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• W2028351490 abstract "To elucidate the exact role of the C-terminal region of GroEL in its functional cycle, the C-terminal 20-amino acid truncated mutant of GroEL was constructed. The steady-state ATPase rate and duration of GroES binding showed that the functional cycle of the truncated GroEL is extended by ∼2 s in comparison with that of the wild type, without interfering with the basic functions of GroEL. We have proposed a model for the functional cycle of GroEL, which consists of two rate-limiting steps of ∼3- and ∼5-s duration (Ueno, T., Taguchi, H., Tadakuma, H., Yoshida, M., and Funatsu, T. (2004) Mol. Cell 14, 423-434g). According to the model, detailed kinetic studies were performed. We found that a 20-residue truncation of the C terminus extends the time until inorganic phosphate is generated and the time for arresting protein folding in the central cavity, i.e. the lifetime of the first rate-limiting step in the functional cycle, to an ∼5-s duration. These results suggest that the integrity of the C-terminal region facilitates the transition from the first to the second rate-limiting state. To elucidate the exact role of the C-terminal region of GroEL in its functional cycle, the C-terminal 20-amino acid truncated mutant of GroEL was constructed. The steady-state ATPase rate and duration of GroES binding showed that the functional cycle of the truncated GroEL is extended by ∼2 s in comparison with that of the wild type, without interfering with the basic functions of GroEL. We have proposed a model for the functional cycle of GroEL, which consists of two rate-limiting steps of ∼3- and ∼5-s duration (Ueno, T., Taguchi, H., Tadakuma, H., Yoshida, M., and Funatsu, T. (2004) Mol. Cell 14, 423-434g). According to the model, detailed kinetic studies were performed. We found that a 20-residue truncation of the C terminus extends the time until inorganic phosphate is generated and the time for arresting protein folding in the central cavity, i.e. the lifetime of the first rate-limiting step in the functional cycle, to an ∼5-s duration. These results suggest that the integrity of the C-terminal region facilitates the transition from the first to the second rate-limiting state. The chaperonin GroEL is an essential molecular chaperone that mediates protein folding with its cofactor GroES in Escherichia coli. GroEL is composed of 14 identical 57-kDa subunits arranged in two heptameric rings stacked back-to-back, each contains a cavity. GroES consists of a dome-shaped heptameric ring of identical 10-kDa subunits and interacts with one or both GroEL rings in an ATP-regulated manner. The substrate protein is encapsulated in the GroEL cavity underneath GroES, where it folds during the time of ATP hydrolysis (1Hartl F.U. Hayer-Hartl M. Science. 2002; 295: 1852-1858Crossref PubMed Scopus (2789) Google Scholar, 2Horwich A.L. Fenton W.A. Chapman E. Farr G.W. Annu. Rev. Cell Dev. Biol. 2007; 23: 115-145Crossref PubMed Scopus (337) Google Scholar). Each subunit of GroEL is divided into the following three distinct domains: apical, intermediate, and equatorial domains. The apical domain forms the entrance to the GroEL cavity and includes the residues involved in the binding to substrate proteins and GroES. The intermediate domain connects the equatorial and apical domains of each subunit. The equatorial domain, which is composed of N- and C-terminal regions, is involved in intra- and inter-ring contacts and contains the ATP-binding site (3Braig K. Otwinowski Z. Hegde R. Boisvert D.C. Joachimiak A. Horwich A.L. Sigler P.B. Nature. 1994; 371: 578-586Crossref PubMed Scopus (1192) Google Scholar, 4Xu Z. Horwich A.L. Sigler P.B. Nature. 