Fluorescence measurements detect changes in scallop myosin regulatory domain

Authors


L. Nyitray, Department of Biochemistry, Eötvös Loránd University, Puskin u. 3., H-1088 Budapest, Hungary. Fax: + 36 1 266 7830; Tel: + 36 1 266 7858; E-mail: nyitray@cerberus.elte.hu.

Abstract

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.

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