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• W2068370467 abstract "We investigated the effects of high hydrostatic pressure in the range of 1–3 kilobars on tetradecameric GroEL, heptameric GroES, and the GroEL-GroES complex. Unlike GroEL monomers formed by urea dissociation, which can be reassembled back to the tetradecamer, the pressure-dissociated monomers do not reassemble readily. This indicates an alteration of their native structures, an example of conformational drift. Pressure versus time profiles and kinetics of the dissociation of both GroEL and GroES at fixed pressures were monitored by light scattering. Unlike GroEL, GroES monomers do reassociate readily. Reaction conditions were varied by adding ATP, Mg2+, ADP, AMP-PNP, and KCl. At any individual pressure, the dissociation process is governed by both thermodynamics and kinetics. This leads to the decrease in the yield of monomers at lower pressures. In the presence of Mg2+ and KCl, GroEL is stable up to 3 kilobars. The presence of either ATP or ADP but not AMP-PNP leads to GroEL dissociation at lower pressures. Interestingly, the GroEL-GroES complex is very stable in the range of 1–2.5 kilobars. However, the addition of ADP destabilizes the complex, which dissociates completely at 1.5 kilobars. The results are rationalized in terms of different degrees of cooperativity between individual monomers and heptameric rings in the GroEL tetradecamer. Such allosteric interactions leading to the alteration of quaternary structure of GroEL in the absence of chemical denaturants are important in understanding the mechanism of chaperonin-assisted protein folding by the GroEL-GroES system. We investigated the effects of high hydrostatic pressure in the range of 1–3 kilobars on tetradecameric GroEL, heptameric GroES, and the GroEL-GroES complex. Unlike GroEL monomers formed by urea dissociation, which can be reassembled back to the tetradecamer, the pressure-dissociated monomers do not reassemble readily. This indicates an alteration of their native structures, an example of conformational drift. Pressure versus time profiles and kinetics of the dissociation of both GroEL and GroES at fixed pressures were monitored by light scattering. Unlike GroEL, GroES monomers do reassociate readily. Reaction conditions were varied by adding ATP, Mg2+, ADP, AMP-PNP, and KCl. At any individual pressure, the dissociation process is governed by both thermodynamics and kinetics. This leads to the decrease in the yield of monomers at lower pressures. In the presence of Mg2+ and KCl, GroEL is stable up to 3 kilobars. The presence of either ATP or ADP but not AMP-PNP leads to GroEL dissociation at lower pressures. Interestingly, the GroEL-GroES complex is very stable in the range of 1–2.5 kilobars. However, the addition of ADP destabilizes the complex, which dissociates completely at 1.5 kilobars. The results are rationalized in terms of different degrees of cooperativity between individual monomers and heptameric rings in the GroEL tetradecamer. Such allosteric interactions leading to the alteration of quaternary structure of GroEL in the absence of chemical denaturants are important in understanding the mechanism of chaperonin-assisted protein folding by the GroEL-GroES system. adenosine 5′-O-(thiotriphosphate) adenosine 5′-(β,γ-imino)triphosphate kilobar The bacterial chaperonin GroEL and its co-chaperonin GroES are multimeric proteins that assist folding of other proteins by preventing misfolding and aggregation. Their quaternary structures are crucial to the mechanism of chaperonin-assisted protein folding. These two proteins are coexpressed from a common GroE operon in Escherichia coli (1Georgopoulos C.P. Hendrix R.W. Casjens S.R. Kaiser A.D. J. Mol. Biol. 1973; 76: 45-60Crossref PubMed Scopus (274) Google Scholar, 2Sternberg N. J. Mol. Biol. 1973; 76: 1-23Crossref PubMed Scopus (51) Google Scholar, 3Sundaram S. Roth C.M. Yarmush M.L. Biotechnol. Prog. 1998; 14: 773-781Crossref PubMed Scopus (16) Google Scholar). Mutational studies have demonstrated that both chaperonins are essential for protein folding in vivo(4Cheng M.Y. Hartl F.U. Martin J. Pollock R.A. Kalousek F. Neupert W. Hallberg E.M. Hallberg R.L. Horwich A.L. Nature. 