1997; 388: 741-750Crossref PubMed Scopus (1041) Google Scholar). Although the final ∼20 residues in the C-terminal region are known to be structurally flexible (3Braig K. Otwinowski Z. Hegde R. Boisvert D.C. Joachimiak A. Horwich A.L. Sigler P.B. Nature. 1994; 371: 578-586Crossref PubMed Scopus (1192) Google Scholar), they project from the equatorial domain into the central space and form the dividing wall of each ring, as indicated by structural studies (5Chen S. Roseman A.M. Hunter A.S. Wood S.P. Burston S.G. Ranson N.A. Clarke A.R. Saibil H.R. Nature. 1994; 371: 261-264Crossref PubMed Scopus (325) Google Scholar, 6Thiyagarajan P. Henderson S.J. Joachimiak A. Structure (Lond.). 1996; 4: 79-88Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Recent studies have renewed interest in the functional role of the C-terminal region. An intriguing feature is the presence of Gly-Gly-Met (GGM) repeated sequences at the C terminus of GroEL. The sequence is strongly conserved among most GroEL homologues (7Brocchieri L. Karlin S. Protein Sci. 2000; 9: 476-486Crossref PubMed Scopus (143) Google Scholar), but its significance is unknown. Earlier studies have shown that up to 27 amino acids can be deleted from the C terminus without affecting E. coli viability in ideal growing conditions (8Burnett B.P. Horwich A.L. Low K.B. J. Bacteriol. 1994; 176: 6980-6985Crossref PubMed Google Scholar, 9McLennan N.F. Girshovich A.S. Lissin N.M. Charters Y. Masters M. Mol. Microbiol. 1993; 7: 49-58Crossref PubMed Scopus (59) Google Scholar, 10McLennan N.F. McAteer S. Masters M. Mol. Microbiol. 1994; 14: 309-321Crossref PubMed Scopus (23) Google Scholar). Recently, Tang et al. (11Tang Y.C. Chang H.C. Roeben A. Wischnewski D. Wischnewski N. Kerner M.J. Hartl F.U. Hayer-Hartl M. Cell. 2006; 125: 903-914Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar, 12Tang Y.C. Chang H.C. Chakraborty K. Hartl F.U. Hayer-Hartl M. EMBO J. 2008; 27: 1458-1468PubMed Google Scholar) showed that modulating the length of the C-terminal region can alter the folding rate of substrate proteins within the GroEL cavity. On the contrary, Farr et al. (13Farr G.W. Fenton W.A. Horwich A.L. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 5342-5347Crossref PubMed Scopus (44) Google Scholar) reported no acceleration in folding rate in GroEL mutants with elongated GGM repeat(s). Elad et al. (14Elad N. Farr G.W. Clare D.K. Orlova E.V. Horwich A.L. Saibil H.R. Mol. Cell. 2007; 26: 415-426Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar) suggested the C-terminal region is a potential interaction site with the substrate proteins. Machida et al. (15Machida K. Kono-Okada A. Hongo K. Mizobata T. Kawata Y. J. Biol. Chem. 2008; 283: 6886-6896Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar) mentioned that the hydrophilic residues in the C-terminal sequence are critical for the substrate folding inside the central cavity. Given that the C-terminal region is located in the central cavity, it is likely that the region contributes to maintaining the volume and the environment of the central cavity for proper protein folding. Furthermore, it should not be forgotten that the C-terminal region is closely related to the rate of ATP hydrolysis (9McLennan N.F. Girshovich A.S. Lissin N.M. Charters Y. Masters M. Mol. Microbiol. 1993; 7: 49-58Crossref PubMed Scopus (59) Google Scholar, 12Tang Y.C. Chang H.C. Chakraborty K. Hartl F.U. Hayer-Hartl M. EMBO J. 2008; 27: 1458-1468PubMed Google Scholar, 13Farr G.W. Fenton W.A. Horwich A.L. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 5342-5347Crossref PubMed Scopus (44) Google Scholar, 15Machida K. Kono-Okada A. Hongo K. Mizobata T. Kawata Y. J. Biol. Chem. 2008; 283: 6886-6896Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 16Langer T. Pfeifer G. Martin J. Baumeister W. Hartl F.U. EMBO J. 1992; 11: 4757-4765Crossref PubMed Scopus (356) Google Scholar). Farr et al. (13Farr G.W. Fenton W.A. Horwich A.L. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 5342-5347Crossref PubMed Scopus (44) Google Scholar) found that GGM repeat-elongated GroEL mutants exhibit an increased ATPase rate. They also suggested that disturbance of the ATPase rate can affect the folding rate of substrate proteins within the cavity. Therefore, it is important to understand what the effects of the C-terminal region are on the functional cycle of GroEL. To this end, we constructed a C-terminal truncated mutant (termed ELtc20), 3The abbreviations used are:ELtc20GroEL mutant with 20-amino acid truncation from the C terminusLAα-lactalbuminrLAdisulfide bond-reduced form of LAELwtwild-type GroELES98CGroES mutant with a single cysteine added at the C terminus of each subunitIC5bio-ESES98C modified with IC5 and biotinZMWzero-mode waveguideGFPS65T mutant of green fluorescence proteinTMRtetramethylrhodamineBSAbovine serum albuminDTTdithiothreitol. 3The abbreviations used are:ELtc20GroEL mutant with 20-amino acid truncation from the C terminusLAα-lactalbuminrLAdisulfide bond-reduced form of LAELwtwild-type GroELES98CGroES mutant with a single cysteine added at the C terminus of each subunitIC5bio-ESES98C modified with IC5 and biotinZMWzero-mode waveguideGFPS65T mutant of green fluorescence proteinTMRtetramethylrhodamineBSAbovine serum albuminDTTdithiothreitol. which lacks the final 20 amino acid residues, and characterized it. ELtc20 was shown to have the basic functions of GroEL and the extended functional cycle (∼10 s) in comparison with that of the wild type (∼8 s). Previously, we proposed a model for the functional cycle of GroEL with two successive rate-limiting steps of ∼3 and ∼5 s duration (17Ueno T. Taguchi H. Tadakuma H. Yoshida M. Funatsu T. Mol. Cell. 2004; 14: 423-434Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Detailed kinetic studies revealed that the C-terminal truncation extends the lifetime of the first rate-limiting step to ∼5 s. From these results, we suggest the notion that the integrity of the C-terminal region facilitates the transition from the first to the second rate-limiting state in the functional cycle. GroEL mutant with 20-amino acid truncation from the C terminus α-lactalbumin disulfide bond-reduced form of LA wild-type GroEL GroES mutant with a single cysteine added at the C terminus of each subunit ES98C modified with IC5 and biotin zero-mode waveguide S65T mutant of green fluorescence protein tetramethylrhodamine bovine serum albumin dithiothreitol. GroEL mutant with 20-amino acid truncation from the C terminus α-lactalbumin disulfide bond-reduced form of LA wild-type GroEL GroES mutant with a single cysteine added at the C terminus of each subunit ES98C modified with IC5 and biotin zero-mode waveguide S65T mutant of green fluorescence protein tetramethylrhodamine bovine serum albumin dithiothreitol. Regents and Proteins—ATP, phosphoenolpyruvate, pyruvate kinase from rabbit muscle, lactate dehydrogenase from hog muscle, NADH, and malate dehydrogenase from pig heart were purchased from Roche Diagnostics. Bovine apo-α-lactalbumin (LA), pepsin, bovine mitochondrial rhodanese, bovine serum albumin (BSA), glucose oxidase, and catalase were obtained from Sigma. Streptavidin and tetramethylrhodamine (TMR)-5′-maleimide were from Invitrogen. IC5-maleimide and biotin-PEAC5-maleimide were purchased from Dojindo Laboratories. Cy3 monofunctional N-hydroxysuccinimide ester was from GE Healthcare. Preparation of GroEL, GroES, and Substrate Proteins—ELtc20 was generated using the following primer sets: Forward, 5′-TAATACGACTCACTATAGG-3′; Reverse, 5′-GAAAAACGATTTAGAGCTCAAAT-3′ (XhoI site underlined). PCR amplification was conducted with KOD-Plus- (Toyobo) using the plasmid pET-EL as a template (18Motojima F. Makio T. Aoki K. Makino Y. Kuwajima K. Yoshida M. Biochem. Biophys. Res. Commun. 