al., 1991; Dyson et al., 1992; Yang et al., 1995; Cammers-Goodwin et al., 1996; Schoenbrunner et al., 1996). A question that arises, therefore, is whether such solvents can influence the kinetics of the folding of intact proteins, by influencing the interactions which stabilise the structure during the folding process. Here, we report experiments with lysozyme designed to explore this point. Hen lysozyme denatured in 60% (v/v) TFE at pH 5.2, like that previously characterised at pH 2.0 in 50% (v/v) TFE (Buck et al., 1993, 1995, 1996), is partially folded with more a-helical structure than that of the native protein, but few specific tertiary interactions (Figure 1). By contrast with denaturation in TFE solution, denaturation of lysozyme by 6 M GuHCl results in a substantially unfolded state in which little secondary or tertiary structure persists (Figure 1). To examine the influence of the TFE state of lysozyme on its refolding pathway, the protein, partially denatured in 60% (v/v) TFE, or substantially denatured in 6 M GuHCl (both containing the four native disulphide bridges), was diluted 11-fold into 20 mM sodium acetate buffer (pH 5.2) at 25°C. Its refolding was monitored by far UV CD, intrinsic tryptophan fluorescence and hydrogen exchange pulse labelling.
113 The results of the stopped-flow intrinsic fluorescence experiments are shown in Figure 2. When folding is monitored after 11-fold dilution of the initial denatured state, the shape of the refolding traces from both denatured states is similar, being characteristic of that previously observed for the folding of lysozyme denatured in GuHCl (Itzhaki et al., 1994). This suggests that the refolding process from the GuHCl and TFE-denatured states are similar despite the very different properties of the initial denatured states and the different final refolding conditions (Figure 2a, curve 1 and 2b curve 1). The rate of folding from the two denatured states is, however, very different; folding occurs much more rapidly following dilution from TFE. Indeed, the two measurable phases observed by fluorescence occur five and nine times faster than refolding from GuHCl (Table 1). In accord with these results, the rates of refolding measured by the binding of the fluorescent inhibitor 4-methylumbelliferyl-N,N '-diacetyl-b-chitobiose (MeU-diNAG) and by stopped flow CD in the far UV (measured at 222 nm) are also much faster following dilution from TFE. The refolding kinetics from the two denatured states, measured by hydrogen exchange pulse labelling, are shown in Figure 3a and b. The data confirm the above results, revealing that even at the level of the individual residues, the folding pathways from the two denatured states are similar and that refolding following dilution from TFE is a more rapid process. Analysis of the kinetic parameters suggests two factors contribute to the apparent increase in the rate of protection; an increased amplitude of fast protection of amides in the a-domain and an increased rate of protection of the slow phase of amides in the b-domain (a smaller increase in rate of protection of amides in the a-domain also occurs; Table 1). Previous studies
Figure 1. Far (a) and near (b) UV CD spectra of hen lysozyme in the native state (W), in 60% (v/v) TFE (R), and in 6 M GuHCl (w). Spectra were acquired using a JASCO J720 spectropolarimeter, using cuvettes of 1 mm path length for far UV CD and 10 mm for near UV CD, and protein samples of 0.05 mg/ml and 0.5 mg/ml, respectively. The proteins were dissolved in 20 mM sodium acetate buffer (pH 5.2), 25°C. The signal is divided by the total number of residues (129) in each case. A higher TFE concentration was used to generate the TFE-denatured state at pH 5.2, relative to that used previously at pH 2.0, because of the greater stability of the native structure at the higher pH (Pfeil & Privalov, 1976; Yang & Honig, 1993).
114
Figure 2. Refolding of lysozyme under various conditions monitored by stopped-flow fluorescence. a, Refolding from the GuHCl-denatured state. Refolding was initiated by dilution of the denatured state into buffer alone (w, curve 1) and into buffer containing 5.5% (v/v) TFE (W, curve 2). b, Refolding from the TFE-denatured state into buffer alone (w, curve 1) and into buffer containing 0.55 M GuHCl (W, curve 2). Measurements were made at 25°C using an Applied Photophysics SX-17MV stopped-flow instrument. An excitation wavelength of 280 nm was used and the total fluorescence intensity above 320 nm was measured. The concentration of initial denatured protein was 20 mg/ml (in either 60% (v/v) TFE or 6 M GuHCl, pH 5.2) and refolding was initiated by 11-fold dilution of the denatured protein into the refolding buffer described.
