F_oF₁-ATP synthase is the enzyme that provides the 'chemical energy currency' adenosine triphosphate, ATP, for living cells. The formation of ATP is accomplished by a stepwise internal rotation of subunits within the enzyme. Briefly, proton translocation through the membrane-bound F_o part of ATP synthase drives a 10-step rotary motion of the ring of c subunits with respect to the non-rotating subunits a and b. This rotation is transmitted to the γ and ε subunits of the F₁ sector resulting in 120° steps. In order to unravel this symmetry mismatch we monitor subunit rotation by a single-molecule fluorescence resonance energy transfer (FRET) approach using three fluorophores specifically attached to the enzyme: one attached to the F₁ motor, another one to the F_o motor, and the third one to a non-rotating subunit. To reduce photophysical artifacts due to spectral fluctuations of the single fluorophores, a duty cycle-optimized alternating three-laser scheme (DCO-ALEX) has been developed. Simultaneous observation of the stepsizes for both motors allows the detection of reversible elastic deformations between the rotor parts of F_o and F₁.

Date Published: 24 February 2009
PDF: 14 pages
Proc. SPIE 7185, Single Molecule Spectroscopy and Imaging II, 718505 (24 February 2009); doi: 10.1117/12.809610

Published in SPIE Proceedings Vol. 7185:
Single Molecule Spectroscopy and Imaging II
Jörg Enderlein; Zygmunt Karol Gryczynski; Rainer Erdmann, Editor(s)