2000; 267: 842-849Crossref PubMed Scopus (39) Google Scholar). PCR product was digested with NdeI and XhoI, and cloned into the same site of pET21c (Novagen). The construct was verified by DNA sequencing. GroEL and GroES were expressed in E. coli and purified as described previously (18Motojima F. Makio T. Aoki K. Makino Y. Kuwajima K. Yoshida M. Biochem. Biophys. Res. Commun. 2000; 267: 842-849Crossref PubMed Scopus (39) Google Scholar). The concentrations of GroEL and GroES were determined spectrophotometrically at 280 nm (17Ueno T. Taguchi H. Tadakuma H. Yoshida M. Funatsu T. Mol. Cell. 2004; 14: 423-434Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar) and were expressed as molar concentrations of tetradecamer and heptamer in this study. ES98C, a GroES mutant with a cysteine residue added at the C terminus (19Murai N. Makino Y. Yoshida M. J. Biol. Chem. 1996; 271: 28229-28234Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar), was labeled with IC5-maleimide and biotin-PEAC5-maleimide in HKM buffer (25 mm HEPES-KOH, pH 7.4, 100 mm KCl, 5 mm MgCl2). The labeled ES98C (IC5bio-ES) was separated from unreacted reagents by a NAP-5 column (GE Healthcare). The extent of labeling was estimated according to the following procedure. The concentration of IC5 was determined spectrophotometrically at 645 nm with an extinction coefficient of 178,000 m-1 cm-1. The concentration of ES98C was determined using the Lowry method (DC protein assay; Bio-Rad). The molar ratio of IC5 to the ES98C heptamer was 1.8 throughout the study. ELwt and ELtc20 were labeled with Cy3 monofunctional N-hydroxysuccinimide ester in HKM buffer containing 20 mm sodium bicarbonate to raise the pH (∼8.7). Labeling resulted in a stoichiometry of ∼1.1 Cy3 dye molecules per GroEL tetradecamer. Fluorescent-labeled GroEL and ES98C exhibited behavior similar to the wild-type proteins (data not shown). LA (300 μm) was treated with HKM buffer containing 7.5 mm DTT for 15 min at room temperature. The disulfide bond-reduced form of LA (rLA) is known to bind strongly to GroEL (18Motojima F. Makio T. Aoki K. Makino Y. Kuwajima K. Yoshida M. Biochem. Biophys. Res. Commun. 2000; 267: 842-849Crossref PubMed Scopus (39) Google Scholar, 19Murai N. Makino Y. Yoshida M. J. Biol. Chem. 1996; 271: 28229-28234Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Pepsin (300 μm) was dissolved in HKM buffer and was used as denatured pepsin, because it is known to lose its native conformation at a neutral pH and interact with GroEL (20Aoki K. Taguchi H. Shindo Y. Yoshida M. Ogasahara K. Yutani K. Tanaka N. J. Biol. Chem. 1997; 272: 32158-32162Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 21McPhie P. Biochem. Biophys. Res. Commun. 1989; 158: 115-119Crossref PubMed Scopus (9) Google Scholar). Rhodanese (75 μm) was denatured in HKM buffer containing 6 m guanidine hydrochloride for at least 1 h at room temperature. The GFP employed in this study is the S65T mutant (22Heim R. Cubitt A.B. Tsien R.Y. Nature. 