Subunit interactions within an expressed regulatory domain of chicken skeletal myosin. Location of the NH2 terminus of the regulatory light chain by fluorescence resonance energy transfer.
The regulatory domain (RD), or neck region of the myosin head, consists of two classes of light chains that stabilize an alpha-helical segment of the heavy chain. RD from chicken skeletal muscle myosin was prepared in Escherichia coli by coexpression of a 9-kDa heavy chain fragment with the essential light chain. Recombinant regulatory light chain (RLC), wild type or mutant, was added separately to reconstitute the complex. The affinity of RD for divalent cations was determined by measuring the change in fluorescence of a pair of heavy chain tryptophans upon addition of calcium or magnesium. The complex bound divalent cations with high affinity, similar to the association constants determined for native myosin. The intrinsic fluorescence of the tryptophans could be used as a donor to measure the fluorescence resonance energy transfer distance to a single labeled cysteine engineered at position 2 on RLC. Dansylated Cys2 could also serve as a donor by preparing RLC with a second cysteine at position 79 which was labeled with an acceptor probe. These fluorescence resonance energy transfer distances (24-30 A), together with a previous measurement between Cys2 and Cys155 (Wolff-Long, V. L., Tao, T., and Lowey, S. (1995) J. Biol. Chem. 270, 31111-31118) suggest a location for the NH2 terminus of RLC that appears to preclude a direct interaction between the phosphorylatable serine and specific residues in the COOH-terminal domain.
Saraswat
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LD
Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, Massachusetts 02254-9110, USA.
Lowey
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S
D000818:Animals; D002413:Cations, Divalent; D002642:Chick Embryo; D005456:Fluorescent Dyes; D015335:Molecular Probes; D018482:Muscle, Skeletal / Q000378:metabolism*; D018994:Myosin Light Chains / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D009282