43rd ANNUAL DROSOPHILA RESEARCH CONFERENCE
April 10-14, 2002

PROGRAM AND ABSTRACT VOLUME
Workshop Abstract

19W
The converter domain modulates the kinetic properties of Drosophila myosin. Kimberly Palmiter Littlefield¹, Becky M. Sanchez¹, David M. Warshaw², Sanford I. Bernstein¹. 1) Biology Department and Molecular Biology Institute, San Diego State University, CA; 2)Department of Molecular Physiology and Biophysics, University of Vermont, Burlington.

The myosin converter domain couples the chemical energy of ATP hydrolysis to the mechanical motion of muscle contraction. The Drosophila embryonic myosin isoform (EMB) converter domain was genetically substituted into the indirect flight muscle (IFM) myosin (IFI) producing the IFI-EC chimera. Previous data showed that actin filament velocity (v_actin) was attenuated 2.4X in the IFI-EC myosin. We hypothesized that swapping the EMB converter into the IFI myosin affects the mechanical (step size, d) and/or kinetic properties of the protein. Using a dual laser trap assay, IFI or IFI-EC myosin, isolated from the IFM of transgenic flies, was mechanically characterized. d did not differ between the IFI (7.5±3.2nm) and IFI-EC (5.8±2.3nm) myosins. Thus a mechanical change cannot account for the reduced v_actin. The kinetic properties of the IFI, EMB, or IFI-EC myosin were characterized with solution actin-activated ATPase (aaATPase) assays. The maximal aaATPase activity of the IFI-EC myosin was 2X greater than the IFI myosin, which in turn was ~1.3X greater than the EMB myosin suggesting that the kinetics are altered in the IFI-EC myosin. Although the rate limiting steps for v_actin and steady-state aaATPase differ, these data suggest that kinetic changes can account for the reduction in v_actin in the IFI-EC mutant. We aim to elucidate how structural differences in the converter domain impart the observed kinetic differences to the myosin isoforms. We hypothesize that the converter helps regulate the movement of the adjacent light chain binding domain (LCBD), which is thought to be a key element in powerstroke generation. To investigate this, we are binding isolated myosin S1 heads to actin filaments and producing high resolution electron cryo-microscopy image reconstructions during various steps of the ATPase cycle (in collaboration with Ron Milligan, The Scripps Research Institute). Images will be fitted within previously determined crystal structures of myosin molecules. This will elucidate whether alternative converter domains regulate the position of the LCBD to help set myosin's mechanochemical properties during the ATPase cycle.