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Kinetics of Secondary Structure Recovery during the Refolding of Reduced Hen Egg White Lysozyme

1997; Elsevier BV; Volume: 272; Issue: 40 Linguagem: Inglês

10.1074/jbc.272.40.24843

1083-351X

Pascale Roux, Muriel Delepierre, Michel Goldberg, Alain-F. Chaffotte,

Enzyme Structure and Function

We have shown previously that, in less than 4 ms, the unfolded/oxidized hen lysozyme recovered its native secondary structure, while the reduced protein remained fully unfolded. To investigate the role played by disulfide bridges in the acquisition of the secondary structure at later stages of the renaturation/oxidation, the complete refolding of reduced lysozyme was studied. This was done in a renaturation buffer containing 0.5 m guanidinium chloride, 60 μm oxidized glutathione, and 20 μm reduced dithiothreitol, in which the aggregation of lysozyme was minimized and where a renaturation yield of 80% was obtained. The refolded protein could not be distinguished from the native lysozyme by activity, compactness, stability, and several spectroscopic measurements. The kinetics of renaturation were then studied by following the reactivation and the changes in fluorescence and circular dichroism signals. When bi- or triphasic sequential models were fitted to the experimental data, the first two phases had the same calculated rate constants for all the signals showing that, within the time resolution of these experiments, the folding/oxidation of hen lysozyme is highly cooperative, with the secondary structure, the tertiary structure, and the integrity of the active site appearing simultaneously. We have shown previously that, in less than 4 ms, the unfolded/oxidized hen lysozyme recovered its native secondary structure, while the reduced protein remained fully unfolded. To investigate the role played by disulfide bridges in the acquisition of the secondary structure at later stages of the renaturation/oxidation, the complete refolding of reduced lysozyme was studied. This was done in a renaturation buffer containing 0.5 m guanidinium chloride, 60 μm oxidized glutathione, and 20 μm reduced dithiothreitol, in which the aggregation of lysozyme was minimized and where a renaturation yield of 80% was obtained. The refolded protein could not be distinguished from the native lysozyme by activity, compactness, stability, and several spectroscopic measurements. The kinetics of renaturation were then studied by following the reactivation and the changes in fluorescence and circular dichroism signals. When bi- or triphasic sequential models were fitted to the experimental data, the first two phases had the same calculated rate constants for all the signals showing that, within the time resolution of these experiments, the folding/oxidation of hen lysozyme is highly cooperative, with the secondary structure, the tertiary structure, and the integrity of the active site appearing simultaneously. Since Anfinsen's work on the in vitro renaturation of unfolded ribonuclease A (1Anfinsen C.B. Science. 1973; 181: 223-230Crossref PubMed Scopus (5217) Google Scholar), it is commonly accepted that all the information required for a protein to fold properly is contained in its amino acid sequence. However, the code that allows the formation of a fully folded protein from its amino acid sequence has not yet been deciphered. Three models are currently proposed to describe this process. The framework model is a sequential model in which secondary structure elements form first followed by a tighter packing of the molecule (2Kim P.S. Baldwin R.L. Annu. Rev. Biochem. 1982; 51: 459-489Crossref PubMed Scopus (954) Google Scholar). Another model assumes that the polypeptide chain undergoes a rapid collapse driven by hydrophobic forces that would yield an intermediate close to the molten globule (3Dill K. Biochemistry. 1985; 24: 1501-1509Crossref PubMed Scopus (1100) Google Scholar). In the puzzle model (4Harrison S.C. Durbin R. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 4028-4030Crossref PubMed Scopus (177) Google Scholar) structural elements form at different sites on the polypeptide chain, and their formation induces further folding of the whole protein. According to experimental data, it is not yet possible to determine which model most accurately describes the folding mechanism. However, some general features of protein folding have arisen. Stopped-flow circular dichroism studies showed that a large amount of secondary structure is formed in the dead time (milliseconds time range) of the observation. This has been observed for proteins such as α-lactalbumin and lysozyme (5Kuwajima K. Hiraoka Y. Ikegushi M. Sugai S. Biochemistry. 1985; 24: 874-881Crossref PubMed Scopus (288) Google Scholar), dihydrofolate reductase (6Kuwajima K. Garvey E.P. Finn B.E. Matthews C.R. Sugai S. Biochemistry. 1991; 30: 7693-7703Crossref PubMed Scopus (158) Google Scholar), and holocytochrome c (7Elöve G.A. Chaffotte A.-F. Roder H. Goldberg M.E. Biochemistry. 1992; 31: 6876-6883Crossref PubMed Scopus (243) Google Scholar). On the other hand, when using pulsed proton exchange followed by NMR identification of the protected protons, the formation of stable secondary structure elements could be detected over a slower observable time range (8Udgaonkar J.B. Baldwin R.L. Nature. 1988; 335: 694-699Crossref PubMed Scopus (567) Google Scholar, 9Roder H. Elove G.A. Englander S.W. Nature. 1988; 335: 700-704Crossref PubMed Scopus (825) Google Scholar). Using both techniques to observe the folding of the same protein, as was the case for cytochrome c (7Elöve G.A. Chaffotte A.-F. Roder H. Goldberg M.E. Biochemistry. 1992; 31: 6876-6883Crossref PubMed Scopus (243) Google Scholar), lysozyme (10Radford S.E. Dobson M.C. Evans P.A. Nature. 1992; 358: 302-307Crossref PubMed Scopus (730) Google Scholar, 11Chaffotte A.-F. Guillou Y. Goldberg M.E. Biochemistry. 1992; 31: 9694-9702Crossref PubMed Scopus (152) Google Scholar), and interleukin-1β (12Varley P. Gronenborn A.M. Christensen H. Wingfield P.T. Pain R.H. Clore G.M. Science. 1993; 260: 1110-1113Crossref PubMed Scopus (171) Google Scholar), secondary structures could be observed by CD at a stage where pulsed proton exchange/NMR failed to detect protected protons. This apparent contradiction suggested that secondary structure elements could form without providing an efficient protection against proton exchange. This was confirmed by studies on a model peptide that mimics early folding intermediates, the C-terminal F2 domain of the β2 subunit of Escherichia coli tryptophan synthase (13Chaffotte A.-F. Cadieux C. Guillou Y. Goldberg M.E. Biochemistry. 1992; 31: 4303-4308Crossref PubMed Scopus (63) Google Scholar,14Guijarro J.I. Jackson M. Chaffotte A.-F. Delepierre M. Mantsch H.H. Goldberg M.E. Biochemistry. 1995; 34: 2998-3008Crossref PubMed Scopus (56) Google Scholar). These studies showed that in isolated F2 the secondary structure elements were in such a fast equilibrium that their protons were only weakly protected from exchange. Hence the initial secondary structures that are present in very early folding intermediates appear to be poorly stabilized. A major question in solving the folding problem is to understand the relative roles of local versus long range interactions in controlling the folding process. While both types of interactions are obviously linked energetically throughout the folding pathway, and both determine the stabilities of all the folding intermediates (15Creighton T.E. BioEssays. 1988; 8: 57-63Crossref PubMed Scopus (230) Google Scholar), the predominance of one over the other is at the basis of the distinction between the framework, the jigsaw puzzle, and the hydrophobic collapse models. The propensity of short natural amino acid sequences to spontaneously form native-like secondary structures through local interactions has been the focus of much attention and has lent much support to the framework model (16Baldwin R.L. J. Biomol. NMR. 1995; 5: 103-109Crossref PubMed Scopus (299) Google Scholar). Yet, several well documented cases are described in the literature where the protein fails to show any detectable secondary structure when long range interactions are not formed. Thus, in the absence of long range interactions between the heme and the polypeptide chain, apocytochrome c remains completely unfolded and shows no secondary structure under conditions where the holoprotein refolds completely (17Fisher W.R. Taniuchi H. Anfinsen C.B. J. Biol. Chem. 1973; 248: 3188-3195Abstract Full Text PDF PubMed Google Scholar). It thus appears that native tertiary contacts play an important role not only in the late stages of the folding but also to initiate or stabilize the formation of the secondary structure. A similar conclusion was reached for lysozyme, based on the following observations. When the native disulfide bonds are maintained in the denatured state, the protein recovers the majority of its secondary structure in less than 4 ms (11Chaffotte A.