High Order Quaternary Arrangement Confers Increased Structural Stability to Brucella sp. Lumazine Synthase
2004; Elsevier BV; Volume: 279; Issue: 9 Linguagem: Inglês
10.1074/jbc.m312035200
ISSN1083-351X
AutoresVanesa Zylberman, Patricio O. Craig, Sebastián Klinke, Bradford C. Braden, Ana Cauerhff, Fernando A. Goldbaum,
Tópico(s)RNA and protein synthesis mechanisms
ResumoThe penultimate step in the pathway of riboflavin biosynthesis is catalyzed by the enzyme lumazine synthase (LS). One of the most distinctive characteristics of this enzyme is the structural quaternary divergence found in different species. The protein exists as pentameric and icosahedral forms, built from practically the same structural monomeric unit. The pentameric structure is formed by five 18-kDa monomers, each extensively contacting neighboring monomers. The icosahedrical structure consists of 60 LS monomers arranged as 12 pentamers giving rise to a capsid exhibiting icosahedral 532 symmetry. In all lumazine synthases studied, the topologically equivalent active sites are located at the interfaces between adjacent subunits in the pentameric modules. The Brucella sp. lumazine synthase (BLS) sequence clearly diverges from pentameric and icosahedric enzymes. This unusual divergence prompted us to further investigate its quaternary arrangement. In the present work, we demonstrate by means of solution light scattering and x-ray structural analyses that BLS assembles as a very stable dimer of pentamers, representing a third category of quaternary assembly for lumazine synthases. We also describe by spectroscopic studies the thermodynamic stability of this oligomeric protein and postulate a mechanism for dissociation/unfolding of this macromolecular assembly. The higher molecular order of BLS increases its stability 20 °C compared with pentameric lumazine synthases. The decameric arrangement described in this work highlights the importance of quaternary interactions in the stabilization of proteins. The penultimate step in the pathway of riboflavin biosynthesis is catalyzed by the enzyme lumazine synthase (LS). One of the most distinctive characteristics of this enzyme is the structural quaternary divergence found in different species. The protein exists as pentameric and icosahedral forms, built from practically the same structural monomeric unit. The pentameric structure is formed by five 18-kDa monomers, each extensively contacting neighboring monomers. The icosahedrical structure consists of 60 LS monomers arranged as 12 pentamers giving rise to a capsid exhibiting icosahedral 532 symmetry. In all lumazine synthases studied, the topologically equivalent active sites are located at the interfaces between adjacent subunits in the pentameric modules. The Brucella sp. lumazine synthase (BLS) sequence clearly diverges from pentameric and icosahedric enzymes. This unusual divergence prompted us to further investigate its quaternary arrangement. In the present work, we demonstrate by means of solution light scattering and x-ray structural analyses that BLS assembles as a very stable dimer of pentamers, representing a third category of quaternary assembly for lumazine synthases. We also describe by spectroscopic studies the thermodynamic stability of this oligomeric protein and postulate a mechanism for dissociation/unfolding of this macromolecular assembly. The higher molecular order of BLS increases its stability 20 °C compared with pentameric lumazine synthases. The decameric arrangement described in this work highlights the importance of quaternary interactions in the stabilization of proteins. Riboflavin, an essential cofactor for all organisms, is biosynthesized in plants, fungi, and microorganisms. The penultimate step in the pathway is catalyzed by the enzyme lumazine synthase (LS). 