TCBG Seminar

Stepwise Rotation of the Gamma-subunit of EfoF1 ATP Synthase During ATP Synthesis and Hydrolysis - A Single-molecule FRET Approach

Dr. Michael Boersch
Universitat Stuttgart

Monday, February 3, 2003
3:00 pm (CT)
3269 Beckman Institute


H+ ATP synthases catalyze the formation of ATP by coupling two rotary motors within the enzyme. To monitor inter-subunit rotation during ATP hydrolysis, we attached two fluorophores - Cy5 at the rotating gamma-subunit and Tetramethylrhodamine (TMR) at one of the two b-subunits - and reconstituted the enzymes fully functional into liposomes. Fluorescence resonance energy transfer (FRET) was monitored in photon bursts of freely diffusing ATP synthases with a confocal setup for single-molecule detection. Incubation with non-hydrolysable AMPPNP resulted in stable intensity ratios within a burst and three different FRET efficiencies corresponding to the three possible orientations of the gamma-subunit. Upon adding ATP at mM concentrations, a consecutive order of three distinguishable FRET efficiencies was observed indicating a stepwise movement of the gamma-subunit. Under the conditions for ATP synthesis, i.e. energisation of the proteoliposomes by an acid-base-transition, we were recently able to detect the stepwise rotation of the gamma-subunit with this single-molecule FRET approach using Cy5-bis-maleimide as a crosslinker for the two b-subunits and TMR as FRET donor on the gamma-subunit. The direction of rotation is opposite to the direction observed during ATP hydrolysis.

Tea and coffee will be served in R3151 Beckman Institute at 2:15pm.

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