Real-time Monitoring of Conformational Dynamics of the ϵ Subunit in F1-ATPase

Abstract

It has been proposed that C-terminal two α-helices of the ϵ subunit of F1-ATPase can undergo conformational transition between retracted folded-hairpin form and extended form. Here, using F1 from thermophilic Bacillus PS3, we monitored this transition in real time by fluorescence resonance energy transfer (FRET) between a donor dye and an acceptor dye attached to N terminus of the γ subunit and C terminus of the ϵ subunit, respectively. High FRET (extended form) of F1 turned to low FRET (retracted form) by ATP, which then reverted as ATP was hydrolyzed to ADP. 5′-Adenyl-β,γ-imidodiphosphate, ADP + AlF4-, ADP + NaN3, and GTP also caused the retracted form, indicating that ATP binding to the catalytic β subunits induces the transition. The ATP-induced transition from high FRET to low FRET occurred in a similar time scale to the ATP-induced activation of ATPase from inhibition by the ϵ subunit, although detailed kinetics were not the same. The transition became faster as temperature increased, but the extrapolated rate at 65 °C (physiological temperature of Bacillus PS3) was still too slow to assign the transition as an obligate step in the catalytic turnover. Furthermore, binding affinity of ATP to the isolated ϵ subunit was weakened as temperature increased, and the dissociation constant extrapolated to 65 °C reached to 0.67 mm, a consistent value to assume that the ϵ subunit acts as a sensor of ATP concentration in the cell. It has been proposed that C-terminal two α-helices of the ϵ subunit of F1-ATPase can undergo conformational transition between retracted folded-hairpin form and extended form. Here, using F1 from thermophilic Bacillus PS3, we monitored this transition in real time by fluorescence resonance energy transfer (FRET) between a donor dye and an acceptor dye attached to N terminus of the γ subunit and C terminus of the ϵ subunit, respectively. High FRET (extended form) of F1 turned to low FRET (retracted form) by ATP, which then reverted as ATP was hydrolyzed to ADP. 5′-Adenyl-β,γ-imidodiphosphate, ADP + AlF4-, ADP + NaN3, and GTP also caused the retracted form, indicating that ATP binding to the catalytic β subunits induces the transition. The ATP-induced transition from high FRET to low FRET occurred in a similar time scale to the ATP-induced activation of ATPase from inhibition by the ϵ subunit, although detailed kinetics were not the same. The transition became faster as temperature increased, but the extrapolated rate at 65 °C (physiological temperature of Bacillus PS3) was still too slow to assign the transition as an obligate step in the catalytic turnover. Furthermore, binding affinity of ATP to the isolated ϵ subunit was weakened as temperature increased, and the dissociation constant extrapolated to 65 °C reached to 0.67 mm, a consistent value to assume that the ϵ subunit acts as a sensor of ATP concentration in the cell. A rotary motor F1-ATPase (F1) 2The abbreviations used are: F1F1-ATPaseF0F1F0F1-ATP synthaseFRETfluorescence resonance energy transferAMPPNP5′-adenyl-β,γ-imidodiphosphate. is a water-soluble portion of F0F1-ATP synthase, which catalyzes ATP synthesis/hydrolysis coupled with a transmembrane proton translocation (1Boyer P.D. Annu. Rev. Biochem. 1997; 66: 717-749Crossref PubMed Scopus (1617) Google Scholar, 2Yoshida M. Muneyuki E. Hisabori T. Nat. Rev. Mol. Cell. Biol. 2001; 2: 669-677Crossref PubMed Scopus (732) Google Scholar). F1 has a subunit structure of α3β3γδϵ; α and β subunits have a non-catalytic and catalytic nucleotide binding sites, respectively; γ subunit rotates in the α3β3 ring; δ subunit connects the ring to the stator part of F0; and ϵ subunit rotates together with γ subunit as a body. The ϵ subunit (∼14 kDa) has a regulatory function and consists of N-terminal β-sandwitch and C-terminal two α-helices (3Uhlin U. Cox G.B. Guss J.M. Structure (Camb.). 