1The abbreviations used are: LS, lumazine synthase; BLS, Brucella sp. lumazine synthase; CD, circular dichroism; TF, tryptophan fluorescence; ANS, 1-anilino-8-naphthalene sulfonic acid; SLS, static light scattering; SEC, size exclusion chromatography column; LEM, lineal extrapolation method; GdnHCl, guanidine hydrochloride; M̄w, weight average molecular weight; DTT, dithiothreitol; ASA, accessible surface area. 1The abbreviations used are: LS, lumazine synthase; BLS, Brucella sp. lumazine synthase; CD, circular dichroism; TF, tryptophan fluorescence; ANS, 1-anilino-8-naphthalene sulfonic acid; SLS, static light scattering; SEC, size exclusion chromatography column; LEM, lineal extrapolation method; GdnHCl, guanidine hydrochloride; M̄w, weight average molecular weight; DTT, dithiothreitol; ASA, accessible surface area. One of the most distinctive characteristics of this enzyme is the structural quaternary divergence found in different species. The protein exists as pentameric and icosahedral forms, built from practically the same structural monomeric unit. The structure of the monomer consists of four repeated β-strand/α-helix motifs producing a sandwich of four parallel β-strands surrounded by four α-helices, two on each face of the β sheet (1Ladenstein R. Schneider M. Huber R. Bartunik H.D. Wilson K. Schott K. Bacher A. J. Mol. Biol. 1988; 203: 1045-1070Crossref PubMed Scopus (125) Google Scholar). In all LS studied, the topologically equivalent active sites are located at the interfaces between adjacent subunits in the pentameric modules (2Gerhardt S. Haase I. Steinbacher S. Kaiser J.T. Cushman M. Bacher A. Huber R. Fischer M. J. Mol. Biol. 2002; 318: 1317-1329Crossref PubMed Scopus (60) Google Scholar). Bacilliaceae express a bifunctional enzyme complex with lumazine synthase and riboflavin synthase activity. Three α-subunits (riboflavin synthase) enclosed by 60 β-subunits (lumazine synthase) form a protein particle of ∼1 MDa (1Ladenstein R. Schneider M. Huber R. Bartunik H.D. Wilson K. Schott K. Bacher A. J. Mol. Biol. 1988; 203: 1045-1070Crossref PubMed Scopus (125) Google Scholar, 3Bacher A. Methods Enzymol. 1986; 122: 192-199Crossref PubMed Scopus (44) Google Scholar). The three-dimensional structure of the lumazine synthase/riboflavin synthase from Bacillus subtilis complexed with a substrate analogue has been determined (4Ritsert K. Huber R. Turk D. Ladenstein R. Schmidt-Base K. Bacher A. J. Mol. Biol. 1995; 253: 151-167Crossref PubMed Scopus (112) Google Scholar). This structure consists of 60 β-subunits (lumazine synthase monomers) arranged as 12 pentamers giving rise to a capsid exhibiting icosahedral 532 symmetry. Two other icosahedric LS have been described by x-ray crystallography. Spinach and thermophilic bacterial Aquifex aelicus LS also exhibit icosahedral 532 symmetry. The three proteins form 1-MDa spherical capsids of 60 LS subunits with icosahedral symmetry. The icosahedral LS structures superimpose very well, highlighting the conservation of the overall folding and quaternary arrangement despite the low sequence homology between them (5Zhang X. Meining W. Fischer M. Bacher A. Ladenstein R. J. Mol. Biol. 2001; 306: 1099-1114Crossref PubMed Scopus (146) Google Scholar). Four structurally characterized LS assemble as pentamers in their native form and do not further associate to form an icosahedral capsid. These include fungal Magnaporthe grisea, yeast Saccharomyces cerevisiae, and Schizosaccharomyces pombe and bacterial Brucella abortus LS (5Zhang X. Meining W. Fischer M. Bacher A. Ladenstein R. J. Mol. Biol. 2001; 306: 1099-1114Crossref PubMed Scopus (146) Google Scholar). Although the four pentameric enzymes fold in a similar