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  • Single-molecule analysis of F0F1-ATP synthase inhibited by N,N-dicyclohexylcarbodiimide.

Single-molecule analysis of F0F1-ATP synthase inhibited by N,N-dicyclohexylcarbodiimide.

The Journal of biological chemistry (2013-07-31)
Masashi Toei, Hiroyuki Noji
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.

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Product Description

Sigma-Aldrich
DCC, puriss., ≥99.0% (GC)
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20 μg
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50 μg
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Sigma-Aldrich
DCC, 99%
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SKUPack SizeAvailabilityPrice
20 μg
1 Estimated to ship on 25 February 2025
JP¥38,100
50 μg
1 Estimated to ship on 25 February 2025
JP¥68,300

To order products, please contact your local dealer.

Sigma-Aldrich
DCC, 1.0 M in methylene chloride
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SKUPack SizeAvailabilityPrice
20 μg
1 Estimated to ship on 25 February 2025
JP¥38,100
50 μg
1 Estimated to ship on 25 February 2025
JP¥68,300

To order products, please contact your local dealer.