(3) Sliding movements of actin filaments undergoing fluctuations propelled by myosin subfragment-1 flexibly bound to the surface

Makio TOKUNAGA, Atsuko IWANE¹, Kazuo KITAMURA² and Toshio YANAGIDA^1,2 (¹Department of Physiology I, Osaka University Medical School, ²Single Molecule Processes Project, ICORP, JST)

　We have recently demonstrated that when skeletal muscle S1 is specifically attached to a glass surface in its tail end, it can move actin as fast as intact myosin. The specific binding was achieved by replacing a endogenous RLC by a recombinant fusion protein of biotin-dependent transcarboxylase (BDTC, a biotinylated peptide) and chicken gizzard RLC (cgmRLC). S1 was bound to an avidin-coated glass surface through BDTC-cgmRLC. In this work, a flexible peptide, 5G, which is expected to have a random coil structure and ~12 nm in expanded length, was inserted between BDTC and cgmRLC (BDTC-5G-cgmRLC). Thus, S1 was attached to a glass surface via the flexible chain. Insertion of 5G did not affect the actin-activated ATPase activity. Velocity of actin filaments caused by BDTC-5G-cgmRLC-S1 on a glass surface was similar to those by intact myosin and by BDTC-cgmRLC-S1, respectively. It was confirmed that the inserted chain was indeed flexible, using both electron microscopy and video image analysis. The present results don't support the widely accepted lever arm model.