arrangement, the postulated reasons for the lack of icosahedral order differ. Superposition of the pentameric LS shows that the loop connecting the helices α4 and α5 is critical for preventing the formation of capsids. In all icosahedral LS a pentapeptide kink located in this loop is essential for pentamer-pentamer contacts (5Zhang X. Meining W. Fischer M. Bacher A. Ladenstein R. J. Mol. Biol. 2001; 306: 1099-1114Crossref PubMed Scopus (146) Google Scholar, 6Braden B.C. Velikovsky C.A. Cauerhff A.A. Polikarpov I. Goldbaum F.A. J. Mol. Biol. 2000; 297: 1031-1036Crossref PubMed Scopus (51) Google Scholar). The yeast and fungal pentameric enzymes have insertions of different length in this loop that change its overall orientation and disrupt potential contacts to neighboring subunits (7Meining W. Mortl S. Fischer M. Cushman M. Bacher A. Ladenstein R. J. Mol. Biol. 2000; 299: 181-197Crossref PubMed Scopus (82) Google Scholar). We have previously analyzed the divergence in macromolecular assembly between pentameric and icosahedral LS enzymes. In this regard, a high degree of divergence was observed between the sequence from B. abortus LS and the other pentameric structurally characterized members of the family (8Fornasari M.S. Laplagne D.A. Frankel N. Cauerhff A.A. Goldbaum F.A. Echave J. Mol. Biol. Evol. 2003; 10.1093/molbev/msg1244PubMed Google Scholar). BLS has a 3-residue insertion between helices α4 and α5 that contribute to form a continuous undistorted helix, unable to form the capsid-stabilizing kink (6Braden B.C. Velikovsky C.A. Cauerhff A.A. Polikarpov I. Goldbaum F.A. J. Mol. Biol. 2000; 297: 1031-1036Crossref PubMed Scopus (51) Google Scholar). Thus, the BLS structure clearly diverges from pentameric and icosahedric enzymes because of the lack of this critical loop. The different orientation of this straight helix is compensated by a longer loop bridging the helix structure with the contiguous sheet. This unusual divergence prompted us to further investigate the quaternary arrangement of BLS. In the present work, we demonstrate by means of solution light scattering and x-ray structural analysis that BLS assembles as a very stable dimer of pentamers, representing a third category of quaternary assembly for LS. We also describe, by spectroscopic studies, the thermodynamic stability of this oligomeric protein and postulate a mechanism for dissociation/unfolding of its macromolecular assembly. The Brucella sp. LS gene was cloned in pET11a vector (Novagen) as reported previously (9Goldbaum F.A. Velikovsky C.A. Baldi P.C. Mortl S. Bacher A. Fossati C.A. J. Med. Microbiol. 1999; 48: 833-839Crossref PubMed Scopus (59) Google Scholar). The plasmid was used to transform BL21(DE3) strain Escherichia coli-competent cells (Stratagene, La Jolla, CA). Ampicillin-resistant colonies were grown until A600 = 1.0 in LB medium containing 100 μg/ml ampicillin, at 37 °C with agitation (300 rpm). Five milliliters of this culture was diluted to 500 ml and grown to reach an A600 of 1.0. At this point the culture was induced adding 1 mm isopropyl-1-thio-β-d-galactopyranoside and incubated for 4 h at 37 °C with agitation (300 rpm). The bacteria were centrifuged at 15,000 × g during 20 min at 4 °C. BLS protein was successfully expressed as inclusion bodies by transformation of strain BL21(DE3) E. coli-competent cells (9Goldbaum F.A. Velikovsky C.A. Baldi P.C. Mortl S. Bacher A. Fossati C.A. J. Med. Microbiol. 