The α3β3γ subcomplex of the catalytic F1 portion of the F0F1 ATP synthase is a tiny rotary motor driven by hydrolysis of ATP . We have undertaken single molecule spectroscopic experiments to examine the mechanism by which the motor generates sufficient torque to cause the unidirectional rotation of the centrally located γ subunit. To ensure that data collected from single molecules is representative of the whole protein ensemble we have sought to obtain uniform populations of the protein bound to a flat surface in a single orientation with respect to the probing laser beam.
Hybrid α3β3γ sub-complexes of the F1-ATPase were assembled using recombinant α and β subunits from the photosynthetic bacterium Rhodospirillum rubrum and recombinant β and γ subunits from spinach chloroplasts using previously established procedures . Histidine6 tags were engineered at the N-termini of the α-subunits prior to assembly into the α3β3γ complex. The purified complex was bound to mica that had been pre-coated with nickel chloride, and analyzed in a liquid flow cell by atomic force microscopy (AFM). The hexameric ring formed by alternating α and β subunits was clearly evident in AFM images. An additional mass, located in the center of the ring and protruding above the plane of the ring by about 2 nm and away from the substrate surface, was assumed to be the main part of the γ subunit. This orientation of the γ subunit is consistent with the expected orientation of the F1 when bound to the substrate surface by a histidine-nickel interaction. Unlike earlier experiments in which the protein was anchored to a glass surface that had been coated with a nickel - NTA (nitrilotriacetic acid) conjugate , the nickel-coated mica surface used in these experiments appeared to be uniformly flat.
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Cindy L. Berrie
Department of Chemistry, University of Kansas
1027 Malott Hall, University of Kansas, Lawrence, KS 66045 USA
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