have shown that amides in the native-like helices in the TFE-denatured state have protection factors ranging from 20 to more than 200 (Buck et al., 1993, 1995). If this structure were to persist during the kinetic refolding experiments, very different proton occupancy would be expected for these amides in the hydrogen exchange labelling experiment. Within experimental error, however, a uniform proton occupancy of 30% is observed for all amides in the a-domain after a refolding time of 3.5 ms. This suggests that the a-helical structure in the TFE-denatured state must be destabilised and rapidly reorganised after the TFE is diluted to initiate refolding. The results of a second series of experiments in which the two denatured states were refolded under identical conditions are also shown in
Folding of Hen Lysozyme in TFE
Figure 2a (curve 2) and 2b (curve 2) and Figure 3c and d. Under these conditions the folding kinetics from the two denatured states are closely similar (Table 2). The data show that a significant increase (ca 3.5-fold) in the rate of folding of the GuHCl-denatured state is caused by the presence of the low concentrations (5.5%, v/v) of TFE in the refolding buffer. Such concentrations of TFE reduce marginally the stability of the native protein (the midpoints of thermal denaturation measured by far UV CD in the presence and absence of 5.5% (v/v) TFE are 74 (22)°C and 71 (22)°C, respectively), and increase the rate of unfolding of the native protein (H.L. & C.M.D., unpublished results). By contrast, the folding kinetics of the TFE-denatured state are retarded approximately 2.5-fold by the inclusion of 0.55 M GuHCl in the refolding buffer. These two effects account for the differences in rate constants observed when the two denatured states were folded under different conditions. A more detailed analysis of the dependence of the refolding kinetics on the concentration of GuHCl and TFE in the refolding buffer, studied by stopped-flow fluorescence, is shown in Figure 4. Both the fast and slow phases of folding from the GuHCl-denatured state increase with increasing TFE concentrations up to about 1 M (1% (v/v) TFE = 0.137 M). By contrast, the rates of both the fast and slow phases of refolding from the TFE-denatured state decrease with the inclusion of increasing concentrations of GuHCl in the refolding buffer. It is well established that the rates of folding and unfolding are critically dependent on the conditions under which these events take place. Addition of denaturants generally reduces the rate of folding and increases the rate of unfolding (Aune & Tanford, 1969; Segawa & Sugihara, 1984; Matouschek et al., 1990; Schmid, 1992). The finding that the addition of low concentrations of TFE enhances the rate of folding of lysozyme without significantly perturbing the folding pathway is therefore of interest. Under conditions where rapid collapse occurs, the folding process involves searching within a compact state for native-like contacts (Sali et al., 1994). In the case of lysozyme the collapsed state has extensive secondary structure and is stabilised by four disulphide bonds; it is likely therefore that significant barriers can exist for the reorganisation of either correctly or incorrectly folded species (Radford et al., 1992; Radford & Dobson, 1995). TFE is known to enhance intramolecular hydrogen bonding and to decrease hydrophobic interactions within a polypeptide chain (Buck et al., 1993; Thomas & Dill, 1993; Shiraki et al., 1995; Cammers-Goodwin et al., 1996). The acceleration of folding of lysozyme by TFE is therefore consistent with recent suggestions that the slow steps on the major pathway of folding involve predominantly the reorganisation of hydrophobic interactions rather than of hydrogen bonds (Itzhaki & Evans; 1996).
115
Folding of Hen Lysozyme in TFE
Table 1. Stopped-flow fluorescence and hydrogen exchange pulse labelling data for refolding of the TFE and GuHCl-denatured states Reaction A. Stopped-flow intrinsic fluorescence b 60% TFE : 5.5% TFE 6 M GuHCl : 0.55 M GuHCl B. Hydrogen exchange pulse labelling c 60% TFE : 5.5% TFE 6 M GuHCl : 0.55 M GuHCl
a-domain b-domain a-domain b-domain
Fast phase t1 (ms)
Amplitude t1 (%)
Slow phase t2 (ms)
Amplitude t2 (%)
Offseta (%)
3 (20.5) 17 (22)
−50 (23) −58 (23)
19 (22) 170 (220)
47 (23) 42 (22)
— —
<2 <4 <5 <10
76 (23) 28 (24) 36 (25) 25 (26)
32 (27) 37 (27) 64 (210) 200 (250)
18 (22) 60 (24) 45 (25) 54 (25)
7 (21) 13 (23) 20 (24) 20 (25).
All experiments were performed at pH 5.2, 25°C. The offset is the endpoint of the fit. b The amplitudes were normalized to the total measurable fluorescence change. A negative sign indicates that fluorescence quenching occurred in that phase. The figures in parentheses represent the standard deviations about the means of several data sets. c The figures in parentheses represent the errors of the fits to the experimental data. The values for the b-domain were fitted without including data for Trp63, Cys64, Ile78, as they have different kinetic behaviour (Radford et al., 1992). a
Figure 3. Protection of amides from hydrogen exchange refolding from the TFE-denatured state (continuous line) and the GuHCl-denatured state (broken line). The experiments were performed using a Biologic QFM5 quench flow apparatus, essentially as described by Radford et al. (1992). a and b, The protein was refolded by 11-fold dilution into 20 mM sodium acetate buffer (pH 5.2), 25°C. a, Amides in the a-domain (w, + , W and R represent amides in helices A, B, C and D, respectively); b, amides in the b-domain (w and W represent amides in the b-sheet and the long loop, respectively). All the data were fitted to a sum of two exponential functions. No dependence of the proton occupancy on the pulse pH was observed, indicating that parallel pathways exist during both refolding processes. The results of refolding from the 6 M GuHCl-denatured state at 25°C in a and b were interpolated from data at 5°C, 20°C and 35°C (A. Matagne, S.E.R. & C.M.D., unpublished results). c and d, Refolding from GuHCl-(w) and TFE-denatured (W) states under identical conditions (0.55 M GuHCl, 5.5% (v/v) TFE, 20 mM sodium acetate, pH 5.2, 25°C). c, Amides in the a-domain; d, amides in the b-domain. The error bars represent one standard deviation from the mean proton occupancy averaged over all amides monitored in each domain. The data are fitted to a double exponential function. Protection of the b-domain during refolding from the GuHCl-denatured state in d is fitted to a single exponential function.
116
Folding of Hen Lysozyme in TFE
Table 2. Kinetics of refolding from the TFE- and GuHCl-denatured states under identical refolding conditions Initial state A. Stopped-flow intrinsic fluorescence b TFE state GuHCl state B. Hydrogen exchange pulse labelling c TFE state a-domain b-domain GuHCl state a-domain b-domain
Fast phase t1 (ms)
Amplitude t1 (%)
Slow phase t2 (ms)
Amplitude t2 (%)
Offseta (%)
4.0 (20.4) 4.6 (20.4)
−58 (23) −58 (23)
47 (25) 51 (24)
40 (23) 41 (23)
— —
<3 <4
63 (23) 20 (26)
36 (210) 38 (28)
23 (23) 60 (25)
14 (22) 21 (23)
<3 —
53 (23) —
32 (25) 40 (27)
31 (22) 77 (27)
16 (21) 24 (24)
All experiments were at pH 5.2, 25°C; 5.5% (v/v) TFE, 0.55 M GuHCl. a The offset is the endpoint of the fit for hydrogen exchange pulse labelling experiments. b The amplitude were normalized to the total measurable fluorescence change. A negative sign indicates that fluorescence quenching occurred in that phase. The figures in parentheses represent the range about the averages of several data sets. c The figures in parentheses represent the errors of fits. The value for the b-domain were fitted without including data for Trp63, Cys64, Ile78.
