The focus of this proposal is to study the conformational changes within the actomyosin complex using our recently introduced technique of luminescence resonance energy transfer (LRET), an extension of the widely used Fluorescence Resonance Energy Transfer. Preliminary results show that LRET is capable of accurately measuring relatively large distances in actomyosin, and indicates that LRET should be generally applicable to the study of protein conformational changes in large complexes. Actomyosin is a particularly important molecular motor in which protein conformational changes lead to muscle contraction and a variety of subcellular motion in eucaryotes. Here the investigators propose to use LRET to determine two essential parameters in muscle mechanics. First, they will measure distances the S1 myosin head and actin filaments in active muscle. The goal is to determine the fraction of myosin heads which are bound to actin, and generate force, during the actomyosin cycle. This parameter has been extensively investigated by other workers but a definitive answer remains elusive. Second, LRET will be used to detect conformational changes within the S1 head, sing purified proteins and active fibers. Despite the widely held view of muscle mechanics involving conformational changes within S1 as an integral part of the """"""""swinging-crossbridge"""""""" model of muscle mechanics, there have been very few direct measurements of such conformational changes. Specifically, they will look for changes in distance between the regulatory light chain of the S1 head and the nucleotide binding region. If current models are correct, this distance should change from approximately 51A at the beginning of the power stroke, to 65A at the end of the power stroke. These two measurements will be made on muscle under isometric conditions, but they serve as models for similar measurements on muscle undergoing length changes. LRET is suited for these measurements because the technique can directly measure conformational changes in active muscle over the distances of interest, and is relatively non-invasive. To achieve these goals requires, in some cases technical advances in LRET. Initial experiments will be made on our current generation of lanthanide chelate donors and our spectrometer. However, the principal investigator will also develop new reactive forms of lanthanide chelates, which will enable site-specific attachment to S1 and other proteins, the ability to measure large distances (greater then 100A), and the ability to detect multiple conformational states. He will also convert their present cuvette-based LRET spectrometer into a microscope-based spectrometer for measurements on single myofibrils and fibers. This will include adding a CCD (charge-coupled-detector) for measurement of emission spectra of donor and acceptor, which will increase the sensitivity and accuracy of energy transfer measurements.
