Elucidation of Steps in the Capture of a Protein Substrate for Efficient Encapsulation by GroE
2006; Elsevier BV; Volume: 281; Issue: 30 Linguagem: Inglês
10.1074/jbc.m601605200
ISSN1083-351X
AutoresMatthew J. Cliff, Claire Limpkin, Angus Cameron, Steven G. Burston, Anthony R. Clarke,
Tópico(s)Toxin Mechanisms and Immunotoxins
ResumoWe have identified five structural rearrangements in GroEL induced by the ordered binding of ATP and GroES. The first discernable rearrangement (designated T → R1) is a rapid, cooperative transition that appears not to be functionally communicated to the apical domain. In the second (R1 → R2) step, a state is formed that binds GroES weakly in a rapid, diffusion-limited process. However, a second optical signal, carried by a protein substrate bound to GroEL, responds neither to formation of the R2 state nor to the binding of GroES. This result strongly implies that the substrate protein remains bound to the inner walls of the initially formed GroEL·GroES cavity, and is not yet displaced from its sites of interaction with GroEL. In the next rearrangement (R2·GroES → R3·GroES) the strength of interaction between GroEL and GroES is greatly enhanced, and there is a large and coincident loss of fluorescence-signal intensity in the labeled protein substrate, indicating that there is either a displacement from its binding sites on GroEL or at least a significant change of environment. These results are consistent with a mechanism in which the shift in orientation of GroEL apical domains between that seen in the apo-protein and stable GroEL·GroES complexes is highly ordered, and transient conformational intermediates permit the association of GroES before the displacement of bound polypeptide. This ensures efficient encapsulation of the polypeptide within the GroEL central cavity underneath GroES. We have identified five structural rearrangements in GroEL induced by the ordered binding of ATP and GroES. The first discernable rearrangement (designated T → R1) is a rapid, cooperative transition that appears not to be functionally communicated to the apical domain. In the second (R1 → R2) step, a state is formed that binds GroES weakly in a rapid, diffusion-limited process. However, a second optical signal, carried by a protein substrate bound to GroEL, responds neither to formation of the R2 state nor to the binding of GroES. This result strongly implies that the substrate protein remains bound to the inner walls of the initially formed GroEL·GroES cavity, and is not yet displaced from its sites of interaction with GroEL. In the next rearrangement (R2·GroES → R3·GroES) the strength of interaction between GroEL and GroES is greatly enhanced, and there is a large and coincident loss of fluorescence-signal intensity in the labeled protein substrate, indicating that there is either a displacement from its binding sites on GroEL or at least a significant change of environment. These results are consistent with a mechanism in which the shift in orientation of GroEL apical domains between that seen in the apo-protein and stable GroEL·GroES complexes is highly ordered, and transient conformational intermediates permit the association of GroES before the displacement of bound polypeptide. This ensures efficient encapsulation of the polypeptide within the GroEL central cavity underneath GroES. The function of the GroE molecular chaperone is to enhance the efficiency of protein folding, whether the substrate is a newly synthesized protein chain or a denatured molecule that has unfolded in response to environmental conditions (1Sigler P.B. Xu Z. Rye H.S. Burston S.G. Fenton W.A. Horwich A.L. Annu. Rev. Biochem. 1998; 67: 581-608Crossref PubMed Scopus (476) Google Scholar, 2Thirumalai D. Lorimer G.H. Annu. Rev. Biophys. Biomol. Struct. 