Figure 4. The effect of TFE (a) and GuHCl (b) on the rate of refolding of the GuHCl-denatured state (a) and the TFE-denatured state (b). Data were monitored using stopped-flow fluorescence. Data for the fast phase (W) and the slow phase (w) are shown separately. All the experiments were performed at pH 5.2, 25°C. The data were fitted to the equation: log(k) = log(k0 ) + A[denaturant], where k is the observed rate constant and k0 is the rate constant observed during refolding into 0.55 M GuHCl (a) and 5.5% (v/v) TFE (b), and A is a measure of the effect of denaturant on the observed refolding rate.
Acknowledgements We thank Andrew Miranker for assistance with NMR data analysis. S.E.R. was supported by the Royal Society. The research of C.M.D. is supported in part by an International Research Scholars award from the Howard Hughes Medical Institute. The Oxford Centre for Molecular Sciences is supported by the UK EPSRC, BBSRC, and MRC.
References Aune, K. C. & Tanford, C. (1969). Thermodynamics of the denaturation of lysozyme by guanidine hydrochloride. II. Dependence on denaturant concentration at 25°. Biochemistry, 8, 4586–4590. Buck, M., Radford, S. E. & Dobson, C. M. (1993). A partially folded state of HEWL in TFE: structural characterisation and implications for protein folding. Biochemistry, 32, 669–678. Buck, M., Schwalbe, H. & Dobson, C. M. (1995). Characterisation of conformational preferences in a partially folded protein by heteronuclear NMR spectroscopy: assignment and secondary structure analysis of hen egg-white lysozyme in trifluoroethanol. Biochemistry, 34, 13219–13232.
Buck, M., Schwalbe, H. & Dobson, C. M. (1996). Main-chain dynamics of a partially folded protein: 15 N NMR relaxation measurements of hen egg white lysozyme denatured in trifluoroethanol. J. Mol. Biol. 257, 669–683. Cammers-Goodwin, A., Allen, T. J., Oslick, S. L., McClure, K. F., Lee, J. H. & Kemp, D. S. (1996). Mechanism of stabilization of helical conformations of polypeptides by water containing trifluoroethanol. J. Am. Chem. Soc. 118, 3082–3090. Chaffotte, A. F., Guillou, Y. & Goldberg, M. E. (1992). Kinetic resolution of peptide bond and side-chain far-UV circular dichroism during the folding of hen egg white lysozyme. Biochemistry, 31, 9694–9702. Dobson, C. M., Evans, P. A. & Radford, S. E. (1994). Understanding how proteins fold: the lysozyme story so far. Trends Biochem. Sci. 19, 31–37. Dyson, H. J., Merutka, G., Waltho, J. P., Lerner, R. A. & Wright, P. E. (1992). Folding of peptide fragments comprising the complete sequence of proteins. Models for initiation of protein folding. I. Myohemerythrin. J. Mol. Biol. 226, 795–817. Ittah, V. & Haas, E. (1995). Non-local interactions stabilise long range loops in the initial folding intermediates of reduced bovine pancreatic trypsin inhibitor. Biochemistry, 34, 4493–4506.
Folding of Hen Lysozyme in TFE
Itzhaki, L. S. & Evans, P. A. (1996). Solvent isotope effects on the refolding kinetics of hen egg-white lysozyme. Protein Sci. 5, 140–146. Itzhaki, L. S., Evans, P. A., Dobson, C. M. & Radford, S. E. (1994). Tertiary interactions in the folding pathway of hen lysozyme: kinetic studies using fluorescent probes. Biochemistry, 33, 5212–5220. Itzhaki, L. S., Neira, J. L., Ruiz-Sanz, J., Prat Gay, G. de & Fersht, A. R. (1995). Search for nucleation sites in smaller fragments of chymotrypsin inhibitor 2. J. Mol. Biol. 254, 289–304. Jackson, S. E. & Fersht, A. R. (1991). Folding of chymotrypsin inhibitor 2. I. Evidence for a two-state transition. Biochemistry, 30, 10428–10435. Kiefhaber, T. (1995). Kinetic traps in lysozyme folding. Proc. Natl Acad. Sci. USA, 92, 9029–9033. Kim, P. S. & Baldwin, R. L. (1990). Intermediates in the folding reactions of small proteins. Annu. Rev. Biochem. 59, 631–660. Kragelund, B. B., Robinson, C. V., Knudsen, J., Dobson, C. M. & Poulsen, F. M. (1995). Folding of a 4-helix bundle: studies of acyl-coenzyme-A binding-protein. Biochemistry, 34, 7217–7224. Lehrman, S. R., Tuls, J. L. & Lund, M. (1990). Peptide a-helicity in aqueous trifluoroethanol: correlations with predicted a-helicity and the secondary structure of the corresponding regions of bovine growth hormone. Biochemistry, 29, 5590–5596. Matouschek, A., Kellis, J. T., Serrano, L., Bycroft, M. & Fersht, A. R. (1990). Transient folding intermediates characterized by protein engineering. Nature, 346, 440–445. Matthews, C. R. (1993). Pathways of protein folding. Annu. Rev. Biochem. 62, 653–683. Miranker, A. D. & Dobson, C. M. (1996). Collapse and cooperativity in protein folding. Curr. Opin. Struct. Biol. 6, 31–42. Nelson, J. W. & Kallenbach, N. R. (1986). Stabilization of the ribonuclease S-peptide a-helix by trifluoroethanol solutions. Proteins: Struct. Funct. Genet. 1, 211–217. Peng, Z. Y. & Kim, P. S. (1994). A protein dissection study of a molten globule. Biochemistry, 33, 2136–2141. Pfeil, W. & Privalov, P. L. (1976). Thermodynamic investigation of lysozyme. Biophys. Chem. 4, 41–50. Plaxco, K. W. & Dobson, C. M. (1996). Time resolved biophysical methods in the study of protein folding. Curr. Opin. Struct. Biol. 6, 630–636. Prat Gay, G. de, Ruiz-Sanz, J., Neira, J. L., Itzhaki, L. S. & Fersht, A. R. (1995). Folding of a nascent polypeptide chain in vitro: cooperative formation of structure in a protein module. Proc. Natl Acad. Sci. USA, 92, 3683–3686. Radford, S. E. & Dobson, C. M. (1995). Insights into protein folding using physical techniques: studies of lysozyme and a-lactalbumin. Phil. Trans. R. Soc. Lond. B, 348, 17–25.
