This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).
The coiled coil of myosin is among the longest in nature and contains functionally diverse elements that will be characterized using a newly developed gravitational force spectrometer that facilitates the application of subpiconewton forces to specific regions of single molecules. The subpiconewton forces can be applied at multiple orientations, while the absolute length of a single molecule under force can be measured with a precision in the nanometer range. Dynamic changes in this molecular length can also be quantified at speeds in the millisecond time scale. Using this gravitational force spectrometer, several fundamental hypotheses will be tested about the dynamics of different regions in the coiled coil and their roles in the functions of myosin molecules. The first hypothesis examines whether the coiled coil stretches more readily due to forces applied perpendicular rather than parallel to the long axis of the coiled coil. If so, the associated stretching could facilitate the properly oriented binding of the myosin head to actin, since in muscle, the myosin coiled coil is perpendicular to the direction of the crossbridge joining thick and thin filaments. The second hypothesis tests whether the hinge regions are more susceptible to stretching under force than other regions of the coiled coil. If the hinges do stretch preferentially in response to the application of subpiconewton forces, then these elastic elements could be an important relay in the transmission of force from the power stroke in the myosin head to filament sliding and the development of tension in muscle which has been implicated from physiological data of muscle contractions. In the third hypothesis, intermolecular interactions of coiled coils between myosin molecules or myosin binding proteins will be carried out to affect the coiled coil's susceptibility to subpiconewton or piconewton forces. These experiments will determine the extent to which the myosin's coiled coil susceptibility to force can be modulated by binding events that are likely to occur in vivo.
In addition to some of its unique measuring capabilities, the gravitational force spectrometer is more affordable than other equipment suitable for single molecule assays, so a broader availability for its usage may be possible especially in areas of educational instruction. It is feasible to equip student laboratories with multiple workstations of these devices which would enable hands-on experiences with single molecule assays for science students. To facilitate this possibility, instructional laboratory units will be written with introductory and background descriptions, methods, principles of setup and operation, description of data analysis and acquisition, study questions, key words, and learning outcomes. These instructional laboratory units will be evaluated and distributed to encourage the incorporation of single molecule assays into student laboratories which will better prepare future scientists to exploit these developing technologies.