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Reversible Unfolding of Bovine β-Lactoglobulin Mutants without a Free Thiol Group

2003; Elsevier BV; Volume: 278; Issue: 47 Linguagem: Inglês

10.1074/jbc.m308592200

ISSN

1083-351X

Autores

Masanori Yagi, Kazumasa Sakurai, Chitkala Kalidas, Carl A. Batt, Yuji Goto,

Tópico(s)

Carbohydrate Chemistry and Synthesis

Resumo

Bovine β-lactoglobulin (β-lg) has been used extensively as a model for studying protein folding. One of the problems preventing clarification of the folding mechanism is the incomplete reversibility from the unfolded state, probably caused by the thiol-disulfide exchange between a free thiol at Cys-121 and two disulfide bonds. We constructed and expressed three β-lg subtype A mutants in which Cys-121 was replaced by Ala, Ser, or Val (i.e. C121A, C121S, and C121V). We studied the reversibilities of these mutants from urea denaturation using circular dichroism, tryptophan fluorescence, reversed-phase and gel-filtration high performance liquid chromatographies, and SDS-PAGE. The folded structure of each mutant was similar to that of wild-type β-lg. Urea-induced unfolding at pH 7.0 and 3.0 showed that although the C121S mutation notably decreases the stability, the destabilizing effects of the C121A and C121V mutations are less severe. For all of the mutants, complete refolding from the unfolded state in 8 m urea at both pH 7.0 and 3.0 was observed. Kinetics of the formation of the irreversibly unfolded species of wild-type β-lg in 8 m urea at pH 7.0 indicated that, first, an intramolecular thiol-disulfide exchange occurs to produce a mixture of species with non-native disulfide bonds followed by the intermolecular thiol-disulfide exchange producing the oligomers. These results indicate that intramolecular and intermolecular thiol-disulfide exchange reactions cause the low reversibility of wild-type β-lg especially at neutral pH and that the mutation of Cys-121 improves the reversibility, enabling us to study the folding of β-lg more exactly under various conditions. Bovine β-lactoglobulin (β-lg) has been used extensively as a model for studying protein folding. One of the problems preventing clarification of the folding mechanism is the incomplete reversibility from the unfolded state, probably caused by the thiol-disulfide exchange between a free thiol at Cys-121 and two disulfide bonds. We constructed and expressed three β-lg subtype A mutants in which Cys-121 was replaced by Ala, Ser, or Val (i.e. C121A, C121S, and C121V). We studied the reversibilities of these mutants from urea denaturation using circular dichroism, tryptophan fluorescence, reversed-phase and gel-filtration high performance liquid chromatographies, and SDS-PAGE. The folded structure of each mutant was similar to that of wild-type β-lg. Urea-induced unfolding at pH 7.0 and 3.0 showed that although the C121S mutation notably decreases the stability, the destabilizing effects of the C121A and C121V mutations are less severe. For all of the mutants, complete refolding from the unfolded state in 8 m urea at both pH 7.0 and 3.0 was observed. Kinetics of the formation of the irreversibly unfolded species of wild-type β-lg in 8 m urea at pH 7.0 indicated that, first, an intramolecular thiol-disulfide exchange occurs to produce a mixture of species with non-native disulfide bonds followed by the intermolecular thiol-disulfide exchange producing the oligomers. These results indicate that intramolecular and intermolecular thiol-disulfide exchange reactions cause the low reversibility of wild-type β-lg especially at neutral pH and that the mutation of Cys-121 improves the reversibility, enabling us to study the folding of β-lg more exactly under various conditions. Bovine