117 Radford, S. C., Dobson, C. M. & Evans, P. A. (1992). The folding of hen lysozyme involves partially structured intermediates and multiple pathways. Nature, 358, 302–307. Rothwarf, D. M. & Scheraga, H. A. (1996). The role of non-native aromatic and hydrophobic interactions in the folding of hen egg white lysozyme. Biochemistry. In the press. Sali, A., Shakhnovich, E. & Karplus, M. (1994). How does a protein fold? Nature, 369, 248–251. Schindler, T., Herrler, M., Marahiel, M. A. & Schmid, F. X. (1995). Extremely rapid protein-folding in the absence of intermediates. Nature Struct. Biol. 2, 663–673. Schmid, F. X. (1992). Kinetics of unfolding and refolding of single-domain protein. In Protein Folding (Creighton, T. E., ed.), pp. 197–241, Freeman, New York. Schoenbrunner, N., Wey, J., Engels, J., Georg, H. & Kiefhaber, T. (1996). Native-like beta structure in a trifluoroethanol induced partially folded state of the all beta-protein tendamistat. J. Mol. Biol. 260, 432–445. Segawa, S. & Sugihara, M. (1984). Characterization of the transition state of lysozyme unfolding. I. Effect of protein-solvent interactions on the transition state. Biopolymers, 23, 2473–2488. Segawa, S., Fukuno, T., Fujiwara, K. & Noda, Y. (1991). Local structure in unfolded lysozyme and correlation with secondary structures in the native conformation: helix-forming or -breaking propensity of peptide segments. Biopolymers, 31, 497–509. Shin, H.-C., Merutka, G., Waltho, J. P., Wright, P. E. & Dyson, H. J. (1993). Peptide models of protein folding initiation sites. 3. The G-H helical hairpin of myoglobin. Biochemistry, 32, 6356–6364. Shiraki, K., Nishikawa, K. & Goto, Y. (1995). Trifluoroethanol-induced stabilization of the a-helical structure of b-lactoglobulin: implication for non-hierarchical protein folding. J. Mol. Biol. 245, 180–194. Thomas, P. D. & Dill, K. A. (1993). Local and nonlocal interactions in globular proteins and mechanisms of alcohol denaturation. Protein Sci. 2, 2050–2065. Waltho, J. P., Feher, V. A., Merutka, G., Dyson, H. J. & Wright, P. E. (1993). Peptide models of protein folding initiation sites. 1. Secondary structure formation by peptides corresponding to the Gand H-helices of myoglobin. Biochemistry, 32, 6337–6347. Yang, A. S. & Honig, B. (1993). On the pH dependence of protein stability. J. Mol. Biol. 231, 459–474. Yang, J. J., Buck, M., Pitkeathly, M., Kotik, M., Haynie, D. T., Dobson, C. M. & Radford, S. E. (1995). Four peptides which span the entire sequence of hen lysozyme have distinct conformational properties in water and TFE: structural implications for early folding intermediates. J. Mol. Biol. 252, 483–491.
Edited by A. R. Fersht (Received 15 May 1996; received in revised form 8 October 1996; accepted 10 October 1996)
COMMUNICATION
Acceleration of the Folding of Hen Lysozyme by Trifluoroethanol Hui Lu, Matthias Buck, Sheena E. Radford and Christopher M. Dobson* Oxford Centre for Molecular Sciences, New Chemistry Laboratory, University of Oxford, South Parks Road Oxford, OX1 3QT, UK
The refolding of a partially structured state of hen lysozyme formed in 60% (v/v) 2,2,2-trifluoroethanol (TFE) has been studied using hydrogen exchange pulse labelling monitored by 2D 1H NMR, and by stopped flow fluorescence and CD measurements. The results are compared with similar studies of the refolding of the protein denatured in 6 M guanidine hydrochloride (GuHCl). Two conclusions have emerged from these studies. First, provided that the refolding conditions are identical, the two denatured states fold with very similar kinetics, despite the fact the extensive secondary structure is present in the TFE-denatured state but not in the protein denatured in 6 M GuHCl. This arises because of the rapid equilibration of structure in the species formed in the initial stage of folding. Second, whilst addition of GuHCl to the refolding buffer decreases the rate of folding, low concentrations of TFE increase the rate of folding. The result is consistent with slow steps in the refolding of lysozyme being associated primarily with the reorganisation of hydrophobic interactions rather than of hydrogen bonded structure. 7 1997 Academic Press Limited
*Corresponding author
Keywords: rate enhancement; hydrophobic interaction; hydrogen bonding; partially folded states; kinetic traps
Investigation of the mechanism by which a protein attains its native three-dimensional structure represents a considerable challenge to experimentalists, not least because of the extreme rapidity of many of the steps involved (Kim & Baldwin, 1990; Matthews, 1993; Plaxco & Dobson, 1996). Although the refolding of some small proteins appears highly cooperative and effectively two state (Jackson & Fersht, 1991; Prat Gay et al., 1995; Kragelund et al., 1995; Schindler et al., 1995), refolding of other proteins involves the transient population of partially folded states (Matthews, 1993; Miranker & Dobson, 1996). One example of the latter class of proteins is lysozyme, studies Present addresses: M. Buck, Department of Chemistry, Harvard University, 12 Oxford Street, Cambridge, MA 02138, USA; S. E. Radford, Department of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, UK. Abbreviations used: TFE, 2,2,2-trifluroethanol; MeUdiNAG, 4-methylumbelliferyl-N,N'-diacetyl-bchitobiose; ANS, 1-anilino napthalene sulphonate; GuHCl, guanidine hydrochloride. 0022–2836/97/020112–06 $25.00/0/mb960715