1997; 5: 1219-1230Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 4Wilkens S. Capaldi R.A. J. Biol. Chem. 1998; 273: 26645-26651Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). F1-ATPase F0F1-ATP synthase fluorescence resonance energy transfer 5′-adenyl-β,γ-imidodiphosphate. Previous structural studies of F1 indicated two conformations of the ϵ subunit with different arrangement of the two α-helices, that is, retracted folded-hairpin form and partly extended form (Fig. 1, A and B) (5Gibbons C. Montgomery M.G. Leslie A.G. Walker J.E. Nat. Struct. Biol. 2000; 7: 1055-1061Crossref PubMed Scopus (438) Google Scholar, 6Rodgers A.J. Wilce M.C. Nat. Struct. Biol. 2000; 7: 1051-1054Crossref PubMed Scopus (153) Google Scholar, 7Hausrath A.C. Capaldi R.A. Matthews B.W. J. Biol. Chem. 2001; 276: 47227-47232Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 8Tsunoda S.P. Rodgers A.J. Aggeler R. Wilce M.C. Yoshida M. Capaldi R.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6560-6564Crossref PubMed Scopus (164) Google Scholar). Cross-linking studies suggested the third conformation with fully extended α-helices 3Recently, Y. Shirakihara and M. Yoshida and T. Suzuki determined the crystal structure of F1 that has the ϵ subunit with fully extended α-helices (unpublished data). (Fig. 1C) (9Suzuki T. Murakami T. Iino R. Suzuki J. Ono S. Shirakihara Y. Yoshida M. J. Biol. Chem. 2003; 278: 46840-46846Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). Biochemical data have indicated that the ϵ subunit adopts the extended form in the absence of nucleotide or in the presence of ADP, in which ATPase activity is inhibited, and that ATP counteracts ADP by favoring the retracted form, which is a non-inhibitory conformation (9Suzuki T. Murakami T. Iino R. Suzuki J. Ono S. Shirakihara Y. Yoshida M. J. Biol. Chem. 2003; 278: 46840-46846Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). Thus, it appears that the regulatory function of the ϵ subunit is dependent on the drastic conformational transition that is affected by nucleotide and other factors. However, previous studies have not provided kinetic information on how these dynamic conformational transitions occur in the enzyme at work. Fluorescence resonance energy transfer (FRET) is a powerful technique that enables us to probe conformational dynamics of biological molecular machines (10Stryer L. Annu. Rev. Biochem. 1978; 47: 819-846Crossref PubMed Scopus (1981) Google Scholar). In this study, this technique was applied to the ϵ subunit in F1 from thermophilic Bacillus PS3. Real-time FRET monitoring revealed slow, reversible conformational change of the ϵ subunit, indicating that the ϵ subunit exerts a slow switch-like regulation. In addition, direct binding of ATP to the isolated ϵ subunit was probed by fluorescence change of the dye attached to the ϵ subunit. Submillimolar affinity of the ϵ subunit to ATP at the physiological temperature of Bacillus PS3 (65 °C) suggests a novel function of the ϵ subunit as an ATP concentration sensor in vivo. Protein Preparation—A mutant of the α3β3γ subcomplex (αC193S, βHis-10 at N terminus, γS3C) and mutants of the ϵ subunit (134C or Q107C) of F1 from thermophilic Bacillus PS3 were purified as described (11Shimabukuro K. Yasuda R. Muneyuki E. Hara K.Y. Kinosita Jr., K. Yoshida M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 14731-14736Crossref PubMed Scopus (224) Google Scholar, 12Kato-Yamada Y. Yoshida M. J. Biol. Chem. 2003; 278: 36013-36016Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). ATPase activity of reconstituted α3β3γϵ subcomplex (referred as F1 hereafter) was measured as described previously (9Suzuki T. Murakami T. Iino R. Suzuki J. Ono S. Shirakihara Y. Yoshida M. J. Biol. Chem. 2003; 278: 46840-46846Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). A molecular extinction coefficient of 154,000 at 280 nm was used for the determination of F1 concentration. Reconstitution of Fluorescent-labeled α3β3γϵ Complex—For FRET measurement, α3β3γ subcomplex and ϵ subunit (134C) were reacted with Cy3- and Cy5-maleimide (Amersham Biosciences, dye/protein molar ratio was 2) in PA5 buffer (50 mm HEPES-KOH, 5 mm MgCl2, 100 mm KCl, pH 7.5) for 30 min at 25 °C, respectively, and unreacted dyes