β-lactoglobulin (β-lg), 1The abbreviations used are: β-lgβ-lactoglobulinCDcircular dichroismHPLChigh performance liquid chromatographyNaPisodium phosphateGly-HClglycine-HCl.1The abbreviations used are: β-lgβ-lactoglobulinCDcircular dichroismHPLChigh performance liquid chromatographyNaPisodium phosphateGly-HClglycine-HCl. one of the main protein components of cow's milk but absent in humans, is composed of nine β-strands and one short and one long α-helix (Fig. 1) (1Sawyer L. Kontopidis G. Biochim. Biophys. Acta. 2000; 1482: 136-148Crossref PubMed Scopus (334) Google Scholar, 2Hambling S.G. MacAlpine A.S. Sawyer L. Fox, P.F. Advanced Dairy Chemistry. Elsevier, Amsterdam1992: 141-190Google Scholar, 3Brownlow S. Cabral J.H.M. Cooper R. Flower D.R. Yewdall S.J. Polikarpov I. North A.C.T. Sawyer L. Structure. 1997; 5: 481-495Abstract Full Text Full Text PDF PubMed Scopus (651) Google Scholar, 4Qin B.Y. Bewley M.C. Creamer L.K. Baker H.M. Baker E.N. Jameson G.B. Biochemistry. 1998; 37: 14014-14023Crossref PubMed Scopus (445) Google Scholar, 5Qin B.Y. Creamer L.K. Baker E.N. Jameson G.B. FEBS Lett. 1998; 438: 272-278Crossref PubMed Scopus (137) Google Scholar, 6Kontopidis G. Holt C. Sawyer L. J. Mol. Biol. 2002; 318: 1043-1055Crossref PubMed Scopus (222) Google Scholar). β-lg binds a variety of hydrophobic compounds including retinol and fatty acids at the hydrophobic cavity of the molecule, although the physiological function of β-lg and moreover the ligand binding are unknown (1Sawyer L. Kontopidis G. Biochim. Biophys. Acta. 2000; 1482: 136-148Crossref PubMed Scopus (334) Google Scholar, 6Kontopidis G. Holt C. Sawyer L. J. Mol. Biol. 2002; 318: 1043-1055Crossref PubMed Scopus (222) Google Scholar). β-lg is made of 162 amino acid residues (a molecular weight of 18,400), five of which are cysteine residues, i.e. Cys-66, Cys-106, Cys-119, Cys-121, and Cys-160. Cys-66 and Cys-160 form a disulfide bond near the surface of the protein molecule, Cys-106 and Cys-119 form a disulfide bond inside, and Cys121 retains a free thiol group. Although β-lg exists as a homodimer at neutral pH, it dissociates into monomers at acidic pH but still retains the native structure (7Kuwata K. Hoshino M. Era S. Batt C.A. Goto Y. J. Mol. Biol. 1998; 283: 731-739Crossref PubMed Scopus (96) Google Scholar, 8Kuwata K. Hoshino M. Forge V. Era S. Batt C.A. Goto Y. Protein Sci. 1999; 8: 2541-2545Crossref PubMed Scopus (156) Google Scholar, 9Uhrínová S. Smith M.H. Jameson G.B. Uhrín D. Sawyer L. Barlow P.N. Biochemistry. 2000; 39: 3565-3574Crossref PubMed Scopus (212) Google Scholar, 10Sakai K. Sakurai K. Sakai M. Hoshino M. Goto Y. Protein Sci. 2000; 9: 1719-1729PubMed Google Scholar, 11Sakurai K. Oobatake M. Goto Y. Protein Sci. 2001; 10: 2325-2335Crossref PubMed Scopus (151) Google Scholar, 12Sakurai K. Goto Y. J. Biol. Chem. 2002; 277: 25735-25740Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Because of its stability under acidic pH conditions, β-lg is resistant to digestion in the stomach and is considered one of the allergens for human infant milk allergy (13Wal J.M. Allergy. 1998; 53: 1013-1022Crossref PubMed Scopus (160) Google Scholar). β-lactoglobulin circular dichroism high performance liquid chromatography sodium phosphate glycine-HCl. β-lactoglobulin circular dichroism high performance liquid chromatography sodium phosphate glycine-HCl. In addition, β-lg shows a unique characteristic in its folding. Although β-lg is a predominantly β-sheet protein, Shiraki et al. (14Shiraki K. Nishikawa K. Goto Y. J. Mol. Biol. 1995; 245: 180-194Crossref PubMed Scopus (440) Google Scholar) noticed that the addition of trifluoroethanol induces a drastic conformational change to a predominantly α-helical structure. Secondary structure predictions indicated