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Biochemistry 2002, 41, 326-333
Does β-Lactoglobulin Denaturation Occur via an Intermediate State?† Laura D’Alfonso, Maddalena Collini, and Giancarlo Baldini* UniVersita` degli Studi di Milano-Bicocca and INFM UdR Milano-Bicocca, P.za della Scienza, 3 I-20126 Milano, Italy ReceiVed July 19, 2001; ReVised Manuscript ReceiVed October 10, 2001
ABSTRACT: The denaturation of β-lactoglobulin (BLG) in the presence of urea and GuHCl has been investigated at different pH values with various spectroscopic techniques. The equilibrium denaturation free energy values, obtained by linearly extrapolating the data to vanishing denaturant (∆GDH2O), are compared and discussed. The fit of the spectroscopic data monitoring the denaturation of BLG has been approached, at first, with a two-state model that describes the protein transition from the folded state (at each pH and in the absence of denaturant) to the denatured state, but in particular, along the GuHCl denaturation pathway some evidence is found of the presence of an intermediate state. Time-resolved fluorescence experiments performed on the BLG-ANS (1-anilino-8-naphthalenesulfonate) complex help to understand the results. Fluorescence polarization anisotropy (FPA) measurements accompanying the denaturation process show the presence of a fast rotational diffusion of the ANS probe, and the data are interpreted in terms of local fluctuations of a still structured tract of the denatured protein where the probe is bound.
β-Lactoglobulin (BLG),1 a 162-residue globular protein present in large quantities in the milk of several mammals, belongs to the lipocalin superfamily. This family includes transport proteins such as the retinol binding protein, the odorant binding protein, and the major urinary protein, which all share the common structural feature of a β-barrel calyx, built from eight antiparallel β-sheets, arranged as an ideal site for hydrophobic ligands (1). During recent years, several investigations have been performed on bovine β-lactoglobulin to elucidate its physiological role and its folding mechanism. The spatial structure and conformation of the protein have been determined in the native state (1, 2), in the acid state (3, 4), and under alkaline conditions (2). To gain insight on the BLG folding mechanism, its denaturation process, induced by both chemical agents and temperature, has been studied by means of different techniques: ultrafiltration and dialysis (5-7), tryptophan intrinsic fluorescence (8, 9), calorimetry (10, 11), NMR (12), differential UV absorption (13, 14), density (15), and, mainly, circular dichroism (16-24). The most recent of these works were aimed at determining whether the BLG denaturation mechanism can be adequately described by a two-state model (N S U), as suggested by circular dichroism (24) and NMR (12) measurements on the BLG acid state, or whether a more complex unfolding pathway, with different intermediate steps (21-22, 25), is necessary. To obtain a more detailed description of the transition from the native to the denatured state, the thermodynamic parameters of the process and their depen† This work was supported by a grant from the Istituto Nazionale per la Fisica della Materia, Sezione B. * Corresponding author. Tel: +390264482870. Fax: +390264482894. E-mail:
[email protected]. 1 Abbreviations: BLG, β-lactoglobulin; NMR, nuclear magnetic resonance; GuHCl, guanidine hydrochloride; ANS, 1-anilino-8-naphthalenesulfonate; CD, circular dichroism; UV, ultraviolet; FPA, fluorescence polarization anisotropy; LEM, linear extrapolation model.
dence on the nature of the denaturant agent and on the initial state of the protein must be established. In fact, despite several denaturation studies performed on BLG, an investigation performed on a well-defined BLG variant aimed at clarifying the role of pH and the nature of the denaturant on the denaturation process is still lacking. The aim of the present study then is that of trying to shed some light on the action of denaturing agents according to their chemical composition and of the protein electrostatic charge (dependent on solution pH) in the transition from the native to the denatured state. Two different chemical denaturing agents have been employed, urea and guanidine hydrochloride (GuHCl). The experiments have been performed in the acid state of the protein (pH 2.3), in its native state (pH 6.2), and in a condition where the protein has already undergone the so-called Tanford transition (pH 8.3; for the details of the transition see ref 26). Furthermore, to better understand the nature of the transition process, differential UV absorption, circular dichroism, and steadystate intrinsic fluorescence have been joined by lifetime and fluorescence polarization anisotropy measurements of ANS (1-anilino-8-naphthalenesulfonate) bound to BLG. These techniques give useful information on protein conformational variations localized near the binding sites and on “effective” protein “local” dimension changes induced by the denaturing agents. BLG is reported to possess two different binding sites for the probe ANS: an “external site”, in proximity of an hydrophobic patch on the protein surface, and an “internal site”, located in the BLG calyx (27). The results have been first analyzed by means of a two-state model to describe the transition and employing the linear approximation (LEM) to express the dependence of the denaturation free energy value on denaturant concentration (28, 29). Some evidence of an “ionic” intermediate state, detected at low GuHCl concentrations, is shown, and a qualitative analysis in terms of a three-state model is discussed.