1995; 373: 663-664Crossref PubMed Scopus (1521) Google Scholar). It was expressed and purified as described previously (23Makino Y. Amada K. Taguchi H. Yoshida M. J. Biol. Chem. 1997; 272: 12468-12474Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 24Sakikawa C. Taguchi H. Makino Y. Yoshida M. J. Biol. Chem. 1999; 274: 21251-21256Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). GFP (20 μm) was denatured in HKM buffer containing 100 mm HCl at room temperature for 2 min. Steady-state ATPase Rate Measurements—Steady-state ATPase rate of GroEL was measured at 23 °C with an ATP-regeneration system (17Ueno T. Taguchi H. Tadakuma H. Yoshida M. Funatsu T. Mol. Cell. 2004; 14: 423-434Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). The assay mixture consisted of HKM buffer containing 0.2 mm NADH, 5 mm phosphoenolpyruvate, 100 μg/ml pyruvate kinase, 100 μg/ml lactate dehydrogenase, 2.5 mm DTT, and 1 mm ATP in the presence or absence of 500 nm GroES. 4Unless otherwise stated, wild-type GroES was used in assays. After a 1-min incubation, ATP hydrolysis was initiated by the injection of 100 nm GroEL into the vigorously stirred mixture. After incubation for 200 s, the denatured protein (75 μm rLA, 75 μm denatured pepsin, or 1 μm denatured rhodanese) was diluted into the assay mixture. The decreases in the absorbance at 340 nm, because of oxidation of NADH, were monitored continuously with a spectrophotometer (V-550, Jasco). Steady-state ATPase activity was determined from the slope of the absorbance decrease with time. Measurements were repeated in triplicate. Sample Preparation for Microscopy—Zero-mode waveguides (ZMWs) were fabricated in a 100-nm-thick aluminum film deposited on a concaved quartz coverslip. The detailed procedures are reported elsewhere (25Miyake T. Tanii T. Sonobe H. Akahori R. Shimamoto N. Ueno T. Funatsu T. Ohdomari I. Anal. Chem. 2008; (in press)PubMed Google Scholar). The ZMW holes used in this study were ∼90 nm in diameter and ∼160 nm in depth (Fig. 2A). The coverslip with ZMWs was placed in an oxygen plasma asher to clean the surfaces before use. A flow cell was constructed from the glass slide and coverslip with ZMWs separated by two spaces of ∼50 μm thickness. IC5bio-ES was immobilized on the bottom of ZMWs via biotinylated BSA and streptavidin as described previously (17Ueno T. Taguchi H. Tadakuma H. Yoshida M. Funatsu T. Mol. Cell. 2004; 14: 423-434Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 26Taguchi H. Ueno T. Tadakuma H. Yoshida M. Funatsu T. Nat. Biotechnol. 2001; 19: 861-865Crossref PubMed Scopus (102) Google Scholar). First, 3.0 mg/ml biotinylated BSA was injected into the flow cell. The cell was washed by HKM buffer. Infusion and washing were repeated as follows: infusion of 0.33 mg/ml streptavidin, washing with HKM buffer, infusion of 25 nm IC5bio-ES, washing with HKM buffer and HKM buffer containing 10 nm Cy3-labeled GroEL, the oxygen scavenger system (25 mm glucose, 2.5 μm glucose oxidase, 10 nm catalase, and 10 mm DTT), 75 μm rLA, and 2 mm ATP. Microscopy and Data Analyses—Single molecules in ZMWs were observed by using an epi-illumination configuration in an inverted microscope (IX70, Olympus). The surface-immobilized IC5bio-ES was illuminated with a red solid-state laser (87.5 microwatts, 635 nm; Coherent). Cy3-labeled GroEL molecules were excited with a green solid-state laser (250 microwatts, 532 nm, COMPASS315M-100; Coherent). Fluorescence was collected through an oil-immersion objective (ApoN 60×OTIRFM, NA 1.49; Olympus) on a microscope equipped with a custom-made dichroic mirror (Chroma Technology) and emission filters (605AF80 for Cy3 detection and HQ700/75 for IC5 detection). Fluorescence images were recorded every 200 ms for 5 min with an electron multiplying charge-coupled device (EM-CCD) camera (C9100-13, Hamamatsu Photonics). At least two fields of images were recorded for 5 min in each assay, and statistical analysis was made from 11 to 14 independent assays. Observations were carried out at 23 °C. The positions of IC5bio-ES were marked using a program specifically developed to interface with the Halcon image processor (MVTec Software GmbH) as described previously (26Taguchi H. Ueno T. Tadakuma H. Yoshida M. Funatsu T. Nat. Biotechnol. 