1999; 48: 833-839Crossref PubMed Scopus (59) Google Scholar). The inclusion bodies were solubilized in 50 mm Tris, 5 mm EDTA, 8 m urea pH 8.0 at room temperature overnight with agitation. The solubilized material was refolded by dialysis during 72 h against phosphate-buffered saline containing 1 mm dithiothreitol. This preparation was purified in a Mono-Q column in a fast-protein liquid chromatography apparatus (Amersham Biosciences, Uppsala, Sweden) using a linear gradient of buffer B (50 mm Tris, 1 m NaCl, pH 8.5). The peak enriched with BLS was further purified on a Superdex-200 column with phosphate-buffered saline buffer, 1 mm DTT. The purity of the BLS preparation was determined on SDS-15% (w/v) polyacrylamide gels. Purified BLS was concentrated (10 mg/ml), frozen in liquid N2, and stored at -20 °C. BLS samples were diluted in 50 mm sodium phosphate, pH 7.0, 1 mm DTT, with increasing concentration of denaturants (urea or GdnHCl). All experiments were performed at 25 °C, and samples were incubated at least 2 h before taking CD measurements. Spectra were measured on a spectropolarimeter (JASCO J-810) using either 0.1- or 0.5-cm path length quartz cells. Unfolding was monitored by far-UV CD (260–200 nm) and expressed as the percentage of molar ellipticity at 222 nm as a function of denaturant concentration. The molar ellipticity of the protein incubated without GdnHCl was taken as 100%. For renaturation tests, BLS was first denatured in high concentration of GdnHCl (6 m), and renaturation was induced by overnight dialysis against 50 mm sodium phosphate, pH 7.0, 1 mm DTT. After this treatment, the CD and SLS signals typical of the native protein were recovered. Emission spectra were carried out by excitation of the samples at 295 nm, and data collection from 300 to 450 nm, using 3-nm band passes for both excitation and emission. Experiments were carried out in 50 mm sodium phosphate, pH 7.0, 1 mm DTT in the presence of increasing concentrations of urea or GdnHCl. Samples were incubated for at least 2 h prior to taking fluorescence measurements. All fluorescence emission spectra were measured at 25 °C on a Jasco FP-770 spectrofluorometer. BLS samples were incubated in 50 mm sodium phosphate, 1 mm DTT, pH 7.0 (in the presence or absence of 2 m GdnHCl) or in 50 mm buffer citrate, 1 mm DTT, pH 4.5. Thermal denaturation was conducted by slowly increasing the temperature with a Peltier system (Jasco). The range of temperature scanning was 25–95 °C at a speed of 4 °C/min. Molar ellipticity at 220 nm was measured every 0.5 °C. Fast or slow cooling back to 25 °C (from 95 to 25 °C at a speed of 1 °C/min) did not show a recovery of ellipticity demonstrating the irreversibility of the thermal unfolding. Thus the temperature midpoint of the thermal transition was considered as an apparent Tm. pH-induced BLS dissociation and unfolding was evaluated by diluting protein samples (5 μm monomers) in 50 mm citrate, 1 mm DTT, pH 6.0–2.5 or in 50 mm sodium phosphate, 1 mm DTT, pH 7.5-7.0. After 1–2 h of incubation at room temperature, CD and SLS signals were determined. ANS fluorescence emission was measured in the same buffer conditions as CD and SLS experiments, aggregating to the samples 50 μm ANS. Values were normalized to ANS fluorescence emission in the same buffer. Excitation and emission were set at 365 and 470 nm, respectively. The weight average molecular weight (Mw) of BLS under different conditions was determined on a Precision Detectors PD2010 light-scattering instrument tandemly connected to an high-performance liquid chromatography system and an LKB 2142 differential refractometer. In general, 20–100 μl of LS (0.3–1 mg/ml) was loaded on a Superdex 200 HR-10/30 (24 ml) or a Sephadex G-25 (1 ml) column and eluted with 50 mm phosphate buffer, 1 mm DTT under different pH, urea, GdnHCl, and NaCl conditions. The 90° light scattering and refractive index signals of the eluting material were recorded on a PC computer and analyzed with the Discovery32 software supplied by Precision Detectors. The 90° light scattering