were removed by gel filtration. Labeling ratio of dye to protein was ∼0.5 and >0.9 for α3β3γ(S3C-Cy3) and ϵ(134C-Cy5), respectively (judged by SDS-PAGE). For reconstitution of α3β3γ(S3C-Cy3) ϵ(134C-Cy5) subcomplex, α3β3γ(S3C-Cy3) and ϵ(134C-Cy5) were mixed (molar ratio of ϵ subunit to α3β3γ was 3) and incubated for 3 h at 25°C, and excess ϵ(134C-Cy5) was removed by ultrafiltration. Reconstituted F1 contained 0.66 mol of ADP and 0.26 mol of ATP/mol of F1 and retained 76% steady-state ATPase activity as compared with that of wild type (134 ± 4.0 s-1 measured at 50 °C). From this result we concluded that dye-labeled F1 used in this study was functional. Molecular extinction coefficients of 150,000 at 550 nm (Cy3) and 250,000 at 650 nm (Cy5) were used for determination of concentration. FRET Measurement—FRET from the donor to the acceptor in the F1 was monitored with a fluorescence spectrometer (FP-3000, Hitachi). Donor was selectively excited with the light of 532 nm (bandwidth was 3 nm). For time course measurements, fluorescence intensity of donor was measured at 565 nm (bandwidth was 10 nm). Measurements were carried out in PA5 buffer, and an ATP (GTP) regenerating system (5 mm phosphoenolpyruvate and 0.5 mg/ml pyruvate kinase) was added in some experiments as described. Contamination of ATP in the solutions of AMPPNP and GTP was less than 0.1% (estimated by high performance liquid chromatography). Steady-state fluorescence anisotropy of protein-conjugated Cy3 and Cy5 in the buffer PA5 was measured in a cuvette with a fluorescence spectrometer (FP-3000). Measurement of Affinity between Nucleotides and Isolated ϵ Subunit—The Q107C mutant of the ϵ subunit was reacted with Cy3-maleimide (dye/protein molar ratio was 2), and unreacted dyes were removed by gel filtration. The labeling ratio of dye to protein was >0.9. Nucleotide was added to ϵ(Q107C-Cy3) in PA5 buffer, and fluorescence intensity was measured with a fluorescence spectrometer (FP-3000). Cy3 was excited with the light of 532 nm (bandwidth was 3 nm), and fluorescence intensity was measured at 565 nm (bandwidth was 10 nm). For the measurement of ADP affinity, hexokinase (6.7 units/ml) and glucose (200 mm) were supplemented to the buffer, and contaminated ATP was converted to ADP. Nucleotide-dependent, Reversible Conformational Change of the ϵ Subunit—In a fluorescence spectrum of α3β3γ(S3C-Cy3)ϵ(134C-Cy5) obtained by excitation of donor dye (Cy3) at 532 nm (Fig. 2A, blue solid line), the peak of donor (Cy3, around 570 nm) decreased, as compared with the spectrum of α3β3γ(S3C-Cy3) (blue dashed line). Concomitantly the peak of acceptor (Cy5, around 670 nm) increased, indicating FRET from donor to acceptor with high efficiency. FRET was further confirmed by recovery of donor fluorescence intensity after photobleaching of acceptor by excitation at 670 nm (data not shown). When ATP (1 mm) was added, although the spectra of α3β3γ(S3C-Cy3) (red dashed line) and ϵ(134C-Cy5) (red dotted line) did not change, intensities of donor and acceptor fluorescence of α3β3γ(S3C-Cy3)ϵ(34C-Cy5) (red solid line) were increased and decreased, respectively. This ATP-induced transition from high FRET to low FRET primarily reflects the conformational change of the ϵ subunit because the γ and ϵ subunits rotate together as a body and the γ subunit does not undergo large conformational transition during catalysis. Hereafter, α3β3γ(S3C-Cy3)ϵ(134C-Cy5) subcomplex was referred as F1 in this study and was used for FRET measurement. Apparent FRET efficiency before and after addition of ATP, estimated from the spectra shown in Fig. 2A, was about 70 and 20%, respectively. It should be mentioned that the distance between donor and acceptor could not be estimated precisely from FRET efficiency in our experiments, since steady-state fluorescence anisotropy of Cy3 and Cy5 conjugated with γS3C and ϵ134C was high A high value of the anisotropy that the of the dye conjugated with F1 is and the of the distance with high is However, that the distance distance which FRET is around 5 nm (10Stryer L. Annu. Rev. Biochem. 