that β-lg has a high α-helical propensity determined by local interactions (15Kuroda Y. Hamada D. Tanaka T. Goto Y. Folding Des. 1996; 1: 255-263Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 16Hamada D. Kuroda Y. Tanaka T. Goto Y. J. Mol. Biol. 1995; 254: 737-746Crossref PubMed Scopus (171) Google Scholar). Moreover, the refolding kinetics measured by the far-UV circular dichroism (CD) indicated the accumulation of intermediates with non-native α-helices (17Hamada D. Segawa S. Goto Y. Nat. Struct. Biol. 1996; 3: 868-874Crossref PubMed Scopus (265) Google Scholar). Taken together, Hamada et al. (17Hamada D. Segawa S. Goto Y. Nat. Struct. Biol. 1996; 3: 868-874Crossref PubMed Scopus (265) Google Scholar, 18Hamada D. Goto Y. J. Mol. Biol. 1997; 269: 479-487Crossref PubMed Scopus (128) Google Scholar) proposed that, during the refolding of β-lg, intermediates with non-native α-helices accumulate because of the strong local α-helical preference. However, non-local interaction and overall free energy favor the native β-structure and the slow α-helix-to-β-sheet transition produces the native β-lg. Recent studies with H/D exchange of amide protons combined with the heteronuclear NMR analysis suggested the structure of the intermediate (19Forge V. Hoshino M. Kuwata K. Arai M. Kuwajima K. Batt C.A. Goto Y. J. Mol. Biol. 2000; 296: 1039-1051Crossref PubMed Scopus (90) Google Scholar, 20Kuwata K. Shastry R. Cheng H. Hoshino M. Batt C.A. Goto Y. Roder H. Nat. Struct. Biol. 2001; 8: 151-155Crossref PubMed Scopus (172) Google Scholar). In the rapidly formed intermediate that accumulated several microseconds after the initiation of refolding, several regions showed persistent resistance to exchange. This includes the regions of β strands F, G, and H and the C-terminal helix. In addition, several residues in β strand A indicated a weak protection. The pattern of protection suggested that β strand A assumes a non-native α-helix in the intermediate (20Kuwata K. Shastry R. Cheng H. Hoshino M. Batt C.A. Goto Y. Roder H. Nat. Struct. Biol. 2001; 8: 151-155Crossref PubMed Scopus (172) Google Scholar). Thus, the intermediate is likely to contain both native and non-native structures so that the present picture of the folding of β-lg is a mixture of the hierarchical and non-hierarchical folding. However, we do not understand the role of the non-native α-helical structure in protein folding. Does it accelerate or decelerate the folding of β-lg? The exact folding mechanism of β-lg is important to understand the interplay between the local and non-local interactions, which might play a role in several biologically important processes such as the conformational transition of prion protein (21Cohen F.E. J. Mol. Biol. 1999; 293: 313-320Crossref PubMed Scopus (136) Google Scholar). Despite the interesting characteristics of β-lg in protein folding, the folding experiments with β-lg have been performed mainly at acidic pH (17Hamada D. Segawa S. Goto Y. Nat. Struct. Biol. 1996; 3: 868-874Crossref PubMed Scopus (265) Google Scholar, 19Forge V. Hoshino M. Kuwata K. Arai M. Kuwajima K. Batt C.A. Goto Y. J. Mol. Biol. 2000; 296: 1039-1051Crossref PubMed Scopus (90) Google Scholar, 20Kuwata K. Shastry R. Cheng H. Hoshino M. Batt C.A. Goto Y. Roder H. Nat. Struct. Biol. 2001; 8: 151-155Crossref PubMed Scopus (172) Google Scholar, 22Ragona L. Fogolari F. Romagnoli S. Zetta L. Maubois J.L. Molinari H. J. Mol. Biol. 1999; 293: 953-969Crossref PubMed Scopus (57) Google Scholar, 23Ragona L. Catalano M. Zetta L. Longhi R. Fogolari F. Molinari H. Biochemistry. 2002; 41: 2786-2796Crossref PubMed Scopus (19) Google Scholar). One of the factors preventing the use of neutral or alkaline pH conditions is the low reversibility of the unfolded state (24Burova T.V. Choiset Y. Tran V. Haertlé T. Protein Eng. 1998; 11: 1065-1073Crossref PubMed Google Scholar, 25Cupo J.F. Pace C.N. Biochemistry. 