2001; 30: 245-269Crossref PubMed Scopus (327) Google Scholar, 3Saibil H.R. Ranson N.A. Trends Biochem. Sci. 2002; 27: 627-632Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 4Burston S.G. Walter S. Buchner J. Kiefhaber T. Protein Folding Handbook. Part II. Wiley-Vch Verlag, Weinheim, Germany2004: 683-708Google Scholar). This dual role means that GroE is expressed constitutively to fulfill the former function and is inducible during heat-shock, when its concentration in the Escherichia coli cytoplasm is raised 5-fold (5Mogk A. Tomoyasu T. Goloubinoff P. Rüdiger S. Röder D. Langen H. Bukau B. EMBO J. 1999; 18: 6934-6949Crossref PubMed Scopus (516) Google Scholar). The ability of this chaperone to increase the productive folding yield, irrespective of either the sequence of the chain or the final shape of the folded substrate, is mechanistically intriguing, as is the coupling of this folding activity with the hydrolysis of ATP. The GroE chaperone comprises two protein assemblies. The largest, GroEL (sometimes referred to as Cpn60, a chaperonin with a subunit molecular mass of 60 kDa), is constructed from fourteen identical subunits arranged in two rings, stacked back-to-back, to form two large cavities separated by a central septum but with wide openings allowing access to the environment (6Braig K. Otwinowski Z. Hegde R. Boisvert D.C. Joachimiak A. Horwich A.L. Sigler P.B. Nature. 1994; 371: 578-586Crossref PubMed Scopus (1191) Google Scholar). These basket-like cavities are capable of encompassing an unfolded protein substrate of ∼60 kDa chain molecular mass (7Weissman J.S. Hohl C.M. Kovalenko O. Kashi Y. Chen S. Braig K. Saibil H.R. Fenton W.A. Horwich A.L. Cell. 1995; 83: 577-587Abstract Full Text PDF PubMed Scopus (390) Google Scholar, 8Mayhew M. da Silva A.C. Martin J. Erdjument-Bromage H. Tempst P. Hartl F.-U. Nature. 1996; 379: 420-426Crossref PubMed Scopus (344) Google Scholar). The second protein component, GroES (sometimes referred to as Cpn10, a chaperonin with a subunit molecular mass of 10 kDa), is a single ring of seven members that forms a dome-like structure of a diameter that is matched to the width of the GroEL opening (9Hunt J.F. Weaver A.J. Landry S.J. Gierasch L. Deisenhofer J. Nature. 1996; 379: 37-45Crossref PubMed Scopus (402) Google Scholar). In the presence of adenine nucleotides, GroES binds preferentially to one of the GroEL rings to act as a lid that can close off one of the central cavities to create a capsule (10Saibil H. Dong Z. Wood S. der Mauer A. Nature. 1991; 353: 25-26Crossref PubMed Scopus (82) Google Scholar, 11Xu Z. Horwich A.L. Sigler P.B. Nature. 1997; 388: 741-750Crossref PubMed Scopus (1038) Google Scholar). It is within this capsule, or cage, that substrate proteins are able to fold, relatively unencumbered by interactions with the protein surfaces that constitute its walls (7Weissman J.S. Hohl C.M. Kovalenko O. Kashi Y. Chen S. Braig K. Saibil H.R. Fenton W.A. Horwich A.L. Cell. 1995; 83: 577-587Abstract Full Text PDF PubMed Scopus (390) Google Scholar, 8Mayhew M. da Silva A.C. Martin J. Erdjument-Bromage H. Tempst P. Hartl F.-U. Nature. 1996; 379: 420-426Crossref PubMed Scopus (344) Google Scholar). More salient to the biological function of the machine, the encapsulated protein chain is not free to form interactions with other protein molecules in a similarly unfolded state, a process that might otherwise lead to the formation of inactive aggregates (12Goloubinoff P. Christeller J.T. Gatenby A.A. Lorimer G.H. Nature. 1989; 342: 884-889Crossref PubMed Scopus (546) Google Scholar, 13Badcoe I.G. Smith C.J. Wood S. Halsall D.J. Holbrook J.J. Lund P. Clarke A.R. Biochemistry. 