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Time-dependent FRET with single enzymes: domain motions and catalysis in H(+)-ATP synthases.

Chemphyschem : a European journal of chemical physics and physical chemistry (2011-02-03)
Roland Bienert, Boris Zimmermann, Verena Rombach-Riegraf, Peter Gräber
ABSTRACT

H(+)-ATP synthases are molecular machines which couple transmembrane proton transport with ATP synthesis from ADP and inorganic phosphate by a rotational mechanism. Single-pair fluorescence resonance energy transfer (spFRET) in single molecules is a powerful tool to analyse conformational changes. It is used to investigate subunit movements in H(+)-ATP synthases from E. coli (EF(0)F(1)) and from spinach chloroplasts (CF(0)F(1)) during catalysis. The enzymes are incorporated into liposome membranes, and this allows the generation of a transmembrane pH difference, which is necessary for ATP synthesis. After labelling of appropriate sites on different subunits with fluorescence donor and acceptor, the kinetics of spFRET are measured. Analysis of the E(FRET) traces reveals rotational movement of the ε and γ subunits in 120° steps with opposite directions during ATP synthesis and ATP hydrolysis. The stepped movement is characterized by a 120° step faster than 1 ms followed by a rest period with an average dwell time of 15 ms, which is in accordance with the turnover time of the enzyme. In addition to the three conformational states during catalysis, also an inactive conformation is found, which is observed after catalysis.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Atto 532 NHS ester, BioReagent, suitable for fluorescence, ≥80% (coupling to amines)
Sigma-Aldrich
Atto 532 maleimide, BioReagent, suitable for fluorescence, ≥90% (coupling to thiols)

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