1983; 22: 2654-2658Crossref PubMed Scopus (84) Google Scholar, 26Griko Y.V. Kutyshenko V.P. Biophys. J. 1994; 67: 356-363Abstract Full Text PDF PubMed Scopus (55) Google Scholar). Irreversible denaturation has been thought to arise from thiol-disulfide exchange caused by the free thiol of Cys-121. Indeed, the formation of intermolecular disulfide bridges by heat denaturation at neutral pH has been reported previously (27Schokker E.P. Singh H. Pinder D.N. Norris G.E. Creamer L.K. Int. Dairy J. 1999; 9: 791-800Crossref Scopus (110) Google Scholar, 28Carrotta R. Bauer R. Waninge R. Rischel C. Protein Sci. 2001; 10: 1312-1318Crossref PubMed Scopus (124) Google Scholar, 29Surroca Y. Haverkamp J. Heck A.J. J. Chromatogr. A. 2002; 13: 275-285Crossref Scopus (69) Google Scholar, 30Croguennec T. Bouhallab S Mollé D. O'Kennedy B.T. Mehra R. Biochem. Biophys. Res. Commun. 2003; 301: 465-471Crossref PubMed Scopus (81) Google Scholar). Even at pH 2.1, an incomplete recovery of CD spectra was observed in samples treated with 7 m urea (22Ragona L. Fogolari F. Romagnoli S. Zetta L. Maubois J.L. Molinari H. J. Mol. Biol. 1999; 293: 953-969Crossref PubMed Scopus (57) Google Scholar). The proximity of Cys-121 to Cys-119 may make the thiol-disulfide exchange possible even under acidic conditions. Although site-directed mutagenesis at Cys-121 was attempted previously to prepare a species devoid of the free thiol group, a mutant without Cys-121 was not obtained with the Escherichia coli expression system (31Cho Y. Gu W. Watkins S. Lee S.P. Kim T.R. Brady J.W. Batt C.A. Protein Eng. 1994; 7: 263-270Crossref PubMed Scopus (43) Google Scholar). In this study, we successfully constructed and expressed Cys-121-substituted mutants of bovine β-lg subtype A (i.e. C121A, C121S, and C121V). We studied their structures, resistance to urea denaturation, and reversibility from the unfolded state in 8 m urea at pH 7.0 or 3.0. We also investigated the time-dependent modification in 8 m urea causing the irreversible denaturation of wild-type β-lg. Hereby, we can state that a free thiol at Cys-121 attacks the disulfide bonds and forms incorrect pairs in an unfolded β-lg, decreasing the reversibility of wild-type β-lg, and that the mutants used here enable us to study the folding of β-lg under various conditions. Plasmids for Mutagenesis—The primers used are 5′-C CTG GTC TGC CAG GCC CTG GTC AGG ACC-3′ for the C121A mutation, 5′-CTG GTC TGC CAG TCC CTG GTC AGG AC-3′ for the C121S mutation, and 5′-C CTG GTC TGC CAG GTC CTG GTC AGG ACC-3′ for the C121V mutation. These primers and their complementary primers were purchased from Sigma. We introduced site-directed mutations with these primers by the QuikChange method (Stratagene, La Jolla, CA) into previously constructed plasmids (pPIC11) containing the bovine β-lg subtype A sequence (32Kim T.R. Goto Y. Hirota N. Kuwata K. Denton H. Wu S.Y. Sawyer L. Batt C.A. Protein Eng. 1997; 10: 1339-1345Crossref PubMed Google Scholar). To obtain a larger amount of plasmids, E. coli XL-1 Blue cells were transformed with the plasmids and the transformants were selected using ampicillin on LB plates. After the incubation of E. coli containing the plasmids in test tubes, a Wizard Plus Miniprep kit (Promega, Madison, WI) was employed to extract the plasmids. We verified the sequences of the mutants in the plasmids using a DNA sequencer, the Applied Biosystems Model 310 (Foster City, CA). Protein Expression and Purification—Each plasmid confirmed to contain the desired sequence was linearized using the restriction enzyme AatI (Toyobo, Osaka, Japan) for Pichia pastoris