-F. Guillou Y. Goldberg M.E. Biochemistry. 1992; 31: 9694-9702Crossref PubMed Scopus (152) Google Scholar). In contrast, the species that are present after 4 ms of refolding of the reduced form have a random-coil CD signal in the far UV region (18Goldberg M.E. Guillou Y. Protein Sci. 1994; 3: 883-887Crossref PubMed Scopus (34) Google Scholar). Yet, the reduced lysozyme is able to slowly recover its native structure when the disulfides are allowed to form (19Saxena V.P. Wetlaufer D.B. Biochemistry. 1970; 9: 5015-5022Crossref PubMed Scopus (363) Google Scholar). Therefore, and because of the wealth of information available on the mechanism of its folding, lysozyme appeared as a good model to investigate the influence of long range interactions (i.e. native disulfide bonds) on the early stages of the folding process. The question we would like to answer could be stated in the following manner: does the formation of the secondary structure bring together the cysteines in the proper conformation to form native disulfide bonds, or do the disulfide bridges form first thereby constraining the polypeptide chain in such a state that formation of secondary structure is induced? The strategy we shall employ to answer this question is to compare the kinetics of recovery of secondary structure and disulfide bonds during the oxidative folding of the reduced wild-type lysozyme and of mutant lysozymes missing some of the cysteine pairs. As a first step in this study, we focused on monitoring the recovery of the CD signal throughout the refolding. Since at the protein concentrations required for CD measurements, the reduced unfolded lysozyme rapidly forms aggregates in conventional buffers, we had to design renaturation conditions that would minimize aggregation. In this report, we describe a buffer system that leads to an efficient renaturation of reduced lysozyme and that is compatible with kinetic far UV CD measurements. The renatured lysozyme thus obtained will be shown to be identical to the native protein, and the kinetics of renaturation monitored by far UV CD, by intrinsic fluorescence, and by enzymatic activity will be reported. Hen egg white lysozyme (HEWL) 1The abbreviations used are: HEWL, hen egg white lysozyme; DTT, dithiothreitol; GdnHCl, guanidine hydrochloride; KPi, potassium phosphate; NOESY, nuclear Overhauser effect spectroscopy. and oxidized glutathione (GSSG) were purchased from Boehringer Mannheim GmbH, Germany, reduced dithiothreitol (DTT) and Micrococcus lysodeikticus from Sigma, and guanidine hydrochloride (GdnHCl) and urea from ICN Biomedicals. Reduced/denatured lysozyme was prepared as described by Goldberg et al.(20Goldberg M.E. Rudolph R. Jaenicke R. Biochemistry. 1991; 30: 2790-2797Crossref PubMed Scopus (410) Google Scholar). The lyophilized reduced/denatured lysozyme was dissolved at 10 mg/ml in 6 mGdnHCl, 0.1 m acetic acid, pH 2.5. Refolding was initiated upon a 100-fold dilution, under strong vortex agitation, in renaturation buffer (0.1 m Tris-HCl, pH 8.2, 1 mm EDTA, 20 μm DTT, 60 μmGSSG). The samples were then incubated at 25 °C. Lysozyme activity was measured by mixing 20-μl aliquots of lysozyme solution (0.1 mg/ml) with 0.980 ml of aM. lysodeikticus solution (0.25 mg/ml) in 66 mmmonobasic potassium phosphate (KPi), pH 6.2, equilibrated at 25 °C. The samples were mixed by repeatedly inverting the cuvette for 15 s. The slope of the linear part of the decrease in turbidity, monitored at 450 nm, was taken as the lytic activity. One unit of activity corresponds to an absorbance decrease of 0.0026/min. The concentrations of lysozyme were measured by absorbance at 280 nm using extinction coefficients of 2.63 cm2·mg−1 for the native form and 2.37 cm2·mg−1 for the reduced/denatured form (20Goldberg M.E. Rudolph R. Jaenicke R. Biochemistry. 