| During muscle contraction chemical energy from ATP hydrolysis is converted to relative sliding of actin and myosin filaments and force production. Although the actomyosin system has been extensively studied, the mechanism underlying its mechanochemical energy transduction remains unknown. The work described here reports the development of a novel technique which is capable of measuring the force and displacement produced by the interaction a single actin filament with a single myosin molecule. An actin filament is held by a dual-beam feedback-enhanced laser trap via beads attached near each end and allowed to interact with a single myosin molecule on a microscope coverslip surface. The laser trap apparatus, which uses a high-resolution motion detection system and feedback control to increase the trap stiffness, allows the measurement of nanometer displacements and piconewton forces at millisecond rates. Single myosin molecules studied with this technique produce displacements averaging 11 nm at low load and forces averaging 3-4 pN near isometric conditions. These values are consistent with the conventional swinging crossbridge model of muscle contraction. The majority of this dissertation focuses on the development and characterization of this technique. The remainder discusses applications.; Single myosin molecule measurements have begun to allow studies of factors that influence myosin's mechanochemical cycle. In addition to measuring force and displacement, it is possible to measure the durations of single interactions. Under conditions of limiting ATP, excess ADP, or decreased temperature, the rates of dissociation of actin from myosin are decreased and can be measured. By varying the stiffness of the laser trap which varies the load that myosin must overcome, it has been possible to generate a force-displacement curve for single myosin molecules. Under conditions which disturb weak binding interactions such as increased ionic strength or mutations in actin's myosin binding face, the unitary force and displacement are unchanged suggesting that strong-binding interactions are distinct from weak-binding interactions and can be studied independently. It is also possible to combine this approach with myosin mutagenesis to study structure-function questions, such as whether the myosin neck region acts as a lever arm to amplify a conformational change in its catalytic domain. Finally, this technology has been used to further characterize the mechanical properties of the microtubule-based motor kinesin. |
