Fluorescence measurements detect changes in scallop myosin regulatory domain

Ca2+‐induced conformational changes of scallop myosin regulatory domain (RD) were studied using intrinsic fluorescence. Both the intensity and anisotropy of tryptophan fluorescence decreased significantly upon removal of Ca2+. By making a mutant RD we found that the Ca2+‐induced fluorescence change is due mainly to Trp21 of the essential light chain which is located at the unusual Ca2+‐binding EF‐hand motif of the first domain. This result suggests that Trp21 is in a less hydrophobic and more flexible environment in the Ca2+‐free state, supporting a model for regulation based on the 2 Å resolution structure of scallop RD with bound Ca2+[Houdusse A. and Cohen C. (1996) Structure4, 21–32]. Binding of the fluorescent probe, 8‐anilinonaphthalene‐1‐sulphonate (ANS) to the RD senses the dissociation of the regulatory light chain (RLC) in the presence of EDTA, by energy transfer from a tryptophan cluster (Trp818, 824, 826, 827) on the heavy chain (HC). We identified a hydrophobic pentapeptide (Leu836–Ala840) at the head–rod junction which is required for the effective energy transfer and conceivably is part of the ANS‐binding site. Extension of the HC component of RD towards the rod region results in a larger ANS response, presumably indicating changes in HC–RLC interactions, which might be crucial for the regulatory function of scallop myosin.