2001; 19: 861-865Crossref PubMed Scopus (102) Google Scholar). The duration of GroEL-GroES binding was determined by marking the association and dissociation events of Cy3-labeled GroEL molecules. Nonspecific binding of Cy3-labeled GroEL to the glass surface was observed as a flickering spot with a short duration (<1 s) and occurred randomly at all apertures in the observation field. For each analysis, the nonspecific binding of Cy3-labeled GroEL around the position of GroES (25-27% of the total binding events) was subtracted. Rate constants were determined by fitting to the resulting histograms using Equation 1 (17Ueno T. Taguchi H. Tadakuma H. Yoshida M. Funatsu T. Mol. Cell. 2004; 14: 423-434Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 26Taguchi H. Ueno T. Tadakuma H. Yoshida M. Funatsu T. Nat. Biotechnol. 2001; 19: 861-865Crossref PubMed Scopus (102) Google Scholar),N(t)=C{-exp(-k1t)+exp(-k2t)}(Eq. 1) where N(t) is the number of GroEL molecules bound to GroES at time t, and t is the duration of binding of GroEL to GroES (termed on-time), respectively; in addition, k1 and k2 are the rate constants of a two-step reaction, respectively. C means that C = N·k1·k2/(k1 - k2), where N is the total number of GroEL bound to GroES. Henceforth, data fitting was carried out using the Kaleidagraph program (version 3.6; Synergy Software). Initial Kinetic Measurement of ATP Hydrolysis—The amount of inorganic phosphate (Pi) produced was measured with the malachite green assay at 23 °C (17Ueno T. Taguchi H. Tadakuma H. Yoshida M. Funatsu T. Mol. Cell. 2004; 14: 423-434Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). GroEL (3 μm) was incubated in HKM buffer containing rLA (150 μm) and 10 mm DTT in the presence or absence of GroES (9 μm) for 5 min at 23 °C, while being continuously mixed. The reaction was initiated by the addition of the same volume (35 μl) of HKM buffer containing 0.4 mm ATP. At the indicated times, ice-cold perchloric acid (5%) was added to the mixture and then centrifuged to remove protein precipitates. The supernatant was treated with a malachite green reagent, and the absorbance at 630 nm was measured. Rate constants were determined by fitting to the data using the following equations (Equation 2, in the absence of GroES; Equation 3, in the presence of GroES) (17Ueno T. Taguchi H. Tadakuma H. Yoshida M. Funatsu T. Mol. Cell. 2004; 14: 423-434Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar),[PL]t=7k[EL]t(Eq. 2) [PL]t=7k1[EL](k2t-k1/(k1+k2)(exp{-(k1+k2t)}-1))/(k1+k2)(Eq. 3) where [EL] is the concentration of GroEL tetradecamer, and [Pi]t is the concentration of Pi at time t, respectively; in addition, k, k1, and k2 are the rate constants, respectively. Fluorometric Monitoring of GFP Refolding—The denatured GFP (100 nm) was diluted 200-fold in the refolding buffer (HKM buffer plus 5 mm DTT) containing GroEL (150 nm) and GroES (1.5 μm). After a 50-s incubation, ATP (0.45 mm) was injected into the mixture using a microsyringe to initiate the refolding mediated by GroEL (time 0). Spontaneous refolding was initiated by injecting acid-denatured GFP into the refolding buffer (time 0). The fluorescence of GFP at 512 nm with excitation at 485 nm was continuously monitored with an interval of 0.01 s using a Jasco FP-6500 spectrofluorometer. The reaction mixtures were continuously stirred at 23 °C throughout the assays. The dead time of the mixing was about 400 ms. The resultant fluorescence profiles for the first 30 s in the assay were analyzed as described previously (17Ueno T. Taguchi