title: Single-molecule analysis of F0F1-ATP synthase inhibited by N,N-dicyclohexylcarbodiimide.
abstract: N,N-Dicyclohexylcarbodiimide (DCCD) is a classical inhibitor of the F0F1-ATP synthase (F0F1), which covalently binds to the highly conserved carboxylic acid of the proteolipid subunit (c subunit) in F0. Although it is well known that DCCD modification of the c subunit blocks proton translocation in F0 and the coupled ATP hydrolysis activity of F1, how DCCD inhibits the rotary dynamics of F0F1 remains elusive. Here, we carried out single-molecule rotation assays to characterize the DCCD inhibition of Escherichia coli F0F1. Upon the injection of DCCD, rotations irreversibly terminated with first order reaction kinetics, suggesting that the incorporation of a single DCCD moiety is sufficient to block the rotary catalysis of the F0F1. Individual molecules terminated at different angles relative to the three catalytic angles of F1, suggesting that DCCD randomly reacts with one of the 10 c subunits. DCCD-inhibited F0F1 sometimes showed transient activation; molecules abruptly rotated and stopped after one revolution at the original termination angle, suggesting that hindrance by the DCCD moiety is released due to thermal fluctuation. To explore the mechanical activation of DCCD-inhibited molecules, we perturbed inhibited molecules using magnetic tweezers. The probability of transient activation increased upon a forward forcible rotation. Interestingly, during the termination F0F1, showed multiple positional shifts, which implies that F1 stochastically changes the angular position of its rotor upon a catalytic reaction. This effect could be caused by balancing the angular positions of the F1 and the F0 rotors, which are connected via elastic elements.
authors.affiliation: From the Department of Applied Chemistry, School of Engineering, University of Tokyo, Tokyo 113-8656, Japan.
authors.forename: Masashi
authors.initials: M
authors.lastname: Toei
authors.affiliation: From the Department of Applied Chemistry, School of Engineering, University of Tokyo, Tokyo 113-8656, Japan. Electronic address: hnoji@appchem.t.u-tokyo.ac.jp.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Noji
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004024:Dicyclohexylcarbodiimide / Q000737:chemistry*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011424:Propionibacterium / Q000201:enzymology* / Q000235:genetics; D020836:Protein Structure, Quaternary; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; D021122:Protein Subunits; D004024:Dicyclohexylcarbodiimide; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases
keywords: ATP Synthase; Enzyme Inhibitors; Enzyme Kinetics; Enzyme Mechanisms; Enzyme Structure; F1F0 ATPase; Proton Transport; Single-molecule Biophysics
references.citation: Nat Chem Biol. 2010 Nov;6(11):814-20
references.pmid: 20871600
references.citation: Nature. 2004 Jan 29;427(6973):465-8
references.pmid: 14749837
references.citation: J Bacteriol. 1975 Nov;124(2):870-83
references.pmid: 126994
references.citation: EMBO J. 2009 Sep 16;28(18):2689-96
references.pmid: 19644443
references.citation: Proc Natl Acad Sci U S A. 2003 Dec 9;100(25):14731-6
references.pmid: 14657340
references.citation: J Biol Chem. 2010 Dec 31;285(53):42058-67
references.pmid: 20974856
references.citation: J Biol Chem. 1989 Mar 5;264(7):3896-903
references.pmid: 2521856
references.citation: Trends Biochem Sci. 1997 Nov;22(11):420-3
references.pmid: 9397682