GS115(his4) (Invitrogen) transformation. Electroporation of P. pastoris was performed with a pulse voltage of 1.7 kV using an E. coli pulser (Bio-Rad). The transformed cells suspended in 1 m sorbitol were spread on RDB plates without His, and colonies of His+ transformants appeared in 2 or 3 days. Some were picked from plates of each mutant and incubated in test tubes to assess their expression. The quantity of protein expressed was checked with SDS-PAGE, and one of the strains with the highest level of expression was selected for every mutant and adopted for subsequent mass culture in a 1-liter jar fermenter. For wild-type β-lg, we used the strain constructed previously and stored as glycerol stock (12Sakurai K. Goto Y. J. Biol. Chem. 2002; 277: 25735-25740Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). The broth of mass culture was centrifuged, and the supernatant was filtered with 5.0-μm-pore size Millex-SV (Millipore, Bedford, MA). The filtered supernatant was diluted 5-fold with water and adjusted to pH 3.5 with concentrated HCl. Each type of β-lg in the supernatants was adsorbed by a CM-Sepharose CL-6B (Amersham Biosciences) column equilibrated with 50 mm glycine-HCl (Gly-HCl) buffer (pH 3.5) and eluted with a gradient of 0–1.0 m NaCl. The solutions with β-lg were dialyzed against dilute HCl at pH 2.5 and lyophilized. The purities of the wild-type β-lg, C121A, and C121S were very high judging from the patterns of SDS-PAGE and matrix-assisted laser desorption ionization time-of-flight mass spectroscopy. No glycosylation was detected for all of the mutants as well as the wild-type β-lg as is the case of β-lg subtype A purified from milk. On the other hand, mass spectra of C121V detected the contamination of species without 2–4 residues of the N terminus. Regardless of purification conditions, we could not remove these modified species. However, because the N-terminal deletions seemed not to affect the structure or reversibility, we used the fraction of C121V without further purification. CD Measurements—CD spectra were acquired with a Jasco J-720 CD spectropolarimeter at 20 °C. Samples were prepared in 50 mm sodium phosphate (NaPi) buffer (pH 7.0) and contained 0.1 mg ml-1 of each protein for far-UV measurements using a 0.2-cm cell and 1.0 mg ml-1 for near-UV measurements using a 1.0-cm cell. The concentrations of proteins were determined from the absorbance at 280 nm with molar extinction coefficient at 280 nm (ϵ280) calculated using: ϵ280 = 5690NTrp + 1280NTyr + 120NS-S, where NTrp, NTyr, and NS-S are the numbers of tryptophan, tyrosine, and disulfide bonds, respectively (33Gill S.C. von Hippel P.H. Anal. Biochem. 1989; 182: 319-326Crossref PubMed Scopus (5048) Google Scholar). The data were represented by the mean residue ellipticity, [θ]. Fluorescence Measurements—Tryptophan fluorescence spectra were measured with a Hitachi F-4500 fluorescence spectrophotometer at 20 °C. Samples contained 0.1 mg ml-1 of each protein and were prepared in 50 mm NaPi buffer, pH 7.0, or 50 mm Gly-HCl buffer, pH 3.0, with each concentration of urea. Measurements were performed with excitation at 295 nm using a cell with a 0.5-cm light path. We assumed a two state mechanism of unfolding and a linear dependence of the free energy change of unfolding, ΔGUH2O, upon the urea concentration, [urea] (34Pace C.N. Trends Biochem. Sci. 1990; 15: 14-17Abstract Full Text PDF PubMed Scopus (387) Google Scholar). To estimate the fraction of native species, the fluorescence spectra from 305 to 400 nm were globally fitted to Equation 1,F=α{fNF0M+(1-fN)F9M}(Eq. 1) where F is the fluorescence intensity at every 0.2 nm from 305 to 400 nm for a sample with each concentration of urea, F0 M and F9 M, for 0 m and 9 m urea, respectively; fN is the fractional