1991; 30: 9195-9200Crossref PubMed Scopus (139) Google Scholar, 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, 15Martin J. Langer T. Boteva R. Schramel A. Horwich A.L. Hartl F.-U. Nature. 1991; 352: 36-42Crossref PubMed Scopus (725) Google Scholar, 16Fisher M.T. Biochemistry. 1992; 31: 3955-3963Crossref PubMed Scopus (101) Google Scholar). If these are not recoverable, other than by hydrolytic breakdown and de novo re-synthesis, it is a waste of the synthetic efforts of the cell. Hence, preventing irreversible aggregation, albeit at the cost of ATP hydrolysis, is a useful process. GroEL, GroES, and ATP are the fundamental requirements for the complete, energy-transducing ATPase in the assisted-folding reaction (12Goloubinoff P. Christeller J.T. Gatenby A.A. Lorimer G.H. Nature. 1989; 342: 884-889Crossref PubMed Scopus (546) Google Scholar). Our current understanding of the principle steps that constitute the reaction cycle are summarized in Fig. 1. As far as is currently understood, there are five rules that govern the operation of the cycle: (a) the GroES co-protein cannot bind to a ring devoid of nucleotide (17Chandrasekhar G.N. Tilly K. Woolford C. Hendrix R. Georgopoulos C. J. Biol. Chem. 1986; 261: 12414-12419Abstract Full Text PDF PubMed Google Scholar); (b) GroES binds more rapidly to an ATP-occupied ring than to one occupied by ADP (18Burston S.G. Ranson N.A. Clarke A.R. J. Mol. Biol. 1995; 249: 138-152Crossref PubMed Scopus (165) Google Scholar, 19Hayer-Hartl M.K. Martin J. Hartl F.-U. Science. 1995; 269: 836-841Crossref PubMed Scopus (138) Google Scholar, 20Rye H.S. Roseman A.M. Chen S. Furtak K. Fenton W.A. Saibil H.R. Horwich A.L. Cell. 1999; 97: 325-338Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar); (c) the binding of seven ATP molecules to one ring leads to a weakening of binding to the other, i.e. there is negative homotropic cooperativity between the rings (18Burston S.G. Ranson N.A. Clarke A.R. J. Mol. Biol. 1995; 249: 138-152Crossref PubMed Scopus (165) Google Scholar, 21Yifrach O. Horovitz A. Biochemistry. 1995; 34: 5303-5308Crossref PubMed Scopus (264) Google Scholar); (d) mixed complexes with one ring occupied by ATP and the other by ADP are stable, but cannot undergo the hydrolytic step owing to ADP on one ring acting as a non-competitive inhibitor of hydrolysis on the other (22Kad N.M. Ranson N.A. Cliff M.J. Clarke A.R. J. Mol. Biol. 1998; 278: 267-278Crossref PubMed Scopus (60) Google Scholar); and (e) the binding of GroES to an ATP-occupied ring commits the nucleotide to hydrolysis (23Todd M.J. Viitanen P.V. Lorimer G.H. Science. 1994; 265: 659-666Crossref PubMed Scopus (428) Google Scholar). Much previous work shows that the substrate proteins are transiently ensnared in the GroES-capped cavity during this ATPase cycle and have a dwell time of tens of seconds in which the protein substrate has a probability of folding dictated by the rate constant of the spontaneous process (24Weissman J.S. Kashi Y. Fenton W.A. Horwich A.L. Cell. 1994; 78: 693-702Abstract Full Text PDF PubMed Scopus (331) Google Scholar, 25Rye H.S. Burston S.G. Fenton W.A. Beechem J.M. Xu Z. Sigler P.B. Horwich A.L. Nature. 1997; 388: 792-797Crossref PubMed Scopus (356) Google Scholar). Because encapsulation cannot rely upon the random chance of an unfolded substrate happening to be in the cavity at the same time that the GroES lid comes on, there must be a mechanism by which the substrate is concentrated in the hole before the lid can provide the means of forming the cage. The structural picture that we have at the moment, derived from both x-ray and electron microscopy techniques, shows that in static or, at least, long-lived species, the binding of GroES and substrate are rather exclusive, i.e. they share binding surfaces (10Saibil H. Dong Z. Wood S. der Mauer A. Nature. 