The long-range goal of our research is to elucidate the molecular mechanisms involved in he interaction of myosin, actin and ATP. In the most prevalent hypothesis a myosin head generates force by rotating while attached to actin. Paramagnetic probes are excellent tools for investigating the contractile mechanism, because both their angular distribution and their motion can be measured from their spectra. We will extend our previous studies of probes bound to both actin and myosin head consists of a large mass at its distal end, which attaches to actin. This is connected to the rod by a more slender neck about 10 nm in length. Previous studies of paramagnetic probes on the distal region suggest that it doesn't rotate to generate force, thus implicating the neck region of myosin in the generation of force. The light chains of myosin are located in the neck region. We have recently attached probes to one site on a purified myosin light chain exchanged it into fibers and found that this region is more flexible than the distal end. Similar techniques will be used to find additional probe sites on light chains and to thus characterize the orientation and motion of myosin at a variety of sites. Data from these sites should provide a detailed picture of the motion and orientation of the myosin head during force generation. In another project we will investigate cooperative interactions between the two heads of myosin. We will trap spin-labeled nucleotides on myosin heads, eliminating their interaction with actin, and we will use this preparation to determine the mechanical properties of myosin molecules that are generating force with only one head. We will also probe conformational changes occurring in actin during its interaction with other proteins: myosin, DNAse I and the regulatory proteins. A high resolution structure of an actin-DNAse complex is nearing completion, making it particularly pertinent to determine whether the bond with DNAse alters the conformation of actin. Knowledge of how force is produced and how it is controlled in striated muscle, especially in cardiac muscle, will help define more rational therapies for treating disorders in these muscles.