population of native species, and α is a variable term for fluorescence intensity. fNs were plotted against urea concentration, and thermodynamic parameters were calculated by fitting to Equation 2,y=(a+b[urea])-(c+d[urea])1+exp{-(ΔGUH2O-m[urea])/RT}+(c+d[urea])}(Eq. 2) where y is the observed fN, ΔGUH2O is the free energy change of unfolding in the absence of urea, m is a parameter for cooperativity of unfolding, and (a + b[urea]) and (c + d[urea]) are terms for the base-line dependence on urea concentration. The midpoint concentration of unfolding, cM, is calculated by Equation 3.cM=ΔGUH2O/m(Eq. 3) The fitting was operated with the software Igor (WaveMetrics, Lake Oswego, OR). Analytical Ultracentrifugation—Sedimentation equilibrium experiments were carried out with a Beckman Optima XL-I analytical centrifuge. Samples for each type of β-lg were prepared in 20 mm NaPi buffer, pH 7.0, containing 20 mm NaCl with three types of protein concentrations (0.33, 0.67, and 1.0 mg ml-1). The dimerization constant of monomer-dimer equilibrium, KD, was determined by fitting CT–r plots as described before (10Sakai K. Sakurai K. Sakai M. Hoshino M. Goto Y. Protein Sci. 2000; 9: 1719-1729PubMed Google Scholar, 11Sakurai K. Oobatake M. Goto Y. Protein Sci. 2001; 10: 2325-2335Crossref PubMed Scopus (151) Google Scholar, 12Sakurai K. Goto Y. J. Biol. Chem. 2002; 277: 25735-25740Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 35Teller D.C. Horbett T.A. Richards E.G. Schachman H.K. Ann. N. Y. Acad. Sci. 1969; 164: 66-101Crossref Scopus (109) Google Scholar). Refolding Yield—To measure the reversibility of unfolding, samples in 8 m urea were diluted 30-fold with buffers after a 2-day incubation at 20 °C. The refolding was monitored based on CD and tryptophan fluorescence. For the measurements of time dependence, the incubation time in 8 m urea was varied. The reversibility of unfolding was also measured by reversed-phase HPLC and gel-filtration HPLC. HPLC experiments were performed with a Gilson HPLC system. A C4 column (Waters, Milford, MA) and TSK-Gel G3000SWXL column (Tosoh, Tokyo, Japan) were employed for the reversed-phase and gel-filtration HPLC measurements, respectively. For both measurements, the flow rate was 0.5 ml min-1 and protein elution was monitored from absorbance at 220 nm. Protein samples (3.0 mg ml-1)in50mm NaPi buffer, pH 7.0, with 8 m urea were incubated at 20 °C. For reversed-phase HPLC, samples were diluted 30-fold and 50 μl of each diluted sample was applied to the column equilibrated with 25% (v/v) acetonitrile containing 0.05% (v/v) trifluoroacetic acid. Proteins were eluted with a gradient of acetonitrile from 25 to 55% (v/v). For gel-filtration HPLC, samples were diluted 10-fold and 50 μl of each sample was applied to the column equilibrated with 50 mm NaPi buffer, pH 7.0, containing 100 mm NaCl. SDS-PAGE was carried out to monitor the formation of disulfide bonded oligomers. The unfolded samples incubated in the same condition as used for the reversed-phase or gel-filtration HPLC measurements (3.0 mg ml-1 of each protein, 50 mm NaPi at pH 7.0, 8 m urea, and 20 °C) were diluted 10-fold with 100 mm HCl to quench the thiol-disulfide exchange reaction. For the preparation of the reduced samples, the unfolded samples incubated for 48 h were reduced with 100 mm dithiothreitol at 37 °C for 3 h and then diluted with 100 mm HCl to the same protein concentration as the non-reduced samples. 