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Single molecule analysis of the F1-ATPase rotary motor

Sirisha Kodeboyina, Jill E. Headrick, Mark Richter, and Cindy L. Berrie*

Departments of Molecular Biosciences and Chemistry, University of Kansas,
Lawrence, KS 66045 USA

This is an abstract for a presentation given at the
10th Foresight Conference on Molecular Nanotechnology

 

The α3β3γ subcomplex of the catalytic F1 portion of the F0F1 ATP synthase is a tiny rotary motor driven by hydrolysis of ATP [1]. 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 [2]. 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[3], 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 [1], the nickel-coated mica surface used in these experiments appeared to be uniformly flat.

References

  1. Noji, H., Yasuda, R., Yoshida, M., & Kinosita Jr. K. (1997) Nature 386, 299-302.
  2. Tucker, W.C., Du, Z., Hein, R., Gromet-Elhanan, Z. & Richter, M.L. (2001) Eur.J.Biochem. 268, 2179-2186.
  3. C. R. III, V. M. Keivens, J. E. Hale, K.K. Nakamura, R. A. Jue, S. Cheng, E. D. Melcher, B. Drake, and M. C. Smith (1993) Biophys. J. 64, 919-924.

Abstract in Microsoft Word® format 24,390 bytes


*Corresponding Address:
Cindy L. Berrie
Department of Chemistry, University of Kansas
1027 Malott Hall, University of Kansas, Lawrence, KS 66045 USA
Phone: (785)-864-3089 Fax: (785) 864-5396
Email: cberrie@ukans.edu



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