references.citation: Proc Natl Acad Sci U S A. 2003 Nov 11;100(23):13179-83
references.pmid: 14595019
references.citation: Nature. 2009 May 21;459(7245):364-70
references.pmid: 19458712
references.citation: Nat Commun. 2012 Feb 21;3:687
references.pmid: 22353718
references.citation: J Mol Biol. 2006 Apr 21;358(1):132-44
references.pmid: 16497328
references.citation: J Biol Chem. 1983 Dec 10;258(23):14407-12
references.pmid: 6227624
references.citation: Nat Struct Mol Biol. 2009 Oct;16(10):1068-73
references.pmid: 19783985
references.citation: Nat Chem Biol. 2011 Nov 20;8(1):86-92
references.pmid: 22101603
references.citation: Annu Rev Biochem. 1997;66:717-49
references.pmid: 9242922
references.citation: Science. 1999 Nov 26;286(5445):1722-4
references.pmid: 10576736
references.citation: J Biol Chem. 1999 Dec 17;274(51):36261-6
references.pmid: 10593914
references.citation: J Biol Chem. 1980 Jun 25;255(12):5643-8
references.pmid: 6445905
references.citation: J Biol Chem. 2007 Mar 23;282(12):9001-7
references.pmid: 17234633
references.citation: J Biol Chem. 2009 Jun 26;284(26):17457-64
references.pmid: 19411254
references.citation: Proc Natl Acad Sci U S A. 2008 Nov 18;105(46):17760-5
references.pmid: 19001275
references.citation: Biochim Biophys Acta. 2000 May 31;1458(2-3):482-510
references.pmid: 10838060
references.citation: Nat Commun. 2013;4:1631
references.pmid: 23535652
references.citation: Nat Struct Biol. 2000 Nov;7(11):1055-61
references.pmid: 11062563
references.citation: Nat Chem Biol. 2010 Dec;6(12):891-9
references.pmid: 20972431
references.citation: Biochemistry. 1994 Jan 25;33(3):665-74
references.pmid: 8292594
references.citation: Nat Rev Mol Cell Biol. 2001 Sep;2(9):669-77
references.pmid: 11533724
references.citation: Proc Natl Acad Sci U S A. 2001 Apr 24;98(9):4966-71
references.pmid: 11320246
references.citation: J Biochem. 2011 Jun;149(6):655-64
references.pmid: 21524994
references.citation: Biochim Biophys Acta. 2002 Feb 15;1553(3):188-211
references.pmid: 11997128
references.citation: Proc Natl Acad Sci U S A. 1980 Feb;77(2):785-9
references.pmid: 6444724
references.citation: Proc Natl Acad Sci U S A. 2005 Feb 1;102(5):1333-8
references.pmid: 15668386
references.citation: FEBS Lett. 2000 Mar 31;470(3):244-8
references.pmid: 10745076
references.citation: Proc Natl Acad Sci U S A. 2005 Mar 22;102(12):4288-93
references.pmid: 15758075
references.citation: EMBO J. 2006 Jun 21;25(12):2911-8
references.pmid: 16791136
references.citation: Biochemistry. 1995 Dec 19;34(50):16412-8
references.pmid: 8845368
references.citation: Cell. 2007 Jul 27;130(2):309-21
references.pmid: 17662945
references.citation: Eur Biophys J. 2010 Nov;39(12):1589-96
references.pmid: 20549499
references.citation: Nature. 1994 Aug 25;370(6491):621-8
references.pmid: 8065448
references.citation: Cell. 1998 Jun 26;93(7):1117-24
references.pmid: 9657145
references.citation: J Biol Chem. 1998 Oct 23;273(43):27873-8
references.pmid: 9774398
references.citation: J Bacteriol. 2003 Aug;185(15):4442-9
references.pmid: 12867453
references.citation: Science. 1999 Nov 26;286(5445):1700-5
references.pmid: 10576729
references.citation: Biochemistry. 1995 Dec 12;34(49):16186-93
references.pmid: 8519776
references.citation: FEBS Lett. 1999 Apr 16;449(1):1-6
references.pmid: 10225416
references.citation: EMBO J. 2006 Oct 4;25(19):4596-604
references.pmid: 16977308
references.citation: EMBO J. 2010 Dec 1;29(23):3911-23
references.pmid: 21037553
references.citation: J Biol Chem. 2003 Feb 21;278(8):6066-74
references.pmid: 12473663