1978; 47: 819-846Crossref PubMed Scopus (1981) Google high and low FRET the conformation distance between is nm) and the nm) conformation of the ϵ subunit, respectively (Fig. it was to between (Fig. and (Fig. 1C) conformations in this study, these two conformations were together and were referred as the extended of the change of FRET efficiency by were monitored by the change of the donor fluorescence mm ATP increased donor fluorescence intensity (Fig. line). the ADP (1 mm) did not the change of donor fluorescence (Fig. blue line). AMPPNP (1 which can to F1 but is not hydrolyzed Leslie A.G. R. Walker J.E. PubMed Scopus Google also caused the in donor although the change was than the of ATP (Fig. light blue line). ATP binding but not induces the conformational change from the extended form to the retracted form. ADP + NaN3, which induces and the catalytic of ATP by F1 is by of at the catalytic The binding of ATP to the α subunits the of from the affected catalytic sites, the ATP This is different from the inhibition and is for and ATP from of F1 E. M. Y. Yoshida M. PubMed Scopus Google and ADP + AlF4-, a of ATP K. Montgomery M.G. Leslie A.G. Walker J.E. Struct. 2000; Full Text Full Text PDF Scopus Google also increased the donor fluorescence intensity (Fig. and Furthermore, GTP also the conformational change (Fig. GTP is a of F1 and Yasuda R. Yoshida M. Kinosita Jr., K. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google but has low affinity to the isolated ϵ subunit Fig. Y. Yoshida M. J. Biol. Chem. 2003; 278: 36013-36016Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). The conformational change of the ϵ subunit was When added ATP in the was converted ADP by addition of excess of hexokinase the change was reverted (Fig. direct for reversible conformational of the ϵ subunit that was previously suggested from (9Suzuki T. Murakami T. Iino R. Suzuki J. Ono S. Shirakihara Y. Yoshida M. J. Biol. Chem. 2003; 278: 46840-46846Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). of from and Conformational activity of F1 from thermophilic Bacillus PS3 is by the ϵ subunit but the inhibition is by addition of ATP Y. T. Muneyuki E. Hisabori T. Yoshida M. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). it is that the extended ϵ subunit has on ATPase activity of F1 but the retracted ϵ subunit does not S.P. Rodgers A.J. Aggeler R. Wilce M.C. Yoshida M. Capaldi R.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6560-6564Crossref PubMed Scopus (164) Google Scholar, T. Murakami T. Iino R. Suzuki J. Ono S. Shirakihara Y. Yoshida M. J. Biol. Chem. 2003; 278: 46840-46846Abstract Full Text Full Text PDF PubMed Scopus (133) Google recovery of ATPase activity from inhibition should of donor fluorescence and we compared time ATPase activity of F1 is after of the by addition of ATP (Fig. Y. T. Muneyuki E. Hisabori T. Yoshida M. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). ATP concentration increased, recovery occurred of the recovery could be by a function transition between the and (Fig. line). Apparent rate estimated from the became as ATP concentration increased (Fig. The in donor fluorescence occurred in a time scale similar to ATPase However, the time could be by a function with intensities than by a function (Fig. and This suggests the presence of or low FRET and rate obtained from the function increased in a as ATP The rate a with the rate of recovery of ATPase activity at ATP (Fig. and However, the was (Fig. of Conformational temperature of thermophilic Bacillus PS3 is about 65 °C, and F1 from this ATPase activity around this temperature M. Y. J. Biol. Chem. Full Text PDF PubMed Google Scholar). temperature of the rate of the conformational change of the ϵ subunit was and in donor fluorescence were by addition of ATP (1 mm) and by ATP respectively. in were dependent on a fluorescence it and at 30 °C, and and at for and were by the function at of rate and slow for increased as temperature increased in and the were in the temperature from 30 to °C (Fig. C and The activation from to and high activation energy is consistent with large conformational