Conformational Changes in the Foot Protein of the Sarcoplasmic Reticulum Assessed by Site-Directed Fluorescent Labeling

Ca²⁺ release from sarcoplasmic reticulum during excitation-contraction coupling is likely to be mediated by conformational changes in the foot protein moiety of the triadic vesicles. As a preparative step toward the studies of dynamic conformational changes in the foot protein moiety, we have developed a new method that permits specific labeling of the foot protein moiety of the isolated membranes with a fluorophore. A novel fluorescent cleavable photoaffinity cross-linking reagent, sulfosuccinimidyl 3-((2-(7-azido-4-methylcoumarin-3-acetamido)ethyl)dithio)propionate (SAED), was conjugated with site-directing carriers, polylysine (Ca²⁺-release inducer) and neomycin (Ca²⁺-release blocker). The conjugates were allowed to bind to polylysine and neomycin-binding sites of the heavy fraction of SR (HSR). After photolysis, the cross-linked reagent was cleaved by reduction and the fluorescently labeled HSR was separated from the carriers by centrifugation. These procedures led to specific incorporation of the methylcoumarin acetate (MCA) into the foot protein. Polylysine and neomycin bound to different sites of the foot protein, since neomycin, at release-blocking concentrations, did not interfere with polylysine binding. The fluorescence intensity of the foot protein labeled with the carrier, neomycin, showed biphasic changes as a function of ryanodine concentration (increasing up to 1 μM ryanodine and decreasing above it), while with the carrier polylysine, ryanodine induced no change in fluorescence intensity. In contrast, the fluorescence intensity of the foot protein labeled with each of the two carriers, neomycin and polylysine, showed almost identical calcium dependence (first increasing from 0.1 μM to about 3.0 μM calcium concentration, and then decreasing at higher calcium concentrations). These results suggest that modulation of Ca²⁺ release by ryanodine involves a local conformational change in the neomycin-binding region of the foot protein, while that by Ca²⁺ involves conformational changes not only in the neomycin-binding region but also in the polylysine-binding region.