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 A resolution structure of scallop RD with bound Ca2+ [Houdusse A. and Cohen C. (1996) Structure 4, 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.
Málnási-Csizmadia
A
A
Department of Biochemistry, Eötvös Loránd University, Budapest, Hungary.
Hegyi
G
G
Tölgyesi
F
F
Szent-Györgyi
A G
AG
Nyitray
L
L
D000817:Anilino Naphthalenesulfonates / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology; D002850:Chromatography, Gel; D005456:Fluorescent Dyes; D008974:Mollusca / Q000378:metabolism*; D009154:Mutation; D009218:Myosins / Q000737:chemistry* / Q000235:genetics; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000235:genetics; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry
D000817:Anilino Naphthalenesulfonates; D005456:Fluorescent Dyes; D011994:Recombinant Proteins; C027132:1-anilino-8-naphthalenesulfonate; D014364:Tryptophan; D009218:Myosins; D002118:Calcium