detector was calibrated using bovine serum albumin (M̄w: 66.5 kDa) as a standard. Prior to the injection in a size exclusion chromatography column (SEC), each sample was preincubated for 1–2 h at room temperature in the elution buffer. Thermodynamics evaluation of GdnHCl-induced unfolding of BLS was fitted to a two-step model: N10↔Ku1(2)N5↔Ku25U. The first step (N10 ↔ 2N5) represents the dissociation of decameric BLS (N10) in two folded pentamers (N5), whereas the second step (N5 ↔ 5U) represents the concomitant dissociation and unfolding of the pentameric structure (N5) in monomeric subunits (U). Because both steps are well resolved from each other, their thermodynamic parameters were independently analyzed using the lineal extrapolation method (Equation 1) assuming a two-state transition model for each step (10Greene Jr., R.F. Pace C.N. J. Biol. Chem. 1974; 249: 5388-5393Abstract Full Text PDF PubMed Google Scholar), ΔGU=ΔGH2O-m[D](Eq. 1) where ΔGU is the free energy of unfolding of a protein at a given denaturant concentration, ΔGH2O is the free energy of unfolding in the absence of denaturant, and m the dependence of the free energy on denaturant concentration ([D]). Step 1—The first step was monitored by SLS in the range of 1.5–2.2 m GdnHCl. The concentration at equilibrium of the N10 and N5 species of BLS were calculated using Equations 4 and 5 derived from rearrangement of Equations 2 and 3, (Eq. 1) ccT=ccN10+ccN5(Eq. 1) (Eq. 1) (Eq. 1) where M̄w represents the weight average molecular weight of BLS. ccN10, ccN5, and ccT represent the decamer, pentamer, and total protein concentration in millgrams/ml, respectively. [N10] and [N5] represent the molar concentration and M̄w10 and M̄w5 the molecular weight of the decameric (174.4 kDa) and pentameric (87.2 kDa) species of BLS, respectively. The equilibrium constant KU(1) and the free energy change ΔGU(1) for Step 1 are defined in Equations 6 and 7. KU(1)=[N5]2/[N10](Eq. 1) ΔGU(1)=-RTlnKU(1)(Eq. 1) The ΔGH2O(1) and m(1) values (Equation 1) for this transition were obtained from the extrapolation to zero denaturant concentration and from the slope of the linear regression fit of the ΔGU(1) values calculated as a function of GdnHCl concentration in the range of 1.5 to 2.2 m. The dissociation constant of the decameric arrangement (KD) was estimated from the ΔGH2O value of this transition using the equation, KD = e(-ΔGH2O/(RT)). Step 3—The second step was monitored by CD and FT in the range of 2.4 and 3.5 m GdnHCl. The equilibrium constant KU(2) and the free energy change ΔGU(2) for this transition are defined in Equations 8 and 9. KU(2)=[U]5/[N5](Eq. 1) ΔGU(2)=-RTlnKU(2)(Eq. 1) The total protein concentration in monomer units (PT) and the fractional population in the native (FN) and unfolded (FU) states were calculated using Equations 10, 11, 12, PT=5[N5]+[U](Eq. 1) FN=5[N5]/PT(Eq. 1) FU=1-FN=[U]/PT=(mf[D]+F)-Y/[(mf[D]+F)-(mu[D]-U)](Eq. 1) where Y is the experimental spectroscopic value, [D] is the GdnHCl concentration, F and U are the intercepts, and mf and mu are the slopes of the pre- and post-unfolding baselines, respectively. Combining Equations 1 and 8, 9, 10, 11, 12, we obtain the general equation (Equation 13) as follows. 