1991; 353: 25-26Crossref PubMed Scopus (82) Google Scholar, 26Fenton W.A. Kashi Y. Furtak K. Horwich A.L. Nature. 1994; 371: 614-619Crossref PubMed Scopus (574) Google Scholar, 27Roseman A.M. Chen S. White H.E. Braig K. Saibil H.R. Cell. 1996; 87: 241-251Abstract Full Text Full Text PDF PubMed Scopus (352) Google Scholar, 28Chen L. Sigler P.B. Cell. 1999; 99: 757-768Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). Taken at face value this means that the protein substrate has to dissociate before GroES can bind. If this is true, then there must be a conformational change that obligatorily displaces the protein substrate, which then has no time to diffuse from the locale before GroES rapidly caps the cavity and thus achieves encapsulation. However, an alternative mechanism can be proposed that proceeds through an intermediate state, as yet unseen by structural methods nor inferred from kinetics, in which the protein substrate remains bound to the walls of GroEL at the same time that GroES associates with its seven sites on the apical domains. This pathway postulates that there is a transient conformational intermediate state for GroEL in which the binding of substrate protein chain and GroES are not mutually exclusive, i.e. GroES binds before displacement of the substrate. There then must be a further step in which the substrate is shed from its binding site on the apical domain and allowed to fold unhindered. It is difficult to distinguish between these two plausible mechanisms except by using time-resolved techniques that can report specific events in the millisecond time range. Previous work on the structural rearrangement of GroEL induced by the binding of ATP shows that the picture is complex, even in the absence of GroES, with four distinct transitions before the nucleotide is hydrolyzed (29Yifrach O. Horovitz A. Biochemistry. 1998; 37: 7083-7088Crossref PubMed Scopus (62) Google Scholar, 30Cliff M.J. Kad N. Hay N. Lund P.A. Webb M.R. Burston S.G. Clarke A.R. J. Mol. Biol. 1999; 293: 667-684Crossref PubMed Scopus (65) Google Scholar, 31Inobe T. Arai M. Nakao M. Ito K. Kamagata K. Makio T. Ameniya Y. Kihara H. Kuwajima K. J. Mol. Biol. 2003; 327: 183-191Crossref PubMed Scopus (25) Google Scholar). These re-organizations of GroEL are spread over the several milliseconds to hundreds of milliseconds time-scales and are thus in the time window of stopped-flow techniques. In an attempt to resolve the molecular events that constitute the encapsulation phase of the GroE chaperone mechanism we have used fast-mixing methods combined with fluorescence labeling to determine the effect of GroES on the ATP-induced structural rearrangements in GroEL, including the point at which GroES associates. This is combined with time-resolved data that monitor the response of a label attached to the protein substrate when ATP-induced structural rearrangements occur in GroEL. The combination of these two lines of enquiry is used to correlate the dynamics of the protein substrate and of GroES. Standard Conditions—The standard reaction buffer in all experiments, except where stated otherwise, was 50 mm triethanolamine hydrochloride, pH 7.5, 50 mm KCl, and 20 mm MgCl2. Proteins and Reagents—The W485-GroEL mutant was a kind gift from Dr. Peter Lund (Birmingham, UK). The mutant GroEL and GroES were purified as described previously (30Cliff M.J. Kad N. Hay N. Lund P.A. Webb M.R. Burston S.G. Clarke A.R. J. Mol. Biol. 1999; 293: 667-684Crossref PubMed Scopus (65) Google Scholar, 18Burston S.G. Ranson N.A. Clarke A.R. J. Mol. Biol. 1995; 249: 138-152Crossref PubMed Scopus (165) Google Scholar). Reduced carboxymethylated α-lactalbumin was obtained from Sigma. ATP and ADP were obtained