KINETIC TUNING OF MYOSIN VIA A FLEXIBLE LOOP ADJACENT TO THE NUCLEOTIDE-BINDING POCKET

A surface loop (25/50-kDa loop) near the nucleotide pocket of myosin h as been proposed to be an important element in determining the rate of ADP release from myosin, and as a consequence, the rate of actin-myos in filament sliding (Spudich, J. A. (1991) Nature 372, 515-518). To te st this hypothesis, loops derived from different myosin II isoforms th at display a range of actin filament sliding velocities were inserted into a smooth muscle myosin backbone. Chimeric myosins were produced b y baculovirus/Sf9 cell expression. Although the nature of this loop af fected the rate of ADP release (up to 9-fold), in vitro motility (2.7- fold), and the V-max of actin-activated ATPase activity (up to a-fold) , the properties of each chimera did not correlate with the relative s peed of the myosin from which the loop was derived. Rather, the rate o f ADP release was a function of loop size/flexibility with the larger loops giving faster rates of ADP release. The rate of actin filament t ranslocation was altered by the rate of ADP release, but was not solel y determined by it. Through a combination of solute quenching and tran sient fluorescence measurements, it is concluded that, as the loop get s smaller, access to the nucleotide pocket is more restricted. ATP bin ding becomes less favored, and ADP binding becomes more favored, In ad dition, the rate of ATP hydrolysis is slowed.