MYOSIN CONFORMATIONAL STATES DETERMINED BY SINGLE FLUOROPHORE POLARIZATION

Muscle contraction is powered by the interaction of the molecular moto r myosin with actin. With new techniques for single molecule manipulat ion and fluorescence detection, it is now possible to correlate, withi n the same molecule and in real time, conformational states and mechan ical function of myosin, A spot-confocal microscope, capable of detect ing single fluorophore polarization, was developed to measure orientat ional states in the smooth muscle myosin light chain domain during the process of motion generation. Fluorescently labeled turkey gizzard sm ooth muscle myosin was prepared by removal of endogenous regulatory li ght chain and re-addition of the light chain labeled at cysteine-108 w ith the 6-isomer of iodoacetamidotetramethylrhodamine (6-IATR), Single myosin molecule fluorescence polarization data, obtained in a motilit y assay, provide direct evidence that the myosin light chain domain ad opts at least two orientational states during the cyclic interaction o f myosin with actin, a randomly disordered state, most likely associat ed with myosin whereas weakly bound to actin, and an ordered state in which the light chain domain adopts a finite angular orientation where as strongly bound after the powerstroke.