Abstract
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.
Highlights
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
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
We have focused our efforts over the last few years on preparing Cys mutants of the regulatory light chain (RLC), a subunit which is readily exchanged into myosin, and can provide a means to introduce probes into the COOH-terminal region of the myosin head (Saraswat and Lowey, 1991; Saraswat et al, 1992)
Summary
Resonance energy transfer was used to measure the distances between pairs of cysteines, Cys and Cys155 and Cys and Cys155, in recombinant chicken skeletal myosin regulatory light chains in the free and bound states. We have focused our efforts over the last few years on preparing Cys mutants of the regulatory light chain (RLC), a subunit which is readily exchanged into myosin, and can provide a means to introduce probes into the COOH-terminal region of the myosin head (Saraswat and Lowey, 1991; Saraswat et al, 1992). This approach avoids modification of the reactive heavy chain Cys residues, SH1 and SH2, which can have adverse effects on the enzymatic activity and motor properties of myosin (Root and Reisler, 1992). The advent of genetic engineering has made it possible to introduce reactive residues at any position in the sequence, and one need only know the three-dimensional structure to determine which sites will be most informative for spectro-
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