Dresden 2011 – scientific programme

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BP: Fachverband Biologische Physik

BP 33: Biological Machines \& Motor Proteins

BP 33.4: Talk

Friday, March 18, 2011, 11:15–11:30, ZEU 250

Using single-molecule FRET to determine the stepsize of the rotating c-ring of F_oF₁-ATP synthase with DCO-ALEX — •Eva Hammann, Stefan Ernst, Andrea Zappe, Jörg Wrachtrup, and Michael Börsch — 3.Physikalisches Institut, Universität Stuttgart, Pfaffenwaldring 57, 70569 Stuttgart, Germany

ATP production is essential for life. The enzyme F_oF₁-ATP synthase performs this task by proton-driven internal subunit rotation. F_oF₁-ATP synthases comprise a membrane-embedded F_o part and a protruding F₁ part in the inner membranes. The F₁ part consists of 5 different subunits with the stoichiometry α₃β₃γδє. The α- and β-subunits form three catalytic binding sides for the reaction of ADP and phosphate to ATP and for the reversed ATP hydrolysis direction. The F_o part consists of three different subunits. The a- and b₂- , δ-, α- and β-subunits form a stator part. The 10 c-subunits (in E. coli bacteria) are arranged in a ring and form the rotary motor with the γ- and є-subunits. The driving force is a proton current through the a-subunit and the c-ring. The rotation of the c-ring can be visualized by labeling the a-subunit and one c-subunit with two different fluorophores and measuring steps by single-molecule Förster resonance energy transfer (FRET). For ATP synthesis activity the protein must be reconstituted in an artificial membrane, or for longer observation times, has to be immobilized on a Ni-NTA-surface. Here we show rotary motion of the c-ring by FRET using an duty-cycle optimized alternating laser excitation scheme (DCO-ALEX).