1989; 337: 620-625Crossref PubMed Scopus (675) Google Scholar, 5Fayet O. Ziegelhoffer T. Georgopoulos C. J. Bacteriol. 1989; 171: 1379-1385Crossref PubMed Scopus (546) Google Scholar, 6Horwich A.L. Low K.B. Fenton W.A. Hirshfield I.N. Furtak K. Cell. 1993; 74: 909-917Abstract Full Text PDF PubMed Scopus (294) Google Scholar). GroES is a single rotationally symmetric ring of seven identical 10-kDa subunits with a dome-shaped architecture (7Hunt J.F. Weaver A.J. Landry S.J. Gierasch L. Deisenhofer J. Nature. 1996; 379: 37-45Crossref PubMed Scopus (404) Google Scholar). GroEL is a tetradecamer (14-mer) of 57-kDa subunits arranged in two seven-membered rings stacked back to back to yield a cylindrical structure. There are no tryptophan residues, and each subunit contains three cysteines, Cys138, Cys458, and Cys519. The x-ray crystal structures of GroES (7Hunt J.F. Weaver A.J. Landry S.J. Gierasch L. Deisenhofer J. Nature. 1996; 379: 37-45Crossref PubMed Scopus (404) Google Scholar), GroEL (8Braig K. Adams P.D. Brunger A.T. Nat. Struct. Biol. 1995; 2: 1083-1094Crossref PubMed Scopus (230) Google Scholar), GroEL fully complexed with 14 ATPγS1 molecules (9Boisvert D.C. Wang J. Otwinowski Z. Horwich A.L. Sigler P.B. Nat. Struct. Biol. 1996; 3: 170-177Crossref PubMed Scopus (246) Google Scholar), and the GroEL-GroES-(ADP)7 complex (10Xu Z. Horwich A.L. Sigler P.B. Nature. 1997; 388: 741-750Crossref PubMed Scopus (1044) Google Scholar) are available in the literature. The GroEL crystal structure demonstrates that each monomer is folded into three distinct domains. First, the apical domain faces the solvent and forms the opening to the central channel and is the peptide-binding site; second, a highly helical equatorial domain is the ATP binding site and forms the inter- and intraring contacts; and third, a hingelike intermediate domain links the apical and equatorial domains. The GroEL-assisted protein folding reaction cycle consists of a number of sequential reactions, i.e. (i) binding of the polypeptide at the apical domains of the cis-ring; (ii) binding of seven molecules of ATP and GroES, forming a stable cis assembly and freeing the tightly bound polypeptide into an “Anfinson's cage” where it folds; (iii) hydrolysis of ATP; and (iv) release of ADP, GroES, and the folded polypeptide (10Xu Z. Horwich A.L. Sigler P.B. Nature. 1997; 388: 741-750Crossref PubMed Scopus (1044) Google Scholar, 11Lorimer G. Nature. 1997; 388 (, 723): 720-721Crossref PubMed Scopus (79) Google Scholar, 12Xu Z. Sigler P.B. J. Struct. Biol. 1998; 124: 129-141Crossref PubMed Scopus (102) Google Scholar). In a recent investigation involving a GroEL containing binding-defective mutant apical domains, Horwich et al. (13Farr G.W. Furtak K. Rowland M.B. Ranson N.A. Saibil H.R. Kirchhausen T. Horwich A.L. Cell. 2000; 100: 561-573Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar) demonstrated that binding of polypeptides to the apical domain of GroEL requires a minimum of three binding-proficient apical domains for stringent substrate proteins, such as malate dehydrogenase and Rubisco, while only two were required for binding a less stringent substrate such as rhodanese (13Farr G.W. Furtak K. Rowland M.B. Ranson N.A. Saibil H.R. Kirchhausen T. Horwich A.L. Cell. 2000; 100: 561-573Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). In addition to GroES and ATP, the presence of Mg2+ and K+ is also necessary for the GroEL-assisted folding (14Buchner J. Schmidt M. Fuchs M. Jaenicke R. Rudolph R. Schmid F.X. Kiefhaber T. Biochemistry. 