of which (with the native disulphide bonds intact) have shown at least two distinct types of intermediate states (Radford et al., 1992; Dobson et al., 1994; Radford & Dobson, 1995). Firstly, within the first 4 ms of folding a nativelike component of secondary structure is formed, along with clustering of at least some aromatic residues, a high affinity for 1-anilino napthalene sulphonate (ANS) binding, but little protection against hydrogen exchange (Radford et al., 1992; Chaffotte et al., 1992; Dobson et al., 1994; Itzhaki et al., 1994). More persistently structured states with extensive protection against amide exchange emerge later. These do not yet, however, have persistent native-like tertiary structure. An additional feature of the lysozyme folding pathway is the existence of heterogeneity in folding, which has been attributed to the presence of kinetic traps generated in the initial stages of folding (Radford et al., 1992; Kiefhaber, 1995; Rothwarf & Scheraga, 1996). One of the most interesting issues in the folding of a protein such as lysozyme is the manner in which structure can form in the absence of many of 7 1997 Academic Press Limited
Folding of Hen Lysozyme in TFE
the interactions that stabilise the native state, and the significance of such structure for the process of folding. One approach to this has been the study of peptide fragments of a protein (Shin et al., 1993; Waltho et al., 1993; Peng & Kim, 1994; Prat Gay et al., 1995; Ittah & Haas, 1995; Itzhaki et al., 1995). A common feature of such peptides is that secondary structure, particularly helical structure, can be stabilised by the addition of solvents, such as trifluoroethanol (TFE) that enhance intramolecular hydrogen bonding (Nelson & Kallenbach, 1989; Lehrman et al., 1990; Segawa et al., 1991; Dyson et al., 1992; Yang et al., 1995; Cammers-Goodwin et al., 1996; Schoenbrunner et al., 1996). A question that arises, therefore, is whether such solvents can influence the kinetics of the folding of intact proteins, by influencing the interactions which stabilise the structure during the folding process. Here, we report experiments with lysozyme designed to explore this point. Hen lysozyme denatured in 60% (v/v) TFE at pH 5.2, like that previously characterised at pH 2.0 in 50% (v/v) TFE (Buck et al., 1993, 1995, 1996), is partially folded with more a-helical structure than that of the native protein, but few specific tertiary interactions (Figure 1). By contrast with denaturation in TFE solution, denaturation of lysozyme by 6 M GuHCl results in a substantially unfolded state in which little secondary or tertiary structure persists (Figure 1). To examine the influence of the TFE state of lysozyme on its refolding pathway, the protein, partially denatured in 60% (v/v) TFE, or substantially denatured in 6 M GuHCl (both containing the four native disulphide bridges), was diluted 11-fold into 20 mM sodium acetate buffer (pH 5.2) at 25°C. Its refolding was monitored by far UV CD, intrinsic tryptophan fluorescence and hydrogen exchange pulse labelling.
113 The results of the stopped-flow intrinsic fluorescence experiments are shown in Figure 2. When folding is monitored after 11-fold dilution of the initial denatured state, the shape of the refolding traces from both denatured states is similar, being characteristic of that previously observed for the folding of lysozyme denatured in GuHCl (Itzhaki et al., 1994). This suggests that the refolding process from the GuHCl and TFE-denatured states are similar despite the very different properties of the initial denatured states and the different final refolding conditions (Figure 2a, curve 1 and 2b curve 1). The rate of folding from the two denatured states is, however, very different; folding occurs much more rapidly following dilution from TFE. Indeed, the two measurable phases observed by fluorescence occur five and nine times faster than refolding from GuHCl (Table 1). In accord with these results, the rates of refolding measured by the binding of the fluorescent inhibitor 4-methylumbelliferyl-N,N '-diacetyl-b-chitobiose (MeU-diNAG) and by stopped flow CD in the far UV (measured at 222 nm) are also much faster following dilution from TFE. The refolding kinetics from the two denatured states, measured by hydrogen exchange pulse labelling, are shown in Figure 3a and b. The data confirm the above results, revealing that even at the level of the individual residues, the folding pathways from the two denatured states are similar and that refolding following dilution from TFE is a more rapid process. Analysis of the kinetic parameters suggests two factors contribute to the apparent increase in the rate of protection; an increased amplitude of fast protection of amides in the a-domain and an increased rate of protection of the slow phase of amides in the b-domain (a smaller increase in rate of protection of amides in the a-domain also occurs; Table 1). Previous studies
Figure 1. Far (a) and near (b) UV CD spectra of hen lysozyme in the native state (W), in 60% (v/v) TFE (R), and in 6 M GuHCl (w). Spectra were acquired using a JASCO J720 spectropolarimeter, using cuvettes of 1 mm path length for far UV CD and 10 mm for near UV CD, and protein samples of 0.05 mg/ml and 0.5 mg/ml, respectively. The proteins were dissolved in 20 mM sodium acetate buffer (pH 5.2), 25°C. The signal is divided by the total number of residues (129) in each case. A higher TFE concentration was used to generate the TFE-denatured state at pH 5.2, relative to that used previously at pH 2.0, because of the greater stability of the native structure at the higher pH (Pfeil & Privalov, 1976; Yang & Honig, 1993).
