DISTANCE MEASUREMENTS NEAR THE MYOSIN HEAD-ROD JUNCTION USING FLUORESCENCE SPECTROSCOPY

We reacted a fluorescent probe, N-methyl-2-anilino-6-naphthalenesulion yl chloride (MNS-Cl), with a specific lysine residue of porcine cardia c myosin located in the S-2 region of myosin. We performed fluorescenc e resonance energy transfer (FRET) spectroscopy measurements between t his site and three loci (Cys109, Cys125, and Cys154) located within di fferent myosin light-chain 2s (LC2) bound to the myosin ''head.'' We u sed LC2s from rabbit skeletal muscle myosin (Cys125), chicken gizzard smooth muscle myosin (Cys109), or a genetically engineered mutant of c hicken skeletal muscle myosin (Cys154). The atomic coordinates of thes e LC2 loci can be closely approximated, and the FRET measurements were used to determine the position of the MNS-labeled lysine with respect to the myosin head. The C-terminus of myosin subfragment-1 determined by Rayment et al. ends abruptly after a sharp turn of its predominant ly alpha-helical structure. We have constructed a model based on our F RET distance data combined with the known structure of chicken skeleta l muscle myosin subfragment-1. This model suggests that the loci that bracket the head-rod junction will be useful for evaluating dynamic ch anges in this region.