This project will study the basic design and specialized adaptations of the vertebrate smooth and non-muscle myosin II isoforms. Myosin II is the protein that, through its interaction with actin, is the force generator of skeletal, cardiac and smooth muscle. Vertebrate smooth muscle contains primarily smooth muscle myosin heavy chain isoforms when fully differentiated, with variable expression of non muscle myosin heavy chains. The proportion of non-muscle myosin increase in proliferating smooth muscle. The non-muscle myosin B is the only non-muscle myosin expressed in developing cardiocytes, and the non-muscle myosin II isoform that will be the focus of this study. The two goals of this study are to identify regions of the myosin molecule that are essential to its motor function and to define the domains within the myosin heavy chain that underlie the functional differences between smooth and non-muscle myosins. This project will focus on the impact of the interactions at the actin interface as well as elements involved in transmitting structural rearrangements to the myosin """"""""lever arm"""""""". Additionally, we will pose fundamental questions about the nature of the coupling of movement to the ATPase cycle, head-head cooperatively within the myosin molecule and whether or not myosin can be processive on an actin filament. In order to study the myosin II heavy chain, we will use a heterologous expression system (baculovirus/SF9 cell expression). For all expressed recombinant S1 and HMM fragments, steady state ATPase activity (Vmax and K/ATPase) as well as actin binding (K/binding) will be determined. In addition, stopped flow measurements of S1 will allow determination of the rate of ADP release and the rate of myosin dissociation from actin. In vitro motility measurements will be performed on all HMM constructs. Collaborative experiments with the optical trapping core will allow determination of single molecule force, as well as allow assessment of possible processivity. Thus, we will characterize the impact of variations in the regions under investigation on the enzymatic properties of the myosin motor. In this manner we will began to dissection the functional and structural domains of the myosin motor, with the framework of delineating the principles of myosin design and isoform diversity.
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