1991; 30: 1586-1591Crossref PubMed Scopus (413) Google Scholar, 15Goloubinoff P. Christeller J.T. Gatenby A.A. Lorimer G.H. Nature. 1989; 342: 884-889Crossref PubMed Scopus (552) Google Scholar, 16Laminet A.A. Ziegelhoffer T. Georgopoulos C. Pluckthun A. EMBO J. 1990; 9: 2315-2319Crossref PubMed Scopus (202) Google Scholar, 17Martin J. Langer T. Boteva R. Schramel A. Horwich A.L. Hartl F.U. Nature. 1991; 352: 36-42Crossref PubMed Scopus (726) Google Scholar, 18Viitanen P.V. Lubben T.H. Reed J. Goloubinoff P. O'Keefe D.P. Lorimer G.H. Biochemistry. 1990; 29: 5665-5671Crossref PubMed Scopus (317) Google Scholar). The role of ATP is important both as an energy source and an allosteric effector. It has been suggested that ATP binding displays both intraring positive cooperativity and interring negative cooperativity (19Todd M.J. Viitanen P.V. Lorimer G.H. Science. 1994; 265: 659-666Crossref PubMed Scopus (429) Google Scholar, 20Yifrach O. Horovitz A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1521-1524Crossref PubMed Scopus (55) Google Scholar). The conformational changes attributed to the binding of Mg2+, ADP, and AMP-PNP with GroEL have been investigated from the stability of such complexes as assessed by urea dissociation, followed by both light scattering and intrinsic tyrosine fluorescence (21Gorovits B.M. Horowitz P.M. J. Biol. Chem. 1995; 270: 28551-28556Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). The results indicate that the stabilities decrease in the following order: GroEL-Mg complex > GroEL > GroEL-Mg-AMP-PNP complex > GroEL-Mg-ADP complex. The binding of ATP has been suggested to destabilize the quaternary structure of GroEL (22Horovitz A. Bochkareva E.S. Kovalenko O. Girshovich A.S. J. Mol. Biol. 1993; 231: 58-64Crossref PubMed Scopus (58) Google Scholar). From labeling of the three cysteines (Cys138, Cys458, and Cys519) of the GroEL 14-mer, it was demonstrated that the binding of adenine nucleotides induces specific changes in the conformation of the protein oligomer (23Jai E.A. Horowitz P.M. J. Protein Chem. 1999; 18: 387-396Crossref PubMed Scopus (8) Google Scholar). It is also interesting to note that labeling at Cys458 by fluorescein 5-maleimide (23Jai E.A. Horowitz P.M. J. Protein Chem. 1999; 18: 387-396Crossref PubMed Scopus (8) Google Scholar) or 4,4′-dithiopyridine (24Bochkareva E. Safro M. Girshovich A. J. Biol. Chem. 1999; 274: 20756-20758Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar) leads to the disassembly of GroEL. These conformational changes due to the binding of nucleotides regulate the exposure of hydrophobic surfaces on the 14-mer that have been suggested to be important for the binding of protein and GroES during assisted folding by GroEL. High hydrostatic pressure techniques are increasingly used as tools to study dissociation and unfolding of protein aggregates (25Weber G. Protein Interactions. Chapman and Hall Inc., New York1992Google Scholar, 26Silva J.L. Weber G. Annu. Rev. Phys. Chem. 1993; 44: 89-113Crossref PubMed Scopus (478) Google Scholar, 27Drljaca A. Hubbard C.D. van Eldik R. Asano T. Basilevsky M.V. Le Noble W.J. Chem. Rev. 1998; 98: 2167-2289Crossref PubMed Scopus (314) Google Scholar, 28Mozhaev V.V. Heremans K. Frank J. Masson P. Balny C. Proteins. 