The stiffness of rabbit skeletal actomyosin cross-bridges determined with an optical tweezers transducer

Muscle contraction is brought about by the cyclical interaction of myosin with actin coupled to the breakdown of ATP. The current view of the mechanism is that the bound actomyosin complex (or "cross-bridge") produces force and movement by a change in conformation. This process is known as the "working stroke." We have measured the stiffness and working stroke of a single cross-bridge (kappa(xb), d(xb), respectively) with an optical tweezers transducer, Measurements were made with the "three bead" geometry devised by Finer et al, (1994), in which two beads, supported in optical traps, are used to hold an actin filament in the vicinity of a myosin molecule, which is immobilized on the surface of a third bead. The movements and forces produced by actomyosin interactions were measured by detecting the position of both trapped beads. We measured, and corrected for, series compliance in the system, which otherwise introduces large errors. First, we used video image analysis to measure the long-range, force-extension property of the actin-to-bead connection (kappa(con)), which is the main source of "end compliance." We found that force-extension diagrams were nonlinear and rather variable between preparations, i.e., end compliance depended not only upon the starting tension, but also upon the F-actin-bead pair used. Second, we measured kappa(xb) and kappa(con) during a single cross-bridge attachment by driving one optical tweezer with a sinusoidal oscillation while measuring the position of both beads. In this way, the bead held in the driven optical tweezer applied force to the cross-bridge, and the motion of the other bead measured cross-bridge movement, Under our experimental conditions (at similar to 2 pN of pretension), connection stiffness (kappa(con)) was 0.26 +/- 0.16 pN nm(-1). We found that rabbit heavy meromyosin produced a working stroke of 5.5 nm, and cross-bridge stiffness (kappa(xb)) was 0.69 +/- 0.47 pN nm(-1).