1991; 30: 2790-2797Crossref PubMed Scopus (410) Google Scholar). Reduced/unfolded lysozyme (0.1 mg/ml) was incubated in the renaturation buffer at 25 °C for 24 h. The protein was then dialyzed against 10 mm KPi, pH 5.5, for 48 h and against 50 mm ammonium acetate, pH 5.5, for another 48 h. The ratio between the lysozyme solution volume and the dialysis buffer volume was about 1/100, and the dialysis buffer was changed every 12 h. At that point, the lysozyme was concentrated about 10-fold using an Amicon DIAFLO cell and a YM3 membrane. After subsequent centrifugation at 10,000 × g for 30 min, the supernatant was lyophilized, and the protein was kept at −20 °C. Gel filtration was performed on a Superdex-75 HR 10/30 column hooked up to a Pharmacia FPLC® system. The column was equilibrated and eluted with the renaturation buffer. The column flow rate was 0.5 ml/mn. Native and denatured lysozyme were incubated for 24 h in renaturation buffer. Both samples were then centrifuged at 10,000 × g for 30 min, to eliminate large aggregates, and 200 μl of each supernatant were loaded on the column. Analytical ultracentrifugation was performed in a Beckman XLA ultracentrifuge, equilibrated at 20 °C, using standard double sector cells. Both native and renatured lysozyme were dialyzed against the same renaturation buffer for 20 h and were centrifuged at 55,000 rpm. The lysozyme concentration was around 0.1 mg/ml. Once the final speed was reached, radial scans at 280 nm were recorded at 20-min intervals. After completion of the seventh scan the apparent sedimentation coefficients were calculated using the second moment method of the XLA-Vel program provided with the XLA-Data Analysis Software (Beckman Instruments, Palo Alto, CA). For recording the CD and the fluorescence spectra, the protein was resuspended in 10 mmKPi, pH 7, at a concentration of 0.1 mg/ml. The instruments were the same as those used for the kinetic analysis (see below). All the spectra were the average of three scans and were corrected by subtracting the spectrum of the buffer recorded under the same conditions just before analyzing the protein. The CD spectra were recorded between 190 and 260 nm, with a spacing of 1 nm, and an integration time of 2 s in a 5-mm optical path cell. The fluorescence spectra were recorded with an integration time of 1 s and an interval of 0.5 nm. The emission band pass was 2.125 nm, and the excitation band pass was 4.25 nm. The excitation wavelength was 295 nm, and the spectra were recorded between 310 and 380 nm, using a cuvette with a 4-mm excitation pathlength and a 1-cm emission pathlength. The mass spectrometry experiments were performed on a Platform spectrometer coupled with an electrospray source (Fisons Instruments, Manchester, UK). The flow rate was 5 μl/min, and the proteins were dissolved in a 1:1 mixture of acetonitrile and water containing 0.2% formic acid. Three different samples were used for the NMR experiments as follows: one corresponding to the native lysozyme, one corresponding to the native lysozyme incubated in the renaturing buffer, and the renatured lysozyme sample. All the samples were dialyzed extensively against dilute HCl, pH 3, before use and freeze-dried. The lyophilized powder was dissolved in 40 μl of D2O (Euriso-Top). The lysozyme concentration, as measured from UV absorbance at 280 nm, was 6.2 mm for the native lysozyme, 0.9 mm for the native lysozyme subjected to the renaturing buffer, and 2.8 mm for the renatured lysozyme.1H NMR experiments, using a Nano-NMR probe (Varian), were run at 500 MHz on a Varian Unity spectrometer with an on-line Sun Sparc 2 workstation. The Nano-NMR probe provides high resolution spectra from liquid samples of only 40 μl (21Manzi A. Salimath P.V. Spiro R.C. Keifer P.A. Freeze H.H. J. Biol. Chem. 1995; 270: 9154-9163Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar) as a result of high resolution magnetic susceptibility matching probe technology used in conjunction to magic angle spinning (22Fitch W.L. Detre G. Holmes C.M. Shoolery J.N. Kiefer P.A. J. Org. Chem. 1994; 59: 7955-7956Crossref Scopus (224) Google Scholar). The experimental data were processed using the VNMR 5.1B program. The spectral width was 7200 Hz and the spinning rate around 2 kHz. Spectra were referred to the water signal at 4.68 ppm at 35 °C (relative to 3-trimethylsilyl-(2,2,3,3-2H4)-propionate, the external reference). Quadrature detection was employed in all experiments with the carrier frequency always maintained at the solvent resonance. The two-dimensional 1H NMR spectra were recorded in the phase-sensitive mode (23States D.J. Haberkorn R.A. Rubens D.J. J. Magn. Reson. 