H. Tadakuma H. Yoshida M. Funatsu T. Mol. Cell. 2004; 14: 423-434Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). The rate constants were calculated by fitting to the fluorescence profiles using the following equations (Equation 4, in the absence of GroEL and GroES; Equation 5, in the presence of GroEL and GroES) (17Ueno T. Taguchi H. Tadakuma H. Yoshida M. Funatsu T. Mol. Cell. 2004; 14: 423-434Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar),I(t)=I(∞){1-exp(-kfoldt)}(Eq. 4) I(t)=I(∞)(1-{kfoldexp(-k1t)-k1exp(-kfoldt)}/(kfold-k1))(Eq. 5) in which I(t) and I(∞) are the fluorescence intensities of GFP at time t and the infinite time, respectively; in addition, k1 and kfold are the rate constants at each step, respectively. The rate constants were the average values from three independent experiments. Stopped-flow Anisotropy Measurements—Rhodanese (60 μm) was mixed with a 10-fold amount of TMR-maleimide in TKE buffer (25 mm Tris-HCl, pH 7.4, 50 mm KCl, 0.5 mm EDTA) containing 6 m guanidine-HCl. Unreacted dye was removed by PD-10 column (GE Healthcare), and the extent of the labeling was determined by absorption spectroscopy using the following extinction coefficients (in 6 m guanidine hydrochloride): rhodanese, 49,000 m-1 cm-1 at 280 nm; TMR, 94,000 m-1 cm-1 at 558 nm. Approximately 0.85 mol of TMR were coupled per mol of denatured rhodanese. The denatured TMR-labeled rhodanese (27.8 μm) was diluted 100-fold in TKE buffer with 116 nm GroEL. After incubation for 15 min at room temperature, the aggregated rhodanese was removed by centrifugation. Then the supernatant was applied to a Resource Q column (GE Healthcare), and GroEL complexed with TMR-rhodanese was isolated. Fluorescence anisotropy measurements were carried out at 23 °C using an SX.18MV stopped-flow spectrophotometer equipped with an FP.1 accessory (Applied Photophysics). A solution of 1 μm GroEL complexed with TMR-rhodanese and 5 mm DTT in HKM buffer was loaded into one syringe of the stopped-flow device. A solution of HKM buffer containing 2 μm GroES and 5 mm DTT with or without 10 mm ATP was loaded into the other syringe. Reactions were initiated by mixing equal volumes from each syringe. Excitation was done with vertically polarized light at 515 nm (bandwidth 18.6 nm). Two photomultiplier tubes, both with 550 nm long pass filters (OG550, Schott), were used to monitor the vertical and horizontal polarized emission components simultaneously. Fluorescence anisotropy data were collected with a 50-s log time base. The dead time of the mixing was about 20 ms. The time trajectories of anisotropy shown were the average of 29-34 individual shots. The trajectories were fitted with Equation 6 (17Ueno T. Taguchi H. Tadakuma H. Yoshida M. Funatsu T. Mol. Cell. 2004; 14: 423-434Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar),A(t)=A4+(A1+A4)exp(-k0t)+(A2+A4)k1/(k0/-k1){exp(-k1t)-exp(-k0t)}+(A3-A4)k0k1/(k0-k1)/(k1-k2')/(k0-k2'){(k1-k2')exp(-k0t)-(k0-k2')exp(-k1t)+(k0-k1)exp(-k2't)}(Eq. 6) where A1 - A4 are the anisotropy values at each step, and A(t) is the anisotropy value at time t, respectively; in addition, k0, k1, and k′2 are the rate constants of a three-step reaction, respectively. Truncation of the C-terminal Region Extends the GroEL ATPase Cycle—To examine the role of the C-terminal region in the function cycle of GroEL, we constructed a C-terminal truncated mutant (named as ELtc20), which lacks the final 20 amino acid residues. ELtc20 was expressed in E. coli, and was purified in the same manner as the wild type (termed ELwt). The truncation had no effect on the oligomer formation and slightly decreased the yield in assisted folding of GFP and malate dehydrogenase (data not shown). We first examined the steady-state ATPase rates of ELwt and ELtc20 in the presence or absence of saturating amounts of GroES and substrate proteins (Fig. 1). GroES is known to decrease by about one-half the rate of ATP hydrolysis by GroEL (18Motojima F. Makio T. Aoki K. Makino Y. Kuwajima K. Yoshida M. Biochem. Biophys. Res. Commun. 