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Department of Molecular Physiology and Biophysics, University of Vermont, Health Science Research Facility, Burlington, VT 05405

Address correspondence to David M. Warshaw, Dept. of Molecular Physiology and Biophysics, University of Vermont, Health Science Research Facility, Burlington, VT 05405-0068. Tel.: (802) 656-2540. Fax: (802) 656-0747. email: warshaw{at}physiology.med.uvm.edu; or Kathleen M. Trybus, Dept. of Molecular Physiology and Biophysics, University of Vermont, Health Science Research Facility, Burlington, VT 05405-0068. Tel.: (802) 656-8750. Fax: (802) 656-0747. email: trybus{at}physiology.med.uvm.edu

Each of the heads of the motor protein myosin II is capable of supporting motion. A previous report showed that double-headed myosin generates twice the displacement of single-headed myosin (Tyska, M.J., D.E. Dupuis, W.H. Guilford, J.B. Patlak, G.S. Waller, K.M. Trybus, D.M. Warshaw, and S. Lowey. 1999. Proc. Natl. Acad. Sci. USA. 96:4402–4407). To determine the role of the second head, we expressed a smooth muscle heterodimeric heavy meromyosin (HMM) with one wild-type head, and the other locked in a weak actin-binding state by introducing a point mutation in switch II (E470A). Homodimeric E470A HMM did not support in vitro motility, and only slowly hydrolyzed MgATP. Optical trap measurements revealed that the heterodimer generated unitary displacements of 10.4 nm, strikingly similar to wild-type HMM (10.2 nm) and approximately twice that of single-headed subfragment-1 (4.4 nm). These data show that a double-headed molecule can achieve a working stroke of 10 nm with only one active head and an inactive weak-binding partner. We propose that the second head optimizes the orientation and/or stabilizes the structure of the motion-generating head, thereby resulting in maximum displacement.

Key Words: step size; single molecule; optical trap; molecular motor; cooperativity

Abbreviations used in this paper: FPLC, fast performance liquid chromatography; HMM, heavy meromyosin; Pyr-actin, pyrene-labeled actin; S1-neo, single-headed subfragment-1 with neonatal epitope tag; wt-HMM, wild-type heavy meromyosin.

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