16 μl of each sample then was mixed with 4 μl of 5-fold concentrated sample buffer and applied to the gel. To obtain the intensities of peaks, an image analysis was performed with the software Image-Pro Plus (Media Cybernetics, Silver Spring, MD). Structure of Mutants—We expressed the three mutants (C121A, C121S, and C121V) of bovine β-lg subtype A efficiently in methylotrophic yeast P. pastoris. The final yields of C121A, C121S, and C121V cultured with 1 liter of medium were ∼190, 330, and 160 mg, respectively (Table I). Under the same conditions, the yield of wild-type β-lg was 170 mg (Table I), indicating that the removal of the free thiol group at Cys-121 does not affect the expression yield.Table IExpression yields and dimerization constants of the wild-type β-lg and its mutantsSpeciesYieldKDaErrors are fitting errorsmg liter–1m-1Wild-type1702.46 (±1.07) × 104C121A1901.94 (±0.49) × 104C121S3303.25 (±0.16) × 104C121V1603.19 (±1.07) × 104a Errors are fitting errors Open table in a new tab As was also shown in our previous papers (12Sakurai K. Goto Y. J. Biol. Chem. 2002; 277: 25735-25740Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 14Shiraki K. Nishikawa K. Goto Y. J. Mol. Biol. 1995; 245: 180-194Crossref PubMed Scopus (440) Google Scholar), the far-UV CD spectrum of wild-type β-lg at pH 7.0 had a minimum at 218 nm with an ellipticity of -6000 (Fig. 2a), showing that it is a predominantly β-sheet protein (Fig. 1). The near-UV spectrum with minima at 293 and 285 nm (Fig. 2a) was also consistent with the previous papers (12Sakurai K. Goto Y. J. Biol. Chem. 2002; 277: 25735-25740Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 14Shiraki K. Nishikawa K. Goto Y. J. Mol. Biol. 1995; 245: 180-194Crossref PubMed Scopus (440) Google Scholar). Far- and near-UV CD spectra of three mutants (C121A, C121S, and C121V) were similar to each other and also to those of wild-type β-lg, showing that the mutations of Cys-121 did not change the global conformation (Fig. 2a). In particular, the CD spectra of C121A were indistinguishable from those of wild-type β-lg. The CD spectra were also similar between the wild-type and mutant proteins at pH 2.0 (data not shown). β-lg has two tryptophan residues, one at position 19 on β-strand A and the other at position 61 on β-strand C (Fig. 1). Although Trp-19 facing into the base of the hydrophobic pocket before the bend of the βA strand is fully buried, Trp-61 at the end of the βC strand is relatively exposed to solvent (3Brownlow S. Cabral J.H.M. Cooper R. Flower D.R. Yewdall S.J. Polikarpov I. North A.C.T. Sawyer L. Structure. 1997; 5: 481-495Abstract Full Text Full Text PDF PubMed Scopus (651) Google Scholar, 4Qin B.Y. Bewley M.C. Creamer L.K. Baker H.M. Baker E.N. Jameson G.B. Biochemistry. 1998; 37: 14014-14023Crossref PubMed Scopus (445) Google Scholar, 5Qin B.Y. Creamer L.K. Baker E.N. Jameson G.B. FEBS Lett. 1998; 438: 272-278Crossref PubMed Scopus (137) Google Scholar, 6Kontopidis G. Holt C. Sawyer L. J. Mol. Biol. 2002; 318: 1043-1055Crossref PubMed Scopus (222) Google Scholar, 8Kuwata K. Hoshino M. Forge V. Era S. Batt C.A. Goto Y. Protein Sci. 1999; 8: 2541-2545Crossref PubMed Scopus (156) Google Scholar). At pH 7.0 in the absence of urea, the fluorescence spectrum of wild-type β-lg had a maximum at 329.4 nm, and upon unfolding in 9 m urea, the maximum shifted to 348.6 nm with an accompanying increase in fluorescence intensity (Fig. 3a). On the other hand, at pH 3.0 the native form had a maximum at 329.0 nm and maximal intensity decreased upon unfolding (Fig. 3b). It is intriguing that the fluorescence intensity of the native form at pH 3.0 is ∼1.5-fold higher than that at pH 7.0, although the exact reason why is unknown (36Mills O.E. Creamer L.K. Biochim. Biophys. Acta. 1975; 379: 618-626Crossref PubMed Scopus (52) Google Scholar, 37Renard D. Lefebvre J. Griffin M.C.A. Griffin W.G. Int. J. Biol. Macromol. 