2000; 267: 842-849Crossref PubMed Scopus (39) Google Scholar). The presence of GroES suppressed the steady-state ATPase rate of ELwt by ∼37% (Fig. 1, open bar). The comparable suppression by GroES (∼35%) was observed in ELtc20 (Fig. 1, filled bar). As reported previously (27Aoki K. Motojima F. Taguchi H. Yomo T. Yoshida M. J. Biol. Chem. 2000; 275: 13755-13758Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 28Rye 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, 29Martin J. Langer T. Boteva R. Schramel A. Horwich A.L. Hartl F.U. Nature. 1991; 352: 36-42Crossref PubMed Scopus (726) Google Scholar), when substrate proteins such as rLA, denatured pepsin, and denatured rhodanese were present, the rate of ATP hydrolysis was enhanced (Fig. 1, open bar). Similarly, the presence of substrate proteins increased in the ATPase rate of ELtc20 (Fig. 1, filled bar). From these results, ELtc20 is considered to have the normal “accelerator” and “brake” for its ATPase rate. However, ELtc20 showed an ∼20% reduction in its steady-state ATPase rate irrespective of the presence of GroES and substrate proteins (p < 0.01, Student's t test). The rates of ELwt and ELtc20 in the presence of GroES and rLA or denatured rhodanese were ∼0.12 and ∼0.10 s-1, respectively. These results suggest that the truncation of the C-terminal region extends the ATPase cycle without impairing the enzyme properties. Truncation of the C-terminal Region Extends the Duration of GroES Binding to GroEL—We previously demonstrated single-molecule imaging of the association and dissociation of the GroEL-GroES complex in the functional GroEL cycle. The dissociation of GroES from GroEL was observed to proceed as a two-step process with rate constants of ∼0.3 and ∼0.2 s-1, respectively (17Ueno T. Taguchi H. Tadakuma H. Yoshida M. Funatsu T. Mol. Cell. 2004; 14: 423-434Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 26Taguchi H. Ueno T. Tadakuma H. Yoshida M. Funatsu T. Nat. Biotechnol. 2001; 19: 861-865Crossref PubMed Scopus (102) Google Scholar) (Fig. 2A). To confirm whether ELtc20 retains the same mechanism to interact with GroES as ELwt, the interaction between ELtc20 and GroES was observed at the single-molecule level (Fig. 2, B-D). In previous studies, biotinylated GroEL was immobilized to the glass surface, and the association and dissociation of fluorescently labeled GroES to and from GroEL were observed (17Ueno T. Taguchi H. Tadakuma H. Yoshida M. Funatsu T. Mol. Cell. 2004; 14: 423-434Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 26Taguchi H. Ueno T. Tadakuma H. Yoshida M. Funatsu T. Nat. Biotechnol. 2001; 19: 861-865Crossref PubMed Scopus (102) Google Scholar). In this study, the interaction was observed between surface-immobilized GroES, a cysteine-introduced variant (ES98C) (19Murai N. Makino Y. Yoshida M. J. Biol. Chem. 1996; 271: 28229-28234Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar) modified with IC5 and biotin (termed IC5bio-ES) and Cy3-labeled GroEL in the presence of saturating amounts of rLA 5Substitution of Asp-490 for cysteine in ELtc20 showed an ∼75% reduction of ATPase activity compared with ELtc20. Moreover, modification of t" @default.
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• W2028351490 title "Effect of the C-terminal Truncation on the Functional Cycle of Chaperonin GroEL" @default.
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