114
Figure 2. Refolding of lysozyme under various conditions monitored by stopped-flow fluorescence. a, Refolding from the GuHCl-denatured state. Refolding was initiated by dilution of the denatured state into buffer alone (w, curve 1) and into buffer containing 5.5% (v/v) TFE (W, curve 2). b, Refolding from the TFE-denatured state into buffer alone (w, curve 1) and into buffer containing 0.55 M GuHCl (W, curve 2). Measurements were made at 25°C using an Applied Photophysics SX-17MV stopped-flow instrument. An excitation wavelength of 280 nm was used and the total fluorescence intensity above 320 nm was measured. The concentration of initial denatured protein was 20 mg/ml (in either 60% (v/v) TFE or 6 M GuHCl, pH 5.2) and refolding was initiated by 11-fold dilution of the denatured protein into the refolding buffer described.
have shown that amides in the native-like helices in the TFE-denatured state have protection factors ranging from 20 to more than 200 (Buck et al., 1993, 1995). If this structure were to persist during the kinetic refolding experiments, very different proton occupancy would be expected for these amides in the hydrogen exchange labelling experiment. Within experimental error, however, a uniform proton occupancy of 30% is observed for all amides in the a-domain after a refolding time of 3.5 ms. This suggests that the a-helical structure in the TFE-denatured state must be destabilised and rapidly reorganised after the TFE is diluted to initiate refolding. The results of a second series of experiments in which the two denatured states were refolded under identical conditions are also shown in
Folding of Hen Lysozyme in TFE
Figure 2a (curve 2) and 2b (curve 2) and Figure 3c and d. Under these conditions the folding kinetics from the two denatured states are closely similar (Table 2). The data show that a significant increase (ca 3.5-fold) in the rate of folding of the GuHCl-denatured state is caused by the presence of the low concentrations (5.5%, v/v) of TFE in the refolding buffer. Such concentrations of TFE reduce marginally the stability of the native protein (the midpoints of thermal denaturation measured by far UV CD in the presence and absence of 5.5% (v/v) TFE are 74 (22)°C and 71 (22)°C, respectively), and increase the rate of unfolding of the native protein (H.L. & C.M.D., unpublished results). By contrast, the folding kinetics of the TFE-denatured state are retarded approximately 2.5-fold by the inclusion of 0.55 M GuHCl in the refolding buffer. These two effects account for the differences in rate constants observed when the two denatured states were folded under different conditions. A more detailed analysis of the dependence of the refolding kinetics on the concentration of GuHCl and TFE in the refolding buffer, studied by stopped-flow fluorescence, is shown in Figure 4. Both the fast and slow phases of folding from the GuHCl-denatured state increase with increasing TFE concentrations up to about 1 M (1% (v/v) TFE = 0.137 M). By contrast, the rates of both the fast and slow phases of refolding from the TFE-denatured state decrease with the inclusion of increasing concentrations of GuHCl in the refolding buffer. It is well established that the rates of folding and unfolding are critically dependent on the conditions under which these events take place. Addition of denaturants generally reduces the rate of folding and increases the rate of unfolding (Aune & Tanford, 1969; Segawa & Sugihara, 1984; Matouschek et al., 1990; Schmid, 1992). The finding that the addition of low concentrations of TFE enhances the rate of folding of lysozyme without significantly perturbing the folding pathway is therefore of interest. Under conditions where rapid collapse occurs, the folding process involves searching within a compact state for native-like contacts (Sali et al., 1994). In the case of lysozyme the collapsed state has extensive secondary structure and is stabilised by four disulphide bonds; it is likely therefore that significant barriers can exist for the reorganisation of either correctly or incorrectly folded species (Radford et al., 1992; Radford & Dobson, 1995). TFE is known to enhance intramolecular hydrogen bonding and to decrease hydrophobic interactions within a polypeptide chain (Buck et al., 1993; Thomas & Dill, 1993; Shiraki et al., 1995; Cammers-Goodwin et al., 1996). The acceleration of folding of lysozyme by TFE is therefore consistent with recent suggestions that the slow steps on the major pathway of folding involve predominantly the reorganisation of hydrophobic interactions rather than of hydrogen bonds (Itzhaki & Evans; 1996).
115
Folding of Hen Lysozyme in TFE
Table 1. Stopped-flow fluorescence and hydrogen exchange pulse labelling data for refolding of the TFE and GuHCl-denatured states Reaction A. Stopped-flow intrinsic fluorescence b 60% TFE : 5.5% TFE 6 M GuHCl : 0.55 M GuHCl B. Hydrogen exchange pulse labelling c 60% TFE : 5.5% TFE 6 M GuHCl : 0.55 M GuHCl
a-domain b-domain a-domain b-domain
Fast phase t1 (ms)
Amplitude t1 (%)
Slow phase t2 (ms)
Amplitude t2 (%)
Offseta (%)
3 (20.5) 17 (22)
−50 (23) −58 (23)
19 (22) 170 (220)
47 (23) 42 (22)
— —
<2 <4 <5 <10
76 (23) 28 (24) 36 (25) 25 (26)
32 (27) 37 (27) 64 (210) 200 (250)
18 (22) 60 (24) 45 (25) 54 (25)
7 (21) 13 (23) 20 (24) 20 (25).
All experiments were performed at pH 5.2, 25°C. The offset is the endpoint of the fit. b The amplitudes were normalized to the total measurable fluorescence change. A negative sign indicates that fluorescence quenching occurred in that phase. The figures in parentheses represent the standard deviations about the means of several data sets. c The figures in parentheses represent the errors of the fits to the experimental data. The values for the b-domain were fitted without including data for Trp63, Cys64, Ile78, as they have different kinetic behaviour (Radford et al., 1992). a
Figure 3. Protection of amides from hydrogen exchange refolding from the TFE-denatured state (continuous line) and the GuHCl-denatured state (broken line). The experiments were performed using a Biologic QFM5 quench flow apparatus, essentially as described by Radford et al. (1992). a and b, The protein was refolded by 11-fold dilution into 20 mM sodium acetate buffer (pH 5.2), 25°C. a, Amides in the a-domain (w, + , W and R represent amides in helices A, B, C and D, respectively); b, amides in the b-domain (w and W represent amides in the b-sheet and the long loop, respectively). All the data were fitted to a sum of two exponential functions. No dependence of the proton occupancy on the pulse pH was observed, indicating that parallel pathways exist during both refolding processes. The results of refolding from the 6 M GuHCl-denatured state at 25°C in a and b were interpolated from data at 5°C, 20°C and 35°C (A. Matagne, S.E.R. & C.M.D., unpublished results). c and d, Refolding from GuHCl-(w) and TFE-denatured (W) states under identical conditions (0.55 M GuHCl, 5.5% (v/v) TFE, 20 mM sodium acetate, pH 5.2, 25°C). c, Amides in the a-domain; d, amides in the b-domain. The error bars represent one standard deviation from the mean proton occupancy averaged over all amides monitored in each domain. The data are fitted to a double exponential function. Protection of the b-domain during refolding from the GuHCl-denatured state in d is fitted to a single exponential function.