from Roche Applied Science. Nucleotide concentrations were confirmed spectrophotometrically using a molar extinction coefficient of 15,300 m-1 cm-1 for adenine at 260 nm. All other reagents were obtained from BDH and were analytical grade. Fluorescent Labeling of CM-LA—A 10 mm stock solution of 5-(dimethylamino)-1-naphthalenesulfonyl chloride (dansyl chloride) in acetonitrile was added to 100 μm CM-LA 5The abbreviations used are: CM-LA, reduced and carboxymethylated α-lactalbumin; HNBB, 2-hydroxy-5-nitrobenzyl bromide. in phosphate buffer, pH 7.5, at a ratio of 1:10. This pH was chosen to selectively label the α-amino group at the N terminus of CM-LA rather than the ϵ-amino group of lysine. The reaction was allowed to proceed for 10 min in the dark before being dialyzed overnight against 100 volumes of 50 mm Tris, pH 7.5, and 10 mg/ml activated charcoal. The extent of labeling was determined by absorbance using a molar extinction coefficient of 3400 m-1 cm-1 at 335 nm. On average one dansyl group was attached per CM-LA polypeptide. Chemical Modification of the Tryptophan Residues in CM-LA—2-Hydroxy-5-nitrobenzyl bromide (HNBB, also known as Koshland Reagent I) reacts in neutral and acid solutions with only tryptophan residues (37Koshland Jr, D.E. Karkhanis Y.D. Latham H.G. J. Am. Chem. Soc. 1964; 86: 1448-1450Crossref Scopus (122) Google Scholar) modifying the indole group, which in turn alters its spectroscopic properties. A 0.1 m stock of HNBB was made in 5% dried acetone before mixing with an equal volume of 200 μm CM-LA in 10 mm sodium phosphate at pH 6.0 in the dark for 2 h. The mixed sample was then dialyzed against 200 volumes of 10 mm sodium phosphate, pH 6.0, in the dark. HNBB activity was confirmed by the loss of tryptophan fluorescence upon excitation at 295 nm. Stopped-flow Time-resolved Tryptophan Fluorescence Measurements—All stopped-flow experiments were performed in the standard buffer using an Applied Photophysics SX-17MV stopped-flow fluorometer. W485-GroEL was excited using monochromatic light at 295 nm, and the resulting fluorescence was selected with a WG320 filter which cuts off all light below 320 nm. All reactions were performed at 25 °C, and at least three transients were averaged for any data point. Fluorescence measurements of the dansyl-CM-LA were made using an excitation wavelength of 340 nm and fluorescence emission selected using a WG420 filter, which cuts off light below 420 nm. Analytical Methods—All data fitting was carried out using the least-squares method of Marquardt (43Marquardt D.W. J. Soc. Ind. Appl. Math. 1963; 11: 431-441Crossref Google Scholar) using the Grafit 5.0 fitting program (Erithacus software). The quadratic tight ligand binding equation (Equation 1) used in Fig. 5 is, F=(E0+Kd+L)+(E0+Kd+L)2−4E0L2E0×(Fmax−Fmin)+Fmin(Eq. 1) where F is the measured fluorescence, Eo is the initial concentration of ligand binding sites, Kd is the dissociation constant, L is the total ligand concentration, and Fmax and Fmin are the maximum and minimum fluorescence intensities observed, respectively. All parameters during the fitting procedures were allowed to float where possible, although it should also be noted that where the magnitude of the rate constants for successive phases are similar, then the determination of both rate constants and fluorescence amplitudes are not accurate. This was particularly the case when determining the rate constants and amplitudes for the R3 → R3* and R3* → R4 transitions at low concentrations of ATP. This affects the degree of accuracy of the value of K½ reported in Fig. 5B. Discrete Events in the Ordered Addition of ATP and GroES to GroEL—The intrinsic fluorescence of an engineered tryptophan (Trp-485) in GroEL can be used to report