1982; 48: 286-292Google Scholar) with 3200 data points in thet 2 dimension and 440 t 1increments. 8, 48, or 64 scans were acquired for the NOESY experiments depending on the concentration with a single mixing time of 150 ms allowing a direct comparison with previously published data (24Redfield C. Dobson C.M. Biochemistry. 1988; 27: 122-136Crossref PubMed Scopus (249) Google Scholar). Zero filling was applied prior to Fourier transformation, and data were processed with shifted sine bell window functions in both dimensions. Low power selective irradiation during the recycling delay and, for NOESY spectra, during the mixing period was used to suppress the residual water peak. The microcalorimetry experiments were performed on a VP-DSC microcalorimeter from MicroCal Inc (Northampton, MA). The renatured and the native lysozymes were dialyzed overnight against 20 mm glycine, pH 2.5, and centrifuged for 30 min at 10,000 × g. The sample concentrations were 0.20 and 0.21 mg/ml, respectively, for the renatured and the native proteins. The samples were equilibrated at 25 °C, introduced into the cell, and incubated for 15 min at 25 °C and 27 p.s.i. before starting the up scan. The temperature range scanned was 25–80 °C, the rate of temperature change was 40 °C/h for both the up scans and the down scans, and the filter period was 20 s. A 1-min pause was introduced at the end of the up scan before starting the down scan. Three independent up scan/down scan experiments were performed with each protein. Each scan with a protein sample was preceded and followed by an identical scan with buffer to establish the base line. After each scan, the corresponding buffer base line was subtracted, and both the up scan and down scan were analyzed using the MicroCal-Origin software for VP-DSC supplied with the machine. Refolding kinetics were studied by CD at three different wavelengths (220, 222, and 228 nm) using a Jobin-Yvon (Longjumeau, France) CD6 spectropolarimeter. The observation cuvette was a cylindrical, 5 mm-path cell, and the sample holder was thermostated at 25 °C. The dead time needed to mix the solutions, fill the cuvette, and start the scan, was about 80 s and was taken into account for data processing. The integration time was 5 s, and the time interval was 10 s. Each experiment was repeated at least 3 times. The files were converted into ASCII files by the ISA/hda ASCII conversion subroutine of the CD6 software, and data processing was achieved by means of the program Fig.P version 2.7 for windows (Biosoft, Cambridge, UK). The signal of the denatured protein in renaturation buffer was calculated from its signal in 6 m GdnHCl as follows. The dependence of the signal on the GdnHCl concentration was accounted for by incubating HEWL in renaturation media containing GdnHCl at concentrations between 3.5 and 6 m, where the protein is completely unfolded. The ellipticities of these solutions were plottedversus the concentration of denaturant and fitted by a linear regression. The extrapolation of the fit to the GdnHCl concentration in the renaturation buffer was used as the signal of the denatured protein under these conditions. The fluorescence kinetics were recorded in a SPEX (Edison, NJ) Fluoromax spectrofluorometer. The recording time was 2–3 h, with a sampling interval of 10 s and an integration time of 5 s. 1 × 1-cm cuvettes were placed in a cell holder thermostated at 25 °C. The excitation band pass was 2.125 nm, and the emission band pass was 4.25 nm. The fluorescence of the denatured protein in the renaturation buffer was calculated as indicated for the CD experiment. The lysozyme was unfolded under non-reducing conditions in the presence of 6m GdnHCl and 0.1 m acetic acid, pH 2.5, and its concentration was adjusted to 8 mg/ml by addition of the same buffer. The CD stopped-flow apparatus used was as described by Chaffotteet al. (11Chaffotte A.-F. Guillou Y. Goldberg M.E. Biochemistry. 