Independent flexible motion of submolecular domains of the Ca²⁺,Mg²⁺-ATPase of sarcoplasmic reticulum measured by time-resolved fluorescence depolarization of site-specifically attached probes

The Ca²⁺-transporting ATPase of rabbit skeletal muscle sarcoplasmic reticulum was sitespecifically labeled with either N-(1-anilinonaphth-4-yl)maleimide (ANM) or 5-[[(iodoacetamido)-ethyl]amino]naphthalene-1-sulfonate (IAEDANS), and the segmental motion of submolecular domains of the ATPase molecule was examined by means of time-resolved and steady-state fluorescence anisotropy measurements. The ANM-binding domain showed wobbling with a rotational relaxation time φ = 69 ns in the absence of free Ca²⁺ without any independent wobbling of the ANM moiety. The IAEDANS-binding domain showed a significantly slower wobbling with φ = 190 ns in the absence of Ca²⁺. The present results demonstrated for the first time that the ATPase molecule is composed of distinct domains whose mobilities are considerably different from each other. The binding of Ca²⁺ to the transport site increased the segmental motion of ANM-labeled domain, leading to a phi; value of 65 ns. Solubilization of the ANM-labeled SR membranes by deoxycholate led to a further increase in the segmental flexibility (φ = 48 ns in the absence of free Ca²⁺), indicating that the mobility of the ANM-binding domain was considerably restricted through interaction with the membrane. The mobility of the ANM-binding domain of solubilized ATPase was also increased to some extent upon binding of Ca²⁺.