5(mf[D]+F)-Y(mf[D]+F)-(mu[D]+U)5PT4+(mf[D]+F)-Y(mf[D]+F)-(mu[D]+U)-1e(ΔGH2O-m(2)[D])/RT=0(Eq. 1) ΔGH2O(2) and m(2) values of Step 2 were obtained fitting the experimental data measured by CD and FT to Equation 13 by a non-linear square fit method. The midpoint of this transition [D]50% was calculated as in Equation 14. [D]50%=[RTln(5/16PT4)+ΔGH2O]/m(Eq. 1) In addition to the previously obtained BLS crystals, which led to the resolution of its three-dimensional structure (6Braden B.C. Velikovsky C.A. Cauerhff A.A. Polikarpov I. Goldbaum F.A. J. Mol. Biol. 2000; 297: 1031-1036Crossref PubMed Scopus (51) Google Scholar), two further crystal forms of BLS were obtained in this work by means of the hanging drop, vapor diffusion method. The first form was diamond-like crystals obtained using 30% (w/v) polyethylene glycol 400, 0.1 m sodium acetate in 0.1 m MES buffer, pH 6.5, which diffracted to 2.9 Å and belongs to the trigonal space group P3121. Furthermore, we obtained plate-like crystals using 12% (w/v) polyethylene glycol 4000, 0.1 m sodium acetate in 0.1 m HEPES buffer, pH 7.5, which diffracted to 3.0 Å and belongs to the monoclinic space group P21. X-ray diffraction data were collected both at our in-house x-ray source, a Bruker M18XH6 MAC Science rotating anode interfaced to a Siemens X-1000 multiwire area detector and at the D03B protein crystallography beamline at the Laboratorio Nacional de Luz Síncrotron, Campinas, Brazil (11Polikarpov I. Oliva G. Castellano E.E. Garratt R.C. Arruda P. Leite A. Craievich A. Nucl. Instruments Methods Physics Res. A. 1998; 405: 159-164Crossref Scopus (59) Google Scholar). Data reduction and processing were carried out with the programs MOSFLM, Scala, and Truncate from the CCP4 suite (12Navaza J.A.C. Acta Crystallogr. Sect. A. 1994; 50: 157-163Crossref Scopus (5027) Google Scholar). Crystal packings were determined using the molecular replacement procedure as implemented in the AMoRe package (12Navaza J.A.C. Acta Crystallogr. Sect. A. 1994; 50: 157-163Crossref Scopus (5027) Google Scholar), with the previously solved BLS structure as a search model. Crystallographic symmetry construction was carried out using the program O (13Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13006) Google Scholar). Total accessible surface area and buried surface areas of interaction (ΔASA) between monomers and pentamers in pentamer and decamer structures, respectively, were calculated with Surface Racer 1.2 program (14Tsodikov O.V. Record Jr., M.T. Sergeev Y.V. J. Comput. Chem. 2002; 23: 600-609Crossref PubMed Scopus (347) Google Scholar) using an implementation of the Lee and Richards (15Lee B. Richards F.M. J. Mol. Biol. 1971; 55: 379-400Crossref PubMed Scopus (5319) Google Scholar) algorithm and a probe radius of 1.7 Å. Intermolecular polar and non-polar interactions were calculated with the Molmol 2k.2 (16Koradi R. Billeter M. Wuthrich K. J. Mol. Graph. 1996; 14 (29-32): 51-55Crossref PubMed Scopus (6477) Google Scholar) and Contacts of Structural Units (17Sobolev V. Sorokine A. Prilusky J. Abola E.E. Edelman M. Bioinformatics. 1999; 15: 327-332Crossref PubMed Scopus (705) Google Scholar) programs. Any pair of atoms is considered to be in contact if the distance between them is less than 4 Å. BLS Is a Stable Dimer of Pentamers in Solution—Previous studies (6Braden B.C. Velikovsky C.A. Cauerhff A.A. Polikarpov I. Goldbaum F.A. J. Mol. Biol. 2000; 297: 1031-1036Crossref PubMed Scopus (51) Google Scholar, 9Goldbaum F.A. Velikovsky C.A. Baldi P.C. Mortl S. Bacher A. Fossati C.A. J. Med. Microbiol. 1999; 48: 833-839Crossref PubMed Scopus (59) Google Scholar) have shown that BLS does not assemble as a 1-MDa capsid oligomer, having a retention time in SEC compatible with an apparent molecular mass of 90 kDa. This behavior suggested that the oligomeric structure of this protein in solution was a pentamer. However, the estimation of the molecular weight of a protein by its retention time on a SEC column is a method prone to artifacts. Additionally, the sequence divergence of BLS, compared with other pentameric LS, prompted us to re-analyze the quaternary structure of the protein by spectroscopic techniques such as static light scattering (SLS). SLS experiments show that the protein has a molecular mass of 