conformational events induced by the binding of ATP (30Cliff M.J. Kad N. Hay N. Lund P.A. Webb M.R. Burston S.G. Clarke A.R. J. Mol. Biol. 1999; 293: 667-684Crossref PubMed Scopus (65) Google Scholar, 31Inobe T. Arai M. Nakao M. Ito K. Kamagata K. Makio T. Ameniya Y. Kihara H. Kuwajima K. J. Mol. Biol. 2003; 327: 183-191Crossref PubMed Scopus (25) Google Scholar). This experiment was performed by rapidly mixing W485-GroEL (hereafter referred to just as GroEL for simplicity) with a solution of ATP using a stopped-flow apparatus and recording time-resolved changes in fluorescence (Fig. 2A, GroEL plus ATP). To investigate the effect of GroES on this reaction the experiment was repeated, this time challenging GroEL with a mixture of ATP and GroES (Fig. 2A, GroEL plus GroES plus ATP). In both reactions the transients observed are complex. ATP alone induces three, easily resolved kinetic phases (two fluorescence quenches and a subsequent fluorescence enhancement), whereas ATP and GroES together induce four kinetic phases (three quenches and a slower enhancement). In both cases they are also preceded by a very rapid signal enhancement, which represents the cooperative T → R transition but which is not fully resolvable at the ATP concentration and temperature used in this experiment (30Cliff M.J. Kad N. Hay N. Lund P.A. Webb M.R. Burston S.G. Clarke A.R. J. Mol. Biol. 1999; 293: 667-684Crossref PubMed Scopus (65) Google Scholar, 31Inobe T. Arai M. Nakao M. Ito K. Kamagata K. Makio T. Ameniya Y. Kihara H. Kuwajima K. J. Mol. Biol. 2003; 327: 183-191Crossref PubMed Scopus (25) Google Scholar). The data in the absence of GroES can be fitted optimally to a four-phase mechanism (30Cliff M.J. Kad N. Hay N. Lund P.A. Webb M.R. Burston S.G. Clarke A.R. J. Mol. Biol. 1999; 293: 667-684Crossref PubMed Scopus (65) Google Scholar); however, the presence of GroES introduces an extra quench phase (see fitting residuals in Fig. 2, B and C). Hence, the full description of the ATP-driven system in the absence of GroES can be written as in Scheme 1, T↔R1↔R2↔R3↔R4SCHEME 1 and the ATP- and GroES-driven system is described by Scheme 2. T↔R1↔R2↔R3↔R3∗↔R4SCHEME 2 Because the final kinetic phase observed in the presence and absence of GroES is a fluorescence enhancement, we have placed the additional kinetic phase observed in the presence of GroES after R3 giving rise to the R3* intermediate species. To determine the point along this pathway at which GroES associates we examined the influence of GroES on each of the kinetic phases. The rate of the fast T → R transition (observable here at 150 μm ATP) was unperturbed by the presence of GroES (Fig. 2D). In addition, the ATP dependence of the first order observed rate constant of the first fluorescence quench phase (the R1 → R2 transition) was also unaffected by the presence of GroES (Fig. 3A). In the presence of GroES kobs for the R1 → R2 transition shows a bi-sigmoid dependence on ATP concentration, as indeed was the case in the absence of GroES (30Cliff M.J. Kad N. Hay N. Lund P.A. Webb M.R. Burston S.G. Clarke A.R. J. Mol. Biol. 1999; 293: 667-684Crossref PubMed Scopus (65) Google Scholar). This was previously assigned to the binding of ATP to first one heptameric ring and then the second heptameric ring, supported by the fact that the second sigmoid could be abolished by binding ADP to one of the rings prior to mixing with ATP (Ref. 30Cliff M.J. Kad N. Hay N. Lund P.A. Webb M.R. Burston S.G. Clarke A.R. J. Mol. Biol. 1999; 293: 667-684Crossref PubMed Scopus (65) Google Scholar and Fig. 3B). This was also the case in the presence of GroES (shown in Fig. 3B and indicated by the dashed line in Fig. 3A). However, upon examining the amplitude of the R1 → R2 phase as a function of the