116
Folding of Hen Lysozyme in TFE
Table 2. Kinetics of refolding from the TFE- and GuHCl-denatured states under identical refolding conditions Initial state A. Stopped-flow intrinsic fluorescence b TFE state GuHCl state B. Hydrogen exchange pulse labelling c TFE state a-domain b-domain GuHCl state a-domain b-domain
Fast phase t1 (ms)
Amplitude t1 (%)
Slow phase t2 (ms)
Amplitude t2 (%)
Offseta (%)
4.0 (20.4) 4.6 (20.4)
−58 (23) −58 (23)
47 (25) 51 (24)
40 (23) 41 (23)
— —
<3 <4
63 (23) 20 (26)
36 (210) 38 (28)
23 (23) 60 (25)
14 (22) 21 (23)
<3 —
53 (23) —
32 (25) 40 (27)
31 (22) 77 (27)
16 (21) 24 (24)
All experiments were at pH 5.2, 25°C; 5.5% (v/v) TFE, 0.55 M GuHCl. a The offset is the endpoint of the fit for hydrogen exchange pulse labelling experiments. b The amplitude were normalized to the total measurable fluorescence change. A negative sign indicates that fluorescence quenching occurred in that phase. The figures in parentheses represent the range about the averages of several data sets. c The figures in parentheses represent the errors of fits. The value for the b-domain were fitted without including data for Trp63, Cys64, Ile78.
Figure 4. The effect of TFE (a) and GuHCl (b) on the rate of refolding of the GuHCl-denatured state (a) and the TFE-denatured state (b). Data were monitored using stopped-flow fluorescence. Data for the fast phase (W) and the slow phase (w) are shown separately. All the experiments were performed at pH 5.2, 25°C. The data were fitted to the equation: log(k) = log(k0 ) + A[denaturant], where k is the observed rate constant and k0 is the rate constant observed during refolding into 0.55 M GuHCl (a) and 5.5% (v/v) TFE (b), and A is a measure of the effect of denaturant on the observed refolding rate.
Acknowledgements We thank Andrew Miranker for assistance with NMR data analysis. S.E.R. was supported by the Royal Society. The research of C.M.D. is supported in part by an International Research Scholars award from the Howard Hughes Medical Institute. The Oxford Centre for Molecular Sciences is supported by the UK EPSRC, BBSRC, and MRC.
References Aune, K. C. & Tanford, C. (1969). Thermodynamics of the denaturation of lysozyme by guanidine hydrochloride. II. Dependence on denaturant concentration at 25°. Biochemistry, 8, 4586–4590. Buck, M., Radford, S. E. & Dobson, C. M. (1993). A partially folded state of HEWL in TFE: structural characterisation and implications for protein folding. Biochemistry, 32, 669–678. Buck, M., Schwalbe, H. & Dobson, C. M. (1995). Characterisation of conformational preferences in a partially folded protein by heteronuclear NMR spectroscopy: assignment and secondary structure analysis of hen egg-white lysozyme in trifluoroethanol. Biochemistry, 34, 13219–13232.
Buck, M., Schwalbe, H. & Dobson, C. M. (1996). Main-chain dynamics of a partially folded protein: 15 N NMR relaxation measurements of hen egg white lysozyme denatured in trifluoroethanol. J. Mol. Biol. 257, 669–683. Cammers-Goodwin, A., Allen, T. J., Oslick, S. L., McClure, K. F., Lee, J. H. & Kemp, D. S. (1996). Mechanism of stabilization of helical conformations of polypeptides by water containing trifluoroethanol. J. Am. Chem. Soc. 118, 3082–3090. Chaffotte, A. F., Guillou, Y. & Goldberg, M. E. (1992). Kinetic resolution of peptide bond and side-chain far-UV circular dichroism during the folding of hen egg white lysozyme. Biochemistry, 31, 9694–9702. Dobson, C. M., Evans, P. A. & Radford, S. E. (1994). Understanding how proteins fold: the lysozyme story so far. Trends Biochem. Sci. 19, 31–37. Dyson, H. J., Merutka, G., Waltho, J. P., Lerner, R. A. & Wright, P. E. (1992). Folding of peptide fragments comprising the complete sequence of proteins. Models for initiation of protein folding. I. Myohemerythrin. J. Mol. Biol. 226, 795–817. Ittah, V. & Haas, E. (1995). Non-local interactions stabilise long range loops in the initial folding intermediates of reduced bovine pancreatic trypsin inhibitor. Biochemistry, 34, 4493–4506.
