Proximity relationships between engineered cysteine residues in chicken skeletal myosin regulatory light chain. A resonance energy transfer study.
Resonance energy transfer was used to measure the distances between pairs of cysteines, Cys2 and Cys155 and Cys73 and Cys155, in recombinant chicken skeletal myosin regulatory light chains in the free and bound states. The fluorescent and nonfluorescent probes N-iodoacetyl-N'-(5-sulfo-1-naphthyl) ethylenediamine and N-(4-dimethylamino-3,5-dinitrophenyl)maleimide were used as the donor and the acceptor, respectively. The distance between Cys2 and Cys155 was measured to be 35 and 30 A in the absence and presence of myosin heavy chain, respectively, suggesting a slightly more compact structure for the light chain in the bound state. The distance between Cys73 and Cys155 measured in a similar manner was 31 and 30 A in the free and bound states, respectively; this latter value is in good agreement with that derived from crystallographic structures. For heavy chain-bound light chains, no measurable distance changes were detected with the binding of ATP or actin. These results show that no gross structural changes occur within the regulatory light chain during the contraction cycle, but that resonance energy transfer between other sites could be used to monitor potential changes in the myosin head upon the binding of nucleotides and actin.
Rosenstiel Basic Medical Sciences Research Center, Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02254, USA.
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D000818:Animals; D001842:Bone and Bones / Q000737:chemistry*; D002645:Chickens; D003545:Cysteine / Q000737:chemistry*; D004735:Energy Transfer; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes; D008301:Maleimides; D015335:Molecular Probes; D009154:Mutation; D018994:Myosin Light Chains / Q000737:chemistry* / Q000235:genetics; D009282:Naphthalenesulfonates
D005456:Fluorescent Dyes; D008301:Maleimides; D015335:Molecular Probes; D018994:Myosin Light Chains; D009282:Naphthalenesulfonates; C016399:N-(4-dimethylamino-3,5-dinitrophenyl)maleimide; C006421:1,5-I-AEDANS; D003545:Cysteine