10.1021/bi0115028 CCC: $22.00 © 2002 American Chemical Society Published on Web 12/05/2001
Intermediate State in BLG Denaturation MATERIALS AND METHODS Lyophilized bovine β-lactoglobulin, genetic variant B (Sigma Chemical Co., lot 13H7150), was dissolved in the proper buffer at the desired concentration. ANS ammonium salt was purchased from Fluka Chemical Co. Protein and dye concentrations were checked photometrically by employing the molar extinction coefficient at the absorption peak 280 ) 17600 cm-1 M-1 and 350 ) 5000 cm-1 M-1, respectively. Phosphate buffers employed in the denaturant agent titration were the following: H3PO4/NaOH, pH 2.3, ionic strength I ) 0.007 M; KH2PO4-Na2HPO4, pH 6.2, I ) 0.010 M; KH2PO4-Na2HPO4/NaOH, pH 8.3, I ) 0.010 M. All of the reagents used in the sample preparation were of analytical grade. The different experiments were performed in the presence of urea and GuHCl at concentrations ranging from 0 to 8 M and to 0 and 5 M, respectively. Fresh samples were prepared at each denaturant concentration by employing denaturant stock solutions at the chosen pH. In the time-resolved fluorescence measurements each denaturant concentration was prepared at least twice to check for data reproducibility. Experiments versus ionic strength were performed by employing different samples for each ionic strength value, and the ionic strength of the solution was obtained by adding to the protein solution proper aliquots of 2 M NaCl prepared in the desired buffer at the chosen pH. The BLG concentration was kept constant during each measurement and equal to about 40 µM for the differential UV absorption, near-UV circular dichroism, extrinsic ANS bound fluorescence, and time-resolved fluorescence measurements. In the far-UV circular dichroism and intrinsic fluorescence measurements the protein concentration was equal to about 5 µM. In the fluorescence experiments the ANS concentration was constant and equal to about 5 µM. Circular dichroism measurements in both spectral regions were performed versus the time elapsed from sample preparation, from 10 min to 48 h, in the presence of different denaturant concentrations (4 and 7.5 M for urea and 2 and 4.5 M for GuHCl), and no significant change was detected in the CD spectra. Measurements were performed about 10 min after sample preparation, typically, but neither sample incubation at the different denaturant concentration nor the time elapsed from sample preparation to measure was found relevant. GuHCl and urea stock solutions were prepared by diluting proper amounts of the denaturant powder (Sigma Chemical Co.) in the desired buffer. Due to the high hygroscopicity of these denaturants the final solution concentration was verified by measuring the solution refraction index by means of an Abbe refractometer having precision of the third decimal point and corresponding to a concentration uncertainty equal to about 0.05 M. Solution parameters, such as density, refraction index, or viscosity, were obtained from the literature (refs 30 and 31 for GuHCl) or by direct measurements. Samples prepared at acid pH employing urea stock solutions showed a small increase in the pH value (up to pH 2.8) when the denaturant concentration was increased. The absence of variation in the protein charge value during the denaturation process is taken as a key factor for the correct analysis of the experimental data, since pH variations
Biochemistry, Vol. 41, No. 1, 2002 327 are known to cause relevant changes in protein spectral properties (27). To keep the pH value (and the protein charge) constant, acid aliquots have been added to the solutions. Two different acid solutions have been employed: 1 M HCl, which leads to the presence of Cl- ions in solution, and 85% w/w H3PO4, which is the same acid employed in the buffer preparation and, therefore, should not alter solution chemical properties. The resulting protein samples at pH 2.3 are labeled as “acid hcl” and “acid hpo”, respectively. Differential UV absorption measurements were performed at 25 °C in the 210-500 nm range in a 10 mm path-length quartz cell (Hellma) on samples containing increasing denaturant concentrations by employing a Perkin-Elmer Lambda 2 spectrophotometer. Spectra of both the protein and the corresponding buffer were recorded, and the latter was subtracted from the former to resolve the information concerning the BLG spectral response. The resulting spectra were normalized for protein concentration, and the difference spectra between the unperturbed state (0 M denaturant concentration) and the increasing concentration of urea and GuHCl were calculated. Particular attention was given to the differential absorption at 293 nm (averaged between 292 and 294 nm) versus denaturant concentration, where the main contribution to the absorption spectrum is due to the protein tryptophans (13). Circular dichroism (CD) spectra in the near-ultraviolet (UV) at wavelengths from 240 to 350 nm and in the far-UV from 200 to 250 nm were recorded at 25 °C by a Jasco 500-A spectropolarimeter. The signal was averaged 30-40 times at each wavelength. Due to the high absorption of the denaturant at wavelengths below 205 nm, the far-UV CD signal down to 200 nm was taken only at lower urea or GuHCl concentrations (