1992; 31: 9694-9702Crossref PubMed Scopus (152) Google Scholar). The stopped-flow module (reservoirs, syringes, mixers, and observation cell) were thermostated at 25 °C. The recording time was 2 s with a sampling period and an integration time of 1 ms. Renaturation was initiated upon mixing of 15 μl of denatured protein (small syringe) with the 585 μl of renaturation buffer (two large syringes). The mixing time was 75 ms, and the final concentration of lysozyme was 0.1 mg/ml. The experiment was repeated 50 times, and the accumulated kinetic data were averaged and analyzed as indicated above for the reduced lysozyme. The renaturation buffer used by Goldberg et al. (20Goldberg M.E. Rudolph R. Jaenicke R. Biochemistry. 1991; 30: 2790-2797Crossref PubMed Scopus (410) Google Scholar) for renaturing reduced lysozyme at 25 °C was progressively optimized, taking into account the two following requirements. (i) Aggregation should be minimized at a lysozyme concentration (0.1 mg/ml) sufficient to give a detectable far UV CD signal. (ii) The disulfide exchange catalysts should not absorb too much light in the far UV so as to permit CD measurements in the 220-nm region. Based on the initial observation by Orsini and Goldberg (25Orsini G. Goldberg M.E. J. Biol. Chem. 1978; 253: 3453-3458Abstract Full Text PDF PubMed Google Scholar), we used moderate concentrations of solubilizing or denaturing agents to minimize aggregation. Although urea and non-detergent sulfobetaines indeed drastically minimized aggregation during the refolding/reoxidation of reduced HEWL (26Goldberg M.E. Expert-Bezancon N. Vuillard L. Rabilloud T. Folding and Design. 1995; 1: 21-27Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar), they could not be used because they absorb light strongly in the far UV. In contrast, GdnHCl absorbs much less in the wavelength range used for far UV CD measurements. Similarly, the classical thiol-disulfide couple described by Saxena and Wetlaufer (19Saxena V.P. Wetlaufer D.B. Biochemistry. 1970; 9: 5015-5022Crossref PubMed Scopus (363) Google Scholar) for the refolding of lysozyme (GSSG/GSH) strongly absorbs light in the far UV. Since GSH, the reduced form of glutathione, is in a 10-fold excess over GSSG, we replaced it by DTT which absorbs light much less in the far UV. By varying the GdnHCl, reduced DTT, and GSSG concentrations, we found that the optimal refolding buffer was 0.1 m Tris-HCl, pH 8.2, 1 mm EDTA, 0.5 m GdnHCl, 60 μmGSSG, and 20 μm DTT, in which the yield of active HEWL was about 80% after 24 h of renaturation. The protein refolded under the conditions described above was characterized and compared with the native enzyme, using the following methods. Both native and refolded lysozyme were centrifuged to eliminate large aggregates and tested for activity. All the activity present in the renatured HEWL preparation (i.e. 74% of the initial activity of the native protein) was recovered in the supernatant, indicating that the aggregates contained only inactive molecules. Moreover the protein in the supernatant and native HEWL had similar specific activities (30,750 units/mg for the native and 29,310 units/mg for the renatured lysozyme in the supernatant). Therefore the renatured mixture contained about 25% of inactive aggregates that were removed by centrifugation and 75% of soluble fully active protein. To characterize further the renatured protein, the samples were analyzed on a gel filtration column (see “Experimental Procedures”). The two proteins had the same elution profile, with a major peak at a volume of 14.5 ml, which contained the protein, and a minor peak at 18 ml, which had an absorbance spectrum similar to that of glutathione. Hence, both native and renatured HEWL eluted in a peak at 14.5 ml, which indicated that they had the same Stokes radius. The proteins were also analyzed by analytical ultracentrifugation. The sedimentation profiles of the two proteins were similar, and the observed sedimentation coefficients were, within experimental error, identical, 2.03 ± 0.05s, for the native lysozyme, and 1.92 ± 0.05s, for the refolded lysozyme. Since both proteins also had identical Stokes radii, it could be concluded that the refolded protein is monomeric, like the native one, and has the same hydrodynamic properties. The renatured lysozyme was further characterized by CD and fluorescence. The CD spectra of the denatured and the native lysozyme were recorded between 190 and 260 nm. The