180 kDa in solution, corresponding to an assembly of two pentamers (decameric arrangement of the 18-kDa polypeptide chain). To characterize this new quaternary arrangement, we studied the thermodynamic stability of BLS, evaluating the unfolding of the protein induced by the common chemical denaturants (urea and GdnHCl) and pH. Preincubation of BLS with increasing concentrations of urea shows that the enzyme remains as a stable dimer of pentamers, with no detectable change in its quaternary structure (180 kDa, determined by SLS, data not shown) as well as in its tertiary and secondary structures as followed by tryptophan fluorescence and CD (Fig. 1). The absence of structural changes, even in 8 m urea, indicates that the quaternary arrangement of BLS is very stable. Conversely, GdnHCl produces a cooperative and reversible change in the tertiary structure reflected by a decrease in tryptophan fluorescence emission (Fig. 1A). This measure senses the environment of Trp-22, the unique tryptophan in BLS monomers that is located on the active site at the monomer-monomer interface (6Braden B.C. Velikovsky C.A. Cauerhff A.A. Polikarpov I. Goldbaum F.A. J. Mol. Biol. 2000; 297: 1031-1036Crossref PubMed Scopus (51) Google Scholar). In addition, GdnHCl incubation (6 m) produces a complete loss of secondary structure of BLS as monitored by CD spectra (Fig. 1B). The differential effect of GdnHCl and urea cannot be explained by their differences in ionic strength. The behavior of BLS in 8.0 m urea and in the presence of 1 m NaCl is superimposable with its described stability in absence of salt (data not shown), implying that unfolding with GdnHCl seems to be due to more specific interactions of the guanidinium cation with the protein (18Botelho M.G. Gralle M. Oliveira C.L. Torriani I. Ferreira S.T. J. Biol. Chem. 2003; 278: 34259-34267Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 19Kohn W.D. Monera O.D. Kay C.M. Hodges R.S. J. Biol. Chem. 1995; 270: 25495-25506Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Thus, we used GdnHCl-induced denaturation to study the mechanism of BLS unfolding. Dissociation of BLS Dimer of Pentamers Is Tight and Precedes the Unfolding of the Pentamer—SLS analysis of the GdnHCl-induced unfolding of BLS shows a biphasic behavior (Fig. 2A). In the first step, observed between 1.5 and 2.2 m GdnHCl, the SLS signal intensity of the protein is reduced to half the value measured in the absence of denaturant. However, no changes in the far-UV CD spectra and tryptophan fluorescence of the protein are observed in this range (Figs. 1A and 2A). Thus, intrinsic tryptophan fluorescence is completely insensitive to the change in the quaternary structure of the protein, indicating that the dissociation does not modify significantly the environment of Trp-22. These results point to the existence of a dissociation phenomenon of the decameric structure of BLS into two pentameric subunits, with no further changes in tertiary and secondary structure. On the other hand, the second step observed between 2.4 and 3.5 m GdnHCl, shows a 5-fold decrease in the SLS signal of BLS concomitantly to the disappearance of its far-UV CD signal at 222 nm, and a significant decrease in the fluorescence intensity of its single tryptophan residue (Figs. 1A and 2A). These results are interpreted as an unfolding and loss of the tertiary and secondary structures of the protein coupled to the dissociation of its pentameric arrangement in five monomeric subunits (18 kDa). The overlapping of the changes observed in the second step by SLS, CD (Fig. 2A) and fluorescence (Fig. 1A) clearly supports our model. These results were further verified by SLS coupled to gel filtration chromatography (Fig. 2B), where it was possible