change of the ϵ subunit. measurement at 65 °C was since the ATP regenerating system and hexokinase were we that the of the was still retained at 65 °C, the extrapolated rate out of two rate for the transitions reached to s-1 and s-1 than that of ATPase Affinity of Nucleotides to the Isolated ϵ previous study using gel that the isolated ϵ subunit ATP with high affinity at 25 °C dissociation constant was to be than 10 Y. Yoshida M. J. Biol. Chem. 2003; 278: 36013-36016Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). However, of the dissociation constant by gel was In this study, we applied fluorescence technique to nucleotide binding to the isolated ϵ subunit. When Cy3 was conjugated with a mutant that has a at the between two α-helices of the ϵ subunit, addition of a high concentration of nucleotide caused an in the fluorescence intensity of Cy3 to the change around the by nucleotide binding (Fig. the change in the fluorescence was concentration of the were by the function the binding (Fig. The dissociation of ATP and ADP to the isolated ϵ subunit at °C, estimated by the were and respectively. The affinity of ATP was about 100 than that of ADP. Affinity of GTP was than that of ATP, and the dissociation constant at °C was that the value of dissociation constant for GTP is a since contaminated ATP in GTP in our could to the ϵ subunit and the Cy3 of the affinity between ATP and the isolated ϵ subunit was also The dissociation constant between ATP and the isolated ϵ subunit increased as temperature increased (Fig. and the of dissociation constant temperature (Fig. The dissociation constant extrapolated to the physiological temperature (65 °C) was 0.67 Conformational Change of the ϵ in F1 a a large conformational change of the ϵ subunit in F1 F0F1-ATP has been suggested from previous structural and studies (3Uhlin U. Cox G.B. Guss J.M. Structure (Camb.). 1997; 5: 1219-1230Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 4Wilkens S. Capaldi R.A. J. Biol. Chem. 1998; 273: 26645-26651Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, C. Montgomery M.G. Leslie A.G. Walker J.E. Nat. Struct. Biol. 2000; 7: 1055-1061Crossref PubMed Scopus (438) Google Scholar, 6Rodgers A.J. Wilce M.C. Nat. Struct. Biol. 2000; 7: 1051-1054Crossref PubMed Scopus (153) Google Scholar, 7Hausrath A.C. Capaldi R.A. Matthews B.W. J. Biol. Chem. 2001; 276: 47227-47232Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 8Tsunoda S.P. Rodgers A.J. Aggeler R. Wilce M.C. Yoshida M. Capaldi R.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6560-6564Crossref PubMed Scopus (164) Google Scholar, T. Murakami T. Iino R. Suzuki J. Ono S. Shirakihara Y. Yoshida M. J. Biol. Chem. 2003; 278: 46840-46846Abstract Full Text Full Text PDF PubMed Scopus (133) Google Y. Yoshida M. Hisabori T. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google FRET measurement for the time us to it in real FRET measurement that in the presence of a of ATP, the change from high FRET to low FRET °C, Fig. Thus, the ATP-induced conformational change of the ϵ subunit in F1 is than the catalytic R. Yoshida M. Kinosita Jr., K. 2001; PubMed Scopus Google that it be of obligate in of the catalytic but it exerts a switch-like or change function of the catalysis. Conformational Change of the ϵ in F1 at the we ATP binding to the β subunit induces an of the γ subunit R. Yoshida M. Kinosita Jr., K. 2001; PubMed Scopus Google Scholar). the of ATP and catalytic the of ADP or occur in at this (11Shimabukuro K. Yasuda R. Muneyuki E. Hara K.Y. Kinosita Jr., K. Yoshida M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 14731-14736Crossref PubMed Scopus (224) Google and the to a a by of ATP it was shown that F1 at the Y. M. Muneyuki E. Hara K.Y. Yasuda R. Kinosita Jr., K. Yoshida M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: PubMed Scopus (164) Google Scholar). that not ATP, but also ADP + and ADP + the transition from high FRET to low FRET (Fig. nucleotide to the Leslie A.G. R. Walker J.E. PubMed Scopus Google Scholar, R. T. K. Kinosita Jr., K. