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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 FoF1-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 FoF1-ATP synthase performs this task by proton-driven internal subunit rotation. FoF1-ATP synthases comprise a membrane-embedded Fo 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 Fo 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).