In striated muscle, tropomyosin (Tm) confers cooperativity to the thin filament-linked regulation of actomyosin ATPase. In smooth muscle Tm is also present in the thin filament, and along with caldesmon and calponin, appears to modulate the primary regulatory mechanism, myosin phosphorylation. The overall objective of this project is to clarify whether Tm plays a similar cooperative role in the smooth muscle regulation, and how caldesmon and calponin modulate this role. Specifically: (1) The fluorescence of several specific labels on Tm and simultaneous measurements of the binding of myosin will be used in titrations with smooth muscle thin filaments. With this assay we will establish that the smooth muscle Tm-actin thin filament equilibrates between the 2 states and determine effects of myosin phosphorylation on the cooperative """"""""turning-on"""""""" of the thin filament and determine how caldesmon and calponin affect the cooperativity of the on-off transition of the Tm- actin filament. (2) Effects of caldesmon and calponin and specific engineered peptides and mutants on the velocity of thin filament movement using in vitro motility assays will be correlated with effects on in vitro actomyosin ATPase activity. (3) Effects of caldesmon on the end-to-end depolymerization of Tm will be determined by using viscosity and ultracentrifugation techniques. The depolymerization effects will be correlated with effects of caldesmon on end-to-end Tm interactions in the thin filament. (4) To obtain specific information about the interaction of caldesmon and calponin with the thin filament we will determine the mode of interaction with titrations using labeled Tm-actin; test if caldesmon and calponin compete with each other and with myosin for binding sites on actin using fluorescence microscopy of labeled proteins incorporated into myofibrils; determine changes in orientation of labeled caldesmon and calponin on the thin filament associated with the binding of HMM and S1 using fluorescence detected linear dichroism (FDLD); and determine changes in orientation of labeled Tm caused by the binding of caldesmon and calponin to the thin filament using FDLD.
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