We use spectroscopic techniques, mainly electron paramagnetic resonance (EPR) spectroscopy, to define the structural changes that occur within the motor proteins, both myosin and kinesin families, during their functional cycles. The light chain domain of myosin is known to alter its orientation during the power stroke, and will be a focus of one line of investigation. We will attach paramagnetic probes to myosin regulatory light chains (LC2) and use them to measure the orientation of the light chain domain (LC domain) of the myosin head in rabbit skeletal muscle fibers to measure the rotation of this region during force generation. Paramagnetic labels will also be attached to the light chains of myosin in smooth muscle. We will explore the roles of ADP and LC2 phosphorylation in the physiological responses of smooth muscle, and in particular in the maintenance of the latch state. We will monitor changes in the conformation of the catalytic domain of myosin in collaboration with Jim Spudich and his laboratory. Paramagnetic probes will be attached to cysteines introduced into specific locations in """"""""cys-lite"""""""" myosin heads (heads from which all native reactive cysteines have been removed). Two regions will be initially investigated, the 50 kD cleft that traverses the catalytic domain from the actin site to the nucleotide pocket, and the converter region which lies at the interface between the catalytic domain and the LC domain. Both regions are thought to undergo conformational changes in response to binding of nucleotides and/or actin. The conformation of these regions will be monitored during interaction with actin and nucleotides. The data will determine what conformations are associated with which nucleotide states, whether multiple conformations are found for specific nucleotides, what energetic differences separate the different conformations, and what conformations are produced by binding to actin. We will use spin-labels to monitor the conformation of the neck region of kinesin, during interaction with nucleotides and microtubules. This project will be carried out in collaboration with Ron Vale and his laboratory, who have developed a """"""""cys-lite"""""""" kinesin dimer, with new cysteines introduced into the neck region. This region is thought to unfold to allow both heads of kinesin to interact simultaneously with a microtubule. This hypothesis will be tested by defining the conformation of this region using the spectra of single probes attached to cysteines introduced into the region, and by measuring the distance between two paramagnetic probes attached to two cysteines. We are using the Computer Graphics Laboratory facilities for building and visualizing our three-dimensional models.
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