Biophysical Journal 90:4195-4203 (2006)

The Rotor Tip Inside a Bearing of a Thermophilic F1-ATPase Is Dispensable for Torque Generation

Mohammad Delawar Hossain * , Shou Furuike *, Yasushi Maki , Kengo Adachi *, M. Yusuf Ali , Mominul Huq , Hiroyasu Itoh  ||, Masasuke Yoshida ** and Kazuhiko Kinosita, Jr. *

* Department of Physics, Faculty of Science and Engineering, Waseda University, Tokyo 169-8555, Japan; Department of Physics, School of Physical Sciences, Shahjalal University of Science and Technology, Sylhet-3114, Bangladesh; Department of Physics, Faculty of Engineering, Bangladesh University of Engineering and Technology, Dhaka-1000, Bangladesh; Department of Biology, Faculty of Science, Niigata University, Niigata 950-2181, Japan; Tsukuba Research Laboratory, Hamamatsu Photonics KK, and || CREST "Creation and Application of Soft Nano-Machine, the Hyperfunctional Molecular Machine" Team 13, Tokodai, Tsukuba 300-2635, Japan; ** Chemical Resources Laboratory, Tokyo Institute of Technology, Yokohama 226-8503, Japan; and ERATO "ATP System", Japan Science and Technology Agency, Yokohama 226-0026, Japan

Correspondence: Address reprint requests to Kazuhiko Kinosita Jr., Dept. of Physics, Faculty of Science and Engineering, Waseda University, 3-4-1 Okubo, Shinjuku-ku, Tokyo 169-8555, Japan. Tel.: 81-3-5952-5871; Fax: 81-3-5952-5877; E-mail: kazuhiko{at}waseda.jp.

F1-ATPase is an ATP-driven rotary molecular motor in which the central g-subunit rotates inside a stator cylinder made of a3ß3 subunits. To elucidate the role of rotor-stator interactions in torque generation, we truncated the g-subunit at its carboxyl terminus, which forms an a helix that penetrates deeply into the stator cylinder. We used an a3ß3 subcomplex of F1-ATPase derived from thermophilic Bacillus PS3 and expressed it in Escherichia coli. We could obtain purified subcomplexes in which 14, 17, or 21 amino-acid residues were deleted. The rotary characteristics of the truncated mutants, monitored by attaching a duplex of 0.49-µm beads to the g-subunit, did not differ greatly from those of the wild-type over the ATP concentrations of 20 nM–2 mM, the most conspicuous effect being ~50% reduction in torque and ~70% reduction in the rate of ATP binding upon deletion of 21 residues. The ATP hydrolysis activity estimated in bulk samples was more seriously affected. The 21-deletion mutant, in particular, was >10-fold less active, but this is likely due to instability of this subcomplex. For torque generation, though not for rapid catalysis, most of the rotor-stator contacts on the deeper half of the penetrating portion of the g-subunit are dispensable.







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Copyright © 2006 by the Biophysical Society.