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: PubMed Scopus Google the E. M. Y. Yoshida M. PubMed Scopus Google and the catalytic K. Montgomery M.G. Leslie A.G. Walker J.E. Struct. 2000; Full Text Full Text PDF Scopus Google respectively, and the it is that transition of α-helices of the ϵ subunit from the extended form to the retracted form can the γ subunit in F1 at the between from and Conformational Change of the ϵ of ATPase from the inhibition by the ϵ subunit and FRET change by ATP addition occurred in a similar time but time did not The real of this is not of be In an to we supplemented a to the of ATPase and FRET measurement and the experiments of 3 and and has been to F1 not to inhibition J.M. PubMed Scopus Google Scholar). The FRET change became faster in the presence of the time course of recovery of ATPase was binding to the isolated ϵ subunit was not affected by at °C). However, the kinetics of ATPase recovery and FRET change still that our F1 could be and some of the that not the ϵ subunit be from the that FRET at the of F1 ATP to the ϵ the isolated ϵ subunit of F1 from thermophilic Bacillus PS3 ATP Y. Yoshida M. J. Biol. Chem. 2003; 278: 36013-36016Abstract Full Text Full Text PDF PubMed Scopus (70) Google and our crystal structure of the has revealed that ATP to the ϵ subunit in a retracted conformation with folded-hairpin Y. and M. we assume that ATP binding to the ϵ subunit the folded-hairpin form of the ϵ subunit in F1 to the from the extended form. In the absence of ATP, the ϵ subunit adopts the extended form in of time and conformational change by be A is that ATP binding to the β subunits the for the ϵ subunit with the extended form to conformational transition to the folded-hairpin form to which ATP and the folded-hairpin form is nucleotide binding on the α subunits not in this change because mutant T. Muneyuki E. M. C. Yoshida M. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google also conformational change by ATP (data not Y. Yoshida M. Hisabori T. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). A of conformational dynamics of the ϵ subunit at the physiological temperature of thermophilic Bacillus PS3 (65 °C) is shown in Fig. It should be that ATP binding affinity of the ϵ subunit at the physiological temperature 0.67 mm at 65 °C) is to the ATP concentration that is in to This affinity can the ATP binding to the ϵ subunit was for thermophilic Bacillus PS3 but not for other as Y. Yoshida M. J. Biol. Chem. 2003; 278: 36013-36016Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). the ϵ subunit from °C have similar affinity to ATP, the binding not be by gel at The ϵ an or a at mm ATP, ϵ subunit the retracted form in a of min after of ATP (Fig. and an of the ϵ subunit was for a (Fig. However, the be different for F0F1-ATP synthase in vivo. In our previous a of the ϵ subunit in the fully extended form in the presence of high of ATP of the ϵ subunit was in the fully extended form at mm (9Suzuki T. Murakami T. Iino R. Suzuki J. Ono S. Shirakihara Y. Yoshida M. J. Biol. Chem. 2003; 278: 46840-46846Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). The was further increased by the proton the compared with the of the conformation of the ϵ subunit in be to the fully extended form in and the ϵ subunit ATP Furthermore, it was shown that reconstitution of the ϵ subunit the α3β3γ subcomplex the efficiency of ATP F1 was in the ATP with Y. T. Y. S. PubMed Scopus Google Scholar). This result that the ϵ subunit acts not as an of ATP but also as a for the ATP as suggested before Y. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the of the ϵ subunit is it is how the proton the conformation of the ϵ subunit and how the ϵ subunit the efficiency of ATP of the of the the of applied to the ϵ subunit, conformation and function in FRET indicated that was conformational change in the N-terminal β-sandwitch part of the ϵ subunit during ATP and by from E. M. M. J. PubMed Scopus Google Scholar). The in the conformational and dynamics of the C-terminal α-helices of the ϵ subunit during ATP and also be probed by the FRET measurement during the of T. K. and for with