two spectra were indistinguishable, with a minimum at around 208 nm. Hence the two enzymes have the same content in secondary structures. The emission fluorescence spectra recorded with an excitation wavelength of 295 nm were also superimposable and showed a maximum of intensity at 340 nm. To ascertain that no chemical change was introduced during denaturation or renaturation, samples of lysozyme at different stages of the unfolding/refolding process were analyzed by mass spectrometry. The mass spectra of native untreated lysozyme, native lysozyme incubated in renaturation buffer, reduced/denatured lysozyme, and refolded lysozyme had the same features. They all showed a major peak whose intensity was arbitrarily set to 100%, and minor peaks whose intensities were between 10 and 20%. Most of these minor peaks did not change in all the spectra. One, however, had the same mass increase upon reduction as the major peak. Its mass of 14,502 Da could not correspond to a lysozyme from which a part only of the signal sequence would have been removed. It might be a modified form of the enzyme that copurified during the industrial purification of HEWL. The mass of the major peak was 14,305 Da for the two native and the renatured lysozyme and 14,311 for the unfolded lysozyme. These values are similar to those expected for the oxidized (14,306 Da) and the reduced (14,314 Da) form of lysozyme, respectively. These results indicate that no chemical change was introduced upon denaturation/renaturation. The spectra obtained in D2O for the three samples exhibit similar features. 63 slowly exchanging amide protons were identified in the renatured lysozyme in agreement with previously published work on native lysozyme (24Redfield C. Dobson C.M. Biochemistry. 1988; 27: 122-136Crossref PubMed Scopus (249) Google Scholar). The NOESY spectrum is comparable to that obtained for the native lysozyme in the same conditions in terms of temperature and mixing time. Fig. 1 displays thed NN connectivities identified in the NOESY spectra for the renatured lysozyme. Extended region ofd NN connectivities are observed for residues 8 to 14/15, 27–38, and 92–100 in agreement with the presence of regular α-helices. Shorter stretches of d NNconnectivities are also observed for residues 40–42, 55–57, 75–78, 82–84, 111–112, and 123–125. These regions correspond either to non-regular helices or tight turns. Extended regions ofd aN sequential connectivities are also observed from residues 42–47, 50–55, and 57–60 in agreement with the presence of triple-stranded anti-parallel β-sheet (data not shown). 2P. Roux, M. E. Goldberg, A.-F. Chaffotte, and M. Delepierre, manuscript in preparation. The stability of the protein was tested using microcalorimetry. Whereas preliminary experiments made with native lysozyme at a scanning rate of 60 °C/min showed a significant difference (1.6 °C) between the transition temperatures observed for the up scan and the down scan, this difference was reduced to less than 1 °C at a scanning rate of 40 °C. With this slower rate, the transition curves of the renatured and the native lysozymes were practically superimposable. Thus, the transition temperatures obtained from the up scans were 58.5 ± 0.1 and 58.4 ± 0.1 °C for native and renatured lysozyme, respectively. The transition temperatures for the down scans were 57.5 ± 0.1 and 57.4 ± 0.1 °C, respectively, and the denaturation enthalpies were 107.6 ± 2.0 and 100.8 ± 1.8 kcal/mol, respectively. Although the differences in the denaturation enthalpies might seem experimentally significant, they could be accounted for by a 3% difference between the real and the measured relative protein concentrations of the two samples, which is well within the experimental error. Altogether, these results indicate that the thermodynamic properties of the renatured lysozyme are indistinguishable from those of the native enzyme, which confirms the identity of the conformations of both proteins. Nonetheless, it should be noted that the values found here for the T m are lower than the one found in the literature (T m = 64, 1 °C, see Ref. 27Privalov G. Kavina V. Freire E. Privalov P.L. Anal. Biochem. 1995; 232: 79-85Crossref PubMed Scopus (174) Google Scholar). This difference could be due to an uncertainty in the pH value since the T m of lyso