Folding of Hen Lysozyme in TFE
Itzhaki, L. S. & Evans, P. A. (1996). Solvent isotope effects on the refolding kinetics of hen egg-white lysozyme. Protein Sci. 5, 140–146. Itzhaki, L. S., Evans, P. A., Dobson, C. M. & Radford, S. E. (1994). Tertiary interactions in the folding pathway of hen lysozyme: kinetic studies using fluorescent probes. Biochemistry, 33, 5212–5220. Itzhaki, L. S., Neira, J. L., Ruiz-Sanz, J., Prat Gay, G. de & Fersht, A. R. (1995). Search for nucleation sites in smaller fragments of chymotrypsin inhibitor 2. J. Mol. Biol. 254, 289–304. Jackson, S. E. & Fersht, A. R. (1991). Folding of chymotrypsin inhibitor 2. I. Evidence for a two-state transition. Biochemistry, 30, 10428–10435. Kiefhaber, T. (1995). Kinetic traps in lysozyme folding. Proc. Natl Acad. Sci. USA, 92, 9029–9033. Kim, P. S. & Baldwin, R. L. (1990). Intermediates in the folding reactions of small proteins. Annu. Rev. Biochem. 59, 631–660. Kragelund, B. B., Robinson, C. V., Knudsen, J., Dobson, C. M. & Poulsen, F. M. (1995). Folding of a 4-helix bundle: studies of acyl-coenzyme-A binding-protein. Biochemistry, 34, 7217–7224. Lehrman, S. R., Tuls, J. L. & Lund, M. (1990). Peptide a-helicity in aqueous trifluoroethanol: correlations with predicted a-helicity and the secondary structure of the corresponding regions of bovine growth hormone. Biochemistry, 29, 5590–5596. Matouschek, A., Kellis, J. T., Serrano, L., Bycroft, M. & Fersht, A. R. (1990). Transient folding intermediates characterized by protein engineering. Nature, 346, 440–445. Matthews, C. R. (1993). Pathways of protein folding. Annu. Rev. Biochem. 62, 653–683. Miranker, A. D. & Dobson, C. M. (1996). Collapse and cooperativity in protein folding. Curr. Opin. Struct. Biol. 6, 31–42. Nelson, J. W. & Kallenbach, N. R. (1986). Stabilization of the ribonuclease S-peptide a-helix by trifluoroethanol solutions. Proteins: Struct. Funct. Genet. 1, 211–217. Peng, Z. Y. & Kim, P. S. (1994). A protein dissection study of a molten globule. Biochemistry, 33, 2136–2141. Pfeil, W. & Privalov, P. L. (1976). Thermodynamic investigation of lysozyme. Biophys. Chem. 4, 41–50. Plaxco, K. W. & Dobson, C. M. (1996). Time resolved biophysical methods in the study of protein folding. Curr. Opin. Struct. Biol. 6, 630–636. Prat Gay, G. de, Ruiz-Sanz, J., Neira, J. L., Itzhaki, L. S. & Fersht, A. R. (1995). Folding of a nascent polypeptide chain in vitro: cooperative formation of structure in a protein module. Proc. Natl Acad. Sci. USA, 92, 3683–3686. Radford, S. E. & Dobson, C. M. (1995). Insights into protein folding using physical techniques: studies of lysozyme and a-lactalbumin. Phil. Trans. R. Soc. Lond. B, 348, 17–25.
117 Radford, S. C., Dobson, C. M. & Evans, P. A. (1992). The folding of hen lysozyme involves partially structured intermediates and multiple pathways. Nature, 358, 302–307. Rothwarf, D. M. & Scheraga, H. A. (1996). The role of non-native aromatic and hydrophobic interactions in the folding of hen egg white lysozyme. Biochemistry. In the press. Sali, A., Shakhnovich, E. & Karplus, M. (1994). How does a protein fold? Nature, 369, 248–251. Schindler, T., Herrler, M., Marahiel, M. A. & Schmid, F. X. (1995). Extremely rapid protein-folding in the absence of intermediates. Nature Struct. Biol. 2, 663–673. Schmid, F. X. (1992). Kinetics of unfolding and refolding of single-domain protein. In Protein Folding (Creighton, T. E., ed.), pp. 197–241, Freeman, New York. Schoenbrunner, N., Wey, J., Engels, J., Georg, H. & Kiefhaber, T. (1996). Native-like beta structure in a trifluoroethanol induced partially folded state of the all beta-protein tendamistat. J. Mol. Biol. 260, 432–445. Segawa, S. & Sugihara, M. (1984). Characterization of the transition state of lysozyme unfolding. I. Effect of protein-solvent interactions on the transition state. Biopolymers, 23, 2473–2488. Segawa, S., Fukuno, T., Fujiwara, K. & Noda, Y. (1991). Local structure in unfolded lysozyme and correlation with secondary structures in the native conformation: helix-forming or -breaking propensity of peptide segments. Biopolymers, 31, 497–509. Shin, H.-C., Merutka, G., Waltho, J. P., Wright, P. E. & Dyson, H. J. (1993). Peptide models of protein folding initiation sites. 3. The G-H helical hairpin of myoglobin. Biochemistry, 32, 6356–6364. Shiraki, K., Nishikawa, K. & Goto, Y. (1995). Trifluoroethanol-induced stabilization of the a-helical structure of b-lactoglobulin: implication for non-hierarchical protein folding. J. Mol. Biol. 245, 180–194. Thomas, P. D. & Dill, K. A. (1993). Local and nonlocal interactions in globular proteins and mechanisms of alcohol denaturation. Protein Sci. 2, 2050–2065. Waltho, J. P., Feher, V. A., Merutka, G., Dyson, H. J. & Wright, P. E. (1993). Peptide models of protein folding initiation sites. 1. Secondary structure formation by peptides corresponding to the Gand H-helices of myoglobin. Biochemistry, 32, 6337–6347. Yang, A. S. & Honig, B. (1993). On the pH dependence of protein stability. J. Mol. Biol. 231, 459–474. Yang, J. J., Buck, M., Pitkeathly, M., Kotik, M., Haynie, D. T., Dobson, C. M. & Radford, S. E. (1995). Four peptides which span the entire sequence of hen lysozyme have distinct conformational properties in water and TFE: structural implications for early folding intermediates. J. Mol. Biol. 252, 483–491.
Edited by A. R. Fersht (Received 15 May 1996; received in revised form 8 October 1996; accepted 10 October 1996)