1998; 22: 41-49Crossref PubMed Scopus (145) Google Scholar). The β-lg mutants showed spectra very similar to those of wild-type β-lg at both pH 7.0 and pH 3.0 (data not shown). These results confirmed that the mutations do not affect the local environment of tryptophan residues. Thus, we succeeded in preparing mutants with a similar overall structure to the wild-type β-lg. Stability of Mutants—The stability of mutant proteins was studied at pH 7.0 and 3.0 by monitoring the change in fluorescence spectra in the presence of various concentrations of urea. Each fluorescence spectrum was fitted to a sum of the native and unfolded states, and the fractions of the native species were plotted against the urea concentration (Fig. 4, a and f). Transition curves were analyzed by a two-state mechanism assuming a linear dependence of the free energy change of unfolding, ΔGU, upon the concentration of urea to obtain ΔGUH2O (ΔGU in the absence of urea), m (the dependence of ΔGU on the concentration of urea), and cM (the midpoint urea concentration) (Table II).Table IIThermodynamic parameters of urea-induced unfolding transition of the wild-type β-lg and its mutants at 20 °CaErrors are fitting errorspHSpeciesΔGUH2OmcMkcal mol—1kcal mol—1m-1m7.0Wild type21.1 (±0.6)5.2 (±0.2)4.09C121A14.6 (±0.3)3.8 (±0.1)3.87C121S8.2 (±0.3)2.9 (±0.1)2.84C121V8.6 (±0.5)1.9 (±0.1)4.623.0Wild type41.2 (±1.2)8.8 (±0.2)4.67C121A36.2 (±1.3)8.7 (±0.3)4.18C121S18.3 (±0.6)5.7 (±0.2)3.21C121V21.1 (±0.3)4.8 (±0.1)4.38a Errors are fitting errors Open table in a new tab β-lg is more stable at acidic pH (Fig. 4f) than at neutral pH (Fig. 4a), although dimers of β-lg stable at neutral pH dissociate into monomers at acidic pH (7Kuwata K. Hoshino M. Era S. Batt C.A. Goto Y. J. Mol. Biol. 1998; 283: 731-739Crossref PubMed Scopus (96) Google Scholar, 8Kuwata K. Hoshino M. Forge V. Era S. Batt C.A. Goto Y. Protein Sci. 1999; 8: 2541-2545Crossref PubMed Scopus (156) Google Scholar, 10Sakai K. Sakurai K. Sakai M. Hoshino M. Goto Y. Protein Sci. 2000; 9: 1719-1729PubMed Google Scholar, 11Sakurai K. Oobatake M. Goto Y. Protein Sci. 2001; 10: 2325-2335Crossref PubMed Scopus (151) Google Scholar). This property was conserved among the mutants. The apparent transition curves (Fig. 4, a and f) as well as the obtained thermodynamic parameters (Table II) showed that wild-type β-lg and C121A have relatively similar values at both neutral and acidic pH. On the other hand, C121S revealed a decrease of cM and m values at both pH 7.0 and 3.0, resulting in remarkably lower values of ΔGUH2O. In the case of C121V, the cM value was similar and slightly higher than that of the wild-type β-lg at pH 3.0 and 7.0, respectively. Because of the decrease in the cooperativity of unfolding, the ΔGUH2O value was decreased significantly at both pH conditions. Analytical Ultracentrifugation—β-lg is a dimer at neutral pH. We determined the dimerization constant (KD) as a probe to monitor the subtle conformational change produced by mutation. Using the data on sedimentation equilibrium, the KD values were calculated for the mutants (Table I). The values of the mutants were similar to the KD of wild-type β-lg. The results showed that the monomer-dimer equilibria of the mutant β-lg species are essentially the same as that of the wild-type β-lg, confirming that the mutations did not affect the overall structure. In accordance with this finding, when retinol binding was examined by the quenching of tryptophan fluorescence upon retinol binding at pH 7.0, the mean dissociation constant was 0.054 μm for wild-type β-lg and 0.037 μm for C121S, indicating that the mutation did not affect the ligand binding. 2C. Kalidas and C. A. Batt, unpublished results. Refolding Measurements—The reversibility of denaturation in 8 m urea at pH 7.0 for 2 days was examined with the wild-type (Fig. 2b) and three β-lg mutants (Fig. 2, c–e)

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