concentration of ATP, it can be seen that it was clearly influenced by the presence of the co-protein (Fig. 3C). The ATP dependence of the amplitude of this phase fits optimally to the Hill equation with an K½ of 13.0 (±0.36) μm and a Hill coefficient of 2.49 (±0.14) in the absence of GroES and K½ of 6.85 (±0.10) μm and a Hill coefficient of 2.28 (±0.06) in the presence of GroES. This effect can only be explained by the binding of GroES to the R2 state, thereby increasing the apparent binding affinity for ATP and shifting the amplitude curve to the left. Thus the above, qualitative analysis leads to the tentative conclusion that GroES interacts with the R2 state. From these data we can thus infer that the reaction scheme (Reaction 1) in the presence of ATP and GroES is as follows. T⋅ATP⇄R1⋅ATP⇄R2⋅ATP⇄R2⋅ATP⋅GroESamp;↑↓ amp;R3⋅ATP⋅GroESamp;↑↓ amp;R3∗⋅ATP⋅GroESamp;↑↓ amp;R4⋅ATP⋅GroESamp;REACTION 1 This interpretation assumes that if GroES binds to a particular state it will affect its optical or kinetic properties. It is possible that GroES binds before the GroEL conformation acquires the R2 conformation, but that the binding is optically and kinetically silent. However, with such a large protein cofactor that is known to have very large effects on the conformation of GroEL, this appears unlikely. Dependence of Observed Rate Constants on the Concentration of ATP—We next assessed the effect of the concentration of ATP on the observed rate constants for the nucleotide-induced rearrangements (Fig. 4), so that we could compare the responses of the system to ATP in the presence and absence of GroES. As described in the previous section, the first enhancement (T → R1) is entirely unaffected by the presence of GroES, and the first quench phase (R1 → R2) is affected only in its amplitude, and there is no effect on rate. The second quench phase, designated (R2 → R3), peaked at a rate constant of ∼14 s-1 in the presence of GroES (Fig. 4A); compared with ∼6 s-1 for the second quench in the absence of GroES (30Cliff M.J. Kad N. Hay N. Lund P.A. Webb M.R. Burston S.G. Clarke A.R. J. Mol. Biol. 1999; 293: 667-684Crossref PubMed Scopus (65) Google Scholar). The third quench phase (R3 → R3*) did not occur at all in the absence of GroES and had a maximum first-order rate constant of ∼1 s-1 at saturating concentrations of ATP (see Fig. 4B). The final kinetic phase, a fluorescence enhancement phase (R3* → R4), showed no variation in rate as ATP concentration increased, being a constant 0.26 s-1 in the presence of GroES (Fig. 4C). This compares with a rate constant of ∼0.6 s-1 for the final enhancement phase in the absence of GroES. However, the amplitude of the R3* → R4 phase increased with the concentration of ATP and showed half-saturation at a concentration of ∼140 μm (see Fig. 4D). Progressive Tightening of the GroEL·GroES Complex after the Initial Interaction—By maintaining a constant concentration of ATP (1 mm) and varying the concentration of GroES we were able to use phase-amplitude analysis to make an estimate of the GroES binding affinity as the GroEL molecule progresses through its ATP-driven rearrangements (Fig. 5). It can be seen that the amplitude of the second quench phase (R2 → R3) is enhanced as the concentration of GroES is increased from sub-stoichiometric to super-stoichiometric levels (Fig. 5A). This phase of the reaction represents the process in Reaction 2. R2⋅ATP+GroES↔R2⋅ATP⋅GroES↔R3⋅ATP⋅GroESREACTION 2 If the first binding step is represented by the following dissociation constant (Equation 1), KdR2(ES)=[R2⋅ATP]⋅[GroES][R2⋅ATP⋅GroES](Eq. 2) and the second by the unimolecular equilibrium constant K(2,3) (Equation 2), where, K(2,3)=[R3⋅ATP⋅GroES][R2⋅ATP⋅GroES](Eq. 3) then the apparent overall binding