to isolate a 90-kDa intermediate at 2 m GdnHCl. The fact that an intermediate of 90 kDa is stable enough to be detected by this methodology suggests that the decameric assembly is composed of two previously associated pentamers. The dissociation of the decamer to folded pentamers is also observed when BLS is incubated at acidic pH (Fig. 3). This phenomenon is evidenced by a 2-fold decrease in the SLS signal intensity in the range of pH 4.0–5.0 as compared with the value measured at pH 7.0. ANS binds to exposed hydrophobic surfaces in partially folded intermediates with higher affinity than to native or completely unfolded proteins (20Semisotnov G.V. Rodionova N.A. Razgulyaev O.I. Uversky V.N. Gripas A.F. Gilmanshin R.I. Biopolymers. 1991; 31: 119-128Crossref PubMed Scopus (1228) Google Scholar, 21Daniel E. Weber G. Biochemistry. 1966; 5: 1893-1900Crossref PubMed Scopus (295) Google Scholar, 22D'Alfonso L. Collini M. Baldini G. Biochim. Biophys. Acta. 1999; 1432: 194-202Crossref PubMed Scopus (57) Google Scholar). This binding result in a marked increase in fluorescence emission compared with the free ANS. We found that the dissociation of BLS at pH in the range 6.0–4.0 is a reversible process that occurs without significant exposure of hydrophobic patches and changes in secondary structure as monitored by ANS fluorescence and CD spectra (Fig. 3). At pHs below 4.0 the protein shows an irreversible unfolding, evidenced by a loss of ellipticity at 222 nm and a dramatic increase in binding to ANS. Clearly, both changes are coupled, implying that at acidic pHs the protein exposes hydrophobic surfaces when secondary structure is lost. The light scattering response cannot be accurately monitored below pH 4.0, presumably because of aggregation. Thermodynamic Stability of BLS Measured by Chemical Denaturation—To describe the thermodynamic stability of BLS, we studied the dissociation and unfolding steps taking advantage of the differential effect of GdnHCl at distinct concentration ranges. Both transitions (Steps 1 and 2) were shown to be highly cooperative and reversible. The dissociation step was characterized by SLS using GdnHCl up to 2.2 m, in a condition that does not disturb the tertiary and secondary structures of the pentamer. This assumption is supported by the facts that the circular dichroism spectrum is not modified (see Fig. 2A) and the intrinsic tryptophan fluorescence does not vary upon addition of GdnHCl (see Fig. 1A). The SLS determination of the equilibrium between the dimer of pentamers and the pentamer gives a ΔG of 90 ± 20 kJ/mol decamer as estimated by the LEM method assuming a two-state transition (see "Experimental Procedures"). This value indicates that the protein remains as a decamer under physiological conditions (estimated KD = 2.48 × 10-16m). On the other hand, the transition from folded pentamer to unfolded monomers is a highly cooperative process that can be measured by GdnHCl denaturation at higher concentrations followed by tryptophan fluorescence (Fig. 1A), SLS, and circular dichroism (Fig. 2A). These signals show a sharp and overlapping change around 2.4–3.5 m GdnHCl. Thus, all three spectroscopic analyses rule out the existence of a populated intermediate during this transition. The dependence of [D]50% with protein concentration (Table I) clearly supports the model of a two-state transition from a folded pentamer to unfolded monomers. Thermodynamic analysis of this equilibrium shows that the ΔG is 330 ± 30 kJ/mol pentamer (Table I; see "Experimental Procedures" for details). In agreement with this analysis, GdnHCl denaturation of BLS previously incubated at pH 5.0 (as a dissociated pentamer, see Fig. 3), gives a ΔG o
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