1996; 24: 81-91Crossref PubMed Scopus (636) Google Scholar) in the absence of externally added chaotropes. The effects of pressure on proteins are generally reversible. The important theories behind this technique and excellent experimental details can be found in several edited books and monographs (25Weber G. Protein Interactions. Chapman and Hall Inc., New York1992Google Scholar, 29Sherman W.F. Stadtmuller A.A. Experimental Techniques in High-pressure Research. Wiley, Chichester, United Kingdom1987Google Scholar, 30Jannasch H.W. Marquis R.E. Zimmerman A.M. Current Perspectives in High Pressure Biology. Academic Press, Inc., Orlando, FL1987Google Scholar, 31van Eldik R. Jonas J. NATO ASI Ser. (Adv. Sci. Inst.) Ser. C Math. Phys. Sci. 197. Reidel, Dordrecht, The Netherlands1987Google Scholar, 32Winter R. Jonas J. NATO ASI Ser. (Adv. Sci. Inst.) Ser. C Math. Phys. Sci. 401. Kluwer Academic Publishers, Dordrecht, The Netherlands1993Google Scholar, 33Markley J.L. Northrop D.B. Royer C. High-pressure Effects in Molecular Biophysics and Enzymology. Oxford University Press, New York1996Crossref Google Scholar, 34Holzapfel W.B. Isaacs N. High-pressure Techniques in Chemistry and Physics. Oxford, Oxford1997Google Scholar). At pressures lower than 4–5 kbar, oligomeric proteins or protein assemblies generally undergo reversible dissociation (35Gross M. Jaenicke R. Eur. J. Biochem. 1994; 221: 617-630Crossref PubMed Scopus (623) Google Scholar) with denaturation (25Weber G. Protein Interactions. Chapman and Hall Inc., New York1992Google Scholar, 35Gross M. Jaenicke R. Eur. J. Biochem. 1994; 221: 617-630Crossref PubMed Scopus (623) Google Scholar, 36Paladini Jr., A.A. Weber G. Biochemistry. 1981; 20: 2587-2593Crossref PubMed Scopus (208) Google Scholar). The resulting monomers may undergo conformational drifts away from their conformations in the oligomer and, therefore, may not reassociate rapidly upon depressurization. The application of higher hydrostatic pressure can cause many single chain proteins to denature. A combination of moderate pressure and low concentrations of chaotropes has been found to be suitable for studying the unfolding of proteins (37Sasahara K. Sakurai M. Nitta K. J. Mol. Biol. 1999; 291: 693-701Crossref PubMed Scopus (53) Google Scholar, 38Sasahara K. Nitta K. Protein Sci. 1999; 8: 1469-1474Crossref PubMed Scopus (23) Google Scholar) and for the recovery of proteins from aggregates (39St John R.J. Carpenter J.F. Randolph T.W. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13029-13033Crossref PubMed Scopus (159) Google Scholar). In addition to providing information on the nature of physical forces involved in the dissociation of oligomeric chaperonins, elucidation of the dissociation mechanism would provide insights into whether such structures would withstand high pressure in bacteria under the depths of the ocean and still be functional for assisting protein folding. Three causes have been suggested for the pressure-induced dissociation of oligomeric proteins (25Weber G. Protein Interactions. Chapman and Hall Inc., New York1992Google Scholar). The first cause is due to imperfect van der Waals contact between the participating monomers and the restriction of amino acid residues approaching too close to each other due to the repulsion of their of their electronic clouds. Such repulsion leads, even with optimal close packing, to creation of small “free volumes” or “dead spaces” (25Weber G. Protein Interactions. Chapman and Hall Inc., New York1992Google Scholar, 36Paladini Jr., A.A. Weber G. Biochemistry. 1981; 20: 2587-2593Crossref PubMed Scopus (208) Google Scholar). Therefore, upon the application of hydrostatic pressure, these small volumes will disappear because of better packing of the solvent against each dissociated subunit/monomer or the unfolded peptide chain. A second cause is due to the existence of salt linkages at the interfaces of monomers/subunits of oligomers, which upon dissociation are exposed and solvated, causing a decrease in volume of the system as a result of solvent electrostriction (25Weber G. Protein Interactions. Chapman and Hall Inc., New York1992Google Scholar). A third cause, which is less well established, is the solvation of nonpolar groups at boundaries of contact between the monomers in the oligomer (25Weber G. Protein Interactions. Chapman and Hall Inc., New York1992Google Scholar). In an earlier investigation, we reported that high hydrostatic pressure can dissociate GroEL tetradecamers (40Gorovits B.M. Raman C.S. Horowitz P.M. J. Biol. Chem. 1995; 270: 2061-2066Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). After depressurization, the monomers reassociated back to the oligomer very slowly with at 12of 150 h at 25 °C. The dissociation and association reactions were facilitated by Mg-ATP only if it was present during pressurization. From their reassociation properties, it has been demonstrated that the monomers formed by pressure dissociation of the 14-mer are different from those formed by the action of 2.5m urea (40Gorovits B.M. Raman C.S. Horowitz P.M. J. Biol. Chem. 1995; 270: 2061-2066Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). In the present investigation, we have studied the effects of high hydrostatic pressure on GroES and GroEL in both the absence and presence of Mg2+ and adenine nucleotides and on the isolated complex GroEL-GroES-(ADP)7. Although other divalent cations such as Ca2+ are known to stabilize macromolecular assemblies (41Bonafe C.F. Villas-Boas M. Suarez M.C. Silva J.L. J. Biol. Chem. 1991; 266: 13210-13216Abstract Full Text PDF PubMed Google Scholar, 42Bonafe C.F. Araujo J.R. Silva J.L. Biochemistry. 1994; 33: 2651-2660Crossref PubMed Scopus (35) Google Scholar), the role of Mg2+ as a functional ligand is unique for the GroEL-GroES system (14Buchner J. Schmidt M. Fuchs M. Jaenicke R. Rudolph R. Schmid F.X. Kiefhaber T. Biochemistry. 1991; 30: 1586-1591Crossref PubMed Scopus (413) Google Scholar, 15Goloubinoff P. Christeller J.T. Gatenby A.A. Lorimer G.H. Nature. 1989; 342: 884-889Crossref PubMed Scopus (552) Google Scholar, 16Laminet A.A. Ziegelhoffer T. Georgopoulos C. Pluckthun A. EMBO J. 1990; 9: 2315-2319Crossref PubMed Scopus (202) Google Scholar, 17Martin J. Langer T. Boteva R. Schramel A. Horwich A.L. Hartl F.U. Nature. 1991; 352: 36-42Crossref PubMed Scopus (726) Google Scholar, 18Viitanen P.V. Lubben T.H. Reed J. Goloubinoff P. O'Keefe D.P. Lorimer G.H. Biochemistry. 1990; 29: 5665-5671Crossref PubMed Scopus (317) Google Scholar). The results are rationalized in terms of different degrees of cooperativity between individual monomers and heptameric rings in the GroEL tetradecamer. GroEL and GroES were purified as described previously (43Staniforth R.A. Cortes A. Burston S.G. Atkinson T. Holbrook J.J. Clarke A.R. FEBS Lett. 1994; 344: 129-135Crossref PubMed Scopus (70) Google Scholar, 44Clark A.C. Hugo E. Frieden C. Biochemistry. 1996; 35: 5893-5901Crossref PubMed Scopus (79) Google Scholar). The GroEL-GroES-(ADP)7 complex was prepared and isolated according to the method of Lorimer et al. (19Todd M.J. Viitanen P.V. Lorimer G.H. Science. 1994; 265: 659-666Crossref PubMed Scopus (429) Google Scholar). Briefly, 10 μm GroEL (14-mer) was added to 20 μm GroES (7-mer) in 50 mm Tris, pH 7.8, 5.0 mmMgCl2, 1.0 mm dithiothreitol, 0.5 mm KCl, 0.1 mm EDTA, and 100 μmATP. The total reaction volume was 100 μl. After 1 min of reaction, 20 μl of 50%glycerol was added to the mixture; the complex was isolated from excess unbound nucleotide and excess GroES using a Sephacryl S-300 gel filtration column (1.0 × 17 cm; bed volume, 13.3 ml). The elution buffer was the same as the reaction buffer but lacking EDTA and nucleotide. The fractions corresponding to the complex were pooled and quantified by Bradford protein determination. The buffer solutions used in the investigation were filtered through 0.2-μm surfactant-free cellulose acetate membrane syringe filters (Nalgene). The 640-nm polystyrene microspheres (latex beads) were from Poly Sciences, Inc. (Warrington, PA). Tris buffer is suitable for pressure experiments because of the small pKadependence upon hydrostatic pressure (45Neuman Jr., R.C. Kauzman W. Zipp A. J. Phys. Chem. 1973; 77: 2687-2691Crossref Scopus (330) Google Scholar). SDS was from Bio-Rad. The high pressure cell and photon counting spectrofluorometer were from ISS Inc. (Champaign, IL). The stainless steel alloy cell with quartz windows can be pressurized up to 3 kbar. Protein samples for the experiments were filled in quartz bottles (1-ml volume) with pressure caps (provided by ISS). These bottles are placed in the metal bottle holder and immersed in the pressurizing fluid (spectroscopic grade ethanol). The high pressure generator was from Advanced Pressure Products (Ithaca, NY). The pressure generator is electronically controlled and programmable to obtain pressure gradients. The temperature of the high pressure cell was maintained by a circulating water bath. Two independent computers controlled the Advanced Pressure Products pressure generator and ISS spectrofluorometer. The pressure gradients were controlled by computer using a program written for the Advanced Pressure Products software. The pressure was increased in 0.1-kbar increments and held for 1 min between the successive steps. The generated data were imported to Origin software (version 6; Microcal Software, Northampton, MA) and analyzed. Kinetics experiments were done after the protein sample in the pressure cell (from ISS) was equilibrated (30 min) to the desired temperature. After equilibration, the fluorometer recording was turned on, followed by the pressure machine. Protein dissociation was followed by monitoring scattering at 400 nm (excitation and emission slits were 2 mm each). To reach the desired pressure, the rate of pressurization was controlled through the Advanced Pressure Products software. In typical experiments, to reach pressures of 1 kbar required 1 min, 2 kbar required 3 min, and 3 kbar required 4 min at a pump speed of 2.0. This introduces severe limitations on following kinetics that would occur in less than the time taken to achieve the target pressure. In some instances, we were able to pressurize and depressurize much faster than the indicated times, but in most cases, rapid pressure change caused damage by shattering the quartz windows or sample bottles. To ensure that the intensity changes were not contributed by dimension changes of the cell due to high pressure, controls were run with latex beads in the sample bottle. The scattering intensity increased slightly under the pressure gradient and then reversed back to the original intensity upon depressurization (data not shown). The data were truncated to take account of the time taken by the pressure cell to reach the target pressure. The rates were evaluated by fitting the data to either mono- or biexponential equations: Y =A1 * exp(−k1 *t) + A2 or Y =A1 * exp(−k1 *t) + A2 * exp(k2 * t) +k3, respectively. The independent variableY was the observed scattering intensity in counts/s after subtracting the scattering due to buffer. The pseudo-first order rate constants k1 and k2 and the amplitudes A1, A2, and A3 were obtained from iterative nonlinear least squares regression of the data using Origin software program (Microcal). The method for nondenaturing gel electrophoresis for the analysis of GroEL monomer and resolution of GroEL14 from GroES7-GroEL14 complexes on native gels has been described in an earlier publication (46Horowitz P.M. Lorimer G.H. Ybarra J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2682-2686Crossref PubMed Scopus (10) Google Scholar). The dissociation and reassociation of GroES by the application of high hydrostatic pressure of 2.5 kbar and depressurization to 1 bar followed by scattering is shown in Fig. 1. The top panel is without Mg2+, whereas in the experiment shown at the bottom panel, the sample contains 10 mm Mg2+. In both cases, the dissociation is fully reversible. The results indicate that Mg2+ has no effect on either the dissociation of GroES heptamer or the reassociation of the monomers. These results suggest that there is no significant conformational drift in the dissociated monomers that would prevent their reassociation. In an earlier study, it was shown that urea could dissociate and unfold GroES in a single, two-state transition as monitored by fluorescence anisotropy of dansyl-labeled GroES, intrinsic fluorescence, bis-ANS binding, sedimentation velocity, and limited proteolysis. From intrinsic fluorescence and sedimentation velocity analysis, it was demonstrated that dissociation by urea and reassociation of the monomers upon removal of the denaturant is reversible (47Seale J.W. Gorovits B.M. Ybarra J. Horowitz P.M. Biochemistry. 1996; 35: 4079-4083Crossref PubMed Scopus (45) Google Scholar). Typical plots for the dissociation of GroEL as a function of high hydrostatic pressure are shown in Fig. 2. The results in the absence and presence of Mg2+ and nucleotides are presented in Table I. The p 12values, which represent the pressure at the midpoint of dissociation transition, are in the following order: Mg2+ = AMP-PNP + Mg2+ > ATPγS = ATP = buffer only > ADP > ATPγS + Mg2+ + KCl > ATP + Mg2+ + KCl > ADP + Mg2+ + KCl. In all of the conditions except when Mg2+ and AMP-PNP + Mg2+ were present, the 14-mer dissociates completely (Fig.2, lower curve). In the presence of Mg2+ or AMP-PNP + Mg2+, the light scattering intensity under the pressure gradient occurs to only about 20%of the scattering intensity relative to that for complete dissociation, and upon depressurization the scattering reverses back to the initial value (Fig. 2, upper curve). This depressurized sample did not show any significant increase in the scattering intensity even after 5–10 h of depressurization.Table IDissociation of GroEL (14-mer) in the absence and presence of Mg2+ and nucleotidesCondition 1-a[GroEL]14-mer = 0.36 μm, [Tris-HCl] = 50 mm, pH 7.8, T = 20 °C. [ATP] = [ADP] = [AMP-PNP] = 1.0 mm; [MgCl2] = 10 mm; [KCl] = 10 mm.p 12CommentkbarBuffer only2.00Complete dissociationWith ATP2.20Complete dissociationWith ADP1.90Complete dissociationWith ATP-γS2.25Complete dissociationWith ATP, MgCl2, KCl1.40Complete dissociationWith ADP, MgCl2, KCl1.15Complete dissociationWith ATP-γS, MgCl2, KCl1.70Complete dissociationWith MgCl22.33Scattering intensity decreased to about 20%of the initial value and reversed back to the initial intensity upon depressurization. 1-bThe reassociation is rapid upon depressurization; therefore, we were unable to analyze the nature of the intermediate by a suitable method.With MgCl2, AMP-PNP2.31Scattering intensity decreased to about 20%of the initial value and reversed back to the initial intensity upon depressurization. 1-bThe reassociation is rapid upon depressurization; therefore, we were unable to analyze the nature of the intermediate by a suitable method.1-a [GroEL]14-mer = 0.36 μm, [Tris-HCl] = 50 mm, pH 7.8, T = 20 °C. [ATP] = [ADP] = [AMP-PNP] = 1.0 mm; [MgCl2] = 10 mm; [KCl] = 10 mm.1-b The reassociation is rapid upon depressurization; therefore, we were unable to analyze the nature of the intermediate by a suitable method. Open table in a new tab The kinetics of reassociation of pressure-dissociated GroEL monomers in the absence of ligands has been studied in our laboratory and has been shown to have a t 12of 150 h at 25 °C (40Gorovits B.M. Raman C.S. Horowitz P.M. J. Biol. Chem. 1995; 270: 2061-2066Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). The observation that ATP + Mg2+ destabilized the 14-mer in the pressure dissociation experiments reported in this investigation is in agreement with the earlier results from urea dissociation (40Gorovits B.M. Raman C.S. Horowitz P.M. J. Biol. Chem. 1995; 270: 2061-2066Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). The stability of GroEL in the presence of Mg2+ has also been seen in the case of its dissociation by urea (21Gorovits B.M. Horowitz P.M. J. Biol. Chem. 1995; 270: 28551-28556Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar), where the U 12values (urea concentration at the midpoint of transition) were in the following order: Mg2+ > AMP-PNP + Mg2+ > buffer only > ADP + Mg2+. Kinetics of dissociation of GroEL samples at 2.5 kbar in the presence of Mg2+ and different nucleotides were monitored by light scattering. The observed rates are summarized in TableII. The dissociation rates are in the following order: Mg2+ = AMP-PNP + Mg2+ > ATP-γS = ATP = ADP > ATPγS + Mg2+ + KCl > ATP + Mg2+ + KCl > buffer only > ADP + Mg2+ + KCl. The general trend is similar to the order of dissociation rates presented in Table I, with the stabilization of the oligomeric structure attributed to the binding of Mg2+and its destabilization when both Mg2+ and an adenine nucleotide were present. The kinetics in the presence of ATPγS + Mg2+ + KCl was biphasic, and both a slow and a fast rate could be evaluated (see Table II). The depressurized samples were analyzed by native 6.5%PAGE. It may be noted that the gel analysis provided a reasonable measure of the formation of monomers because their reassociation rate is extremely slow, with at 12= 150 h (40Gorovits B.M. Raman C.S. Horowitz P.M. J. Biol. Chem. 1995; 270: 2061-2066Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). The results are presented in Fig. 3. In these gels, the monomers, given an equivalent number of subunits, always stain more intensely than the 14-mers. Upon comparing the intensities of the bands for the 14-mer and monomers with the standards (native 14-mer inlane 1 and urea-dissociated monomer inlane 2), it is evident that the GroEL showed maximum stability when either Mg2+ or AMP-PNP + Mg2+ were present in the sample. This is a clear indication that Mg2+ induces subunit interactions that lead to a tight oligomeric structure, which can be destabilized by adding ADP (lane 5) or ATP. The dissociated sample when ATP alone (not shown) was present resembles that of ATP + Mg2+(lane 8). The gel pattern indicates that, although the 14-mer did not dissociate completely at this pressure, significant dissociation occurred, depending upon the nature of nucleotide (see lanes 6 and 8), even in Tris buffer alone without Mg2+ and nucleotides (lane 3).Table IIObserved rates for the dissociation of GroEL (14-mer) in the absence and presence of Mg2+ and nucleotides at 2.5 kbarCondition 2-a[GroEL]14-mer = 0.36 μm, [Tris-HCl] = 50 mm, pH 7.8, T = 20 °C.kobss−1Buffer only4.0 ± 0.04 × 10−3With ATP2.5 ± 0.03 × 10−3With ATPγS2.4 ± 0.03 × 10−3With ADP2.6 ± 0.02 × 10−3With ATP," @default.
• W2068370467 created "2016-06-24" @default.
• W2068370467 creator A5035578813 @default.
• W2068370467 creator A5070526529 @default.
• W2068370467 creator A5080904988 @default.
• W2068370467 date "2001-03-01" @default.
• W2068370467 modified "2023-09-29" @default.
• W2068370467 title "High Hydrostatic Pressure Can Probe the Effects of Functionally Related Ligands on the Quaternary Structures of the Chaperonins GroEL and GroES" @default.
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