affinity reported by the amplitude plot (Kd1ES(app)) is given by Equation 3. Kd1ES(app)=KdR2(ES)(1+K(2,3))(Eq. 4) The amplitude data in Fig. 5A can be fitted to the tight-binding equation with Kd1,ES(app) = 190 (±80) nm, although it must be stated that these data do not accurately resolve this binding affinity, a fact reflected in the degree of error. A similar kind of analysis can be performed for the last two kinetic phases, R3 → R3* and R3* → R4. The fluorescence amplitude of the R3 → R3* phase, again at a fixed ATP concentration of 1 mm, also increases with increasing GroES concentration and can be fitted to determine the apparent affinity of GroES at this stage of the reaction. Although it is difficult to resolve accurate data at low concentrations of GroES, the data in Fig. 5B fit optimally to the tight-binding equation with Kd2,ES(app) = 15.0 (±14.1) nm, ∼10-fold tighter than Kd1,ES(app). Although the effective binding affinity is poorly determined, we take these data as evidence that there is a tightening of the GroES interaction as the GroEL molecule progresses from the R3 to the R3* conformation. The presence of increasing concentrations of GroES also enhanced the amplitude of the R3* → R4 phase (Fig. 5C). The data fit optimally to the tight-binding equation with a Kd3,ES(app) = 4.39 (±2.86) nm. This probably represents a further tightening of the GroEL-GroES interaction as the reaction progresses from R3* → R4, although the errors in fitting the amplitudes of multiexponentials are considerable. The Dynamics of a Bound Polypeptide; the Experimental System—The binding of ATP weakens the affinity of GroEL for polypeptide (13Badcoe I.G. Smith C.J. Wood S. Halsall D.J. Holbrook J.J. Lund P. Clarke A.R. Biochemistry. 1991; 30: 9195-9200Crossref PubMed Scopus (139) Google Scholar, 15Martin J. Langer T. Boteva R. Schramel A. Horwich A.L. Hartl F.-U. Nature. 1991; 352: 36-42Crossref PubMed Scopus (725) Google Scholar, 32Staniforth R.A. Burston S.G. Atkinson T. Clarke A.R. Biochem. J. 1994; 300: 651-658Crossref PubMed Scopus (88) Google Scholar, 41Sparrer H. Lilie H. Buchner J. J. Mol. Biol. 1996; 258: 74-87Crossref PubMed Scopus (63) Google Scholar); therefore, the conundrum arises as to why the initial binding of ATP to a GroEL·protein substrate complex does not cause unfolded polypeptide to be released into the bulk solution before GroES has associated, especially because the binding sites for the two ligands overlap on the GroEL apical domains. Is GroES association so rapid as to prevent significant loss of polypeptide, or has GroEL evolved a mechanism to ensure efficient capture of the polypeptide into the central cavity beneath GroES? One way to address this question is to determine at which point along the ATP-induced allosteric pathway the polypeptide "senses" the conformational rearrangements in GroEL. Complications in the kinetics caused by the refolding of the protein substrate were avoided by using α-lactalbumin, which had been reduced and carboxymethylated (CM-LA). The removal of Ca2+ and the reduction of the disulfide bonds in α-lactalbumin resulted in the protein fully unfolding, and its interaction with GroEL has been well characterized (33Hayer-Hartl M.K. Ewbank J.J. Creighton T.E. Hartl F.-U. EMBO J. 1994; 13: 3192-3202Crossref PubMed Scopus (157) Google Scholar, 34Robinson C.V. Gross M. Eyles S.J. Ewbank J.J. Mayhew M. Hartl F.-U. Dobson C.M. Radford S.E. Nature. 1994; 372: 646-651Crossref PubMed Scopus (194) Google Scholar, 35Okazaki A. Ikura T. Nikaido K. Kuwajima K. Nat. Struct. Biol. 1994; 1: 439-446Crossref PubMed Scopus (104) Google Scholar, 36Yifrach O. Horovitz A. J. Mol. Biol. 1996; 255: 356-361Crossref PubMed Scopus (98) Google Scholar). Modification of the sulfh
Referência(s)