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 fo ot protein moiety of the triadic vesicles. As a preparative step towar d the studies of dynamic conformational changes in the foot protein mo iety, 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, su lfosuccinimidyl methylcoumarin-3-acetamido)ethyl)dithio)propionate (SA ED), was conjugated with site-directing carriers, polylysine (Ca2+-rel ease inducer) and neomycin (Ca2+-release blocker). The conjugates were allowed to bind to polylysine- and neomycin-binding sites of the heav y 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 protei n. Polylysine and neomycin bound to different sites of the foot protei n, since neomycin, at release-blocking concentrations, did not interfe re with polylysine binding. The fluorescence intensity of the foot pro tein labeled with the carrier, neomycin, showed biphasic changes as a function of ryanodine concentration (increasing up to 1-mu-M ryanodine and decreasing above it), while with the carrier polylysine, ryanodin e induced no change in fluorescence intensity. In contrast, the fluore scence intensity of the foot protein labeled with each of the two carr iers, neomycin and polylysine, showed almost identical calcium depende nce (first increasing from 0.1-mu-M to about 3.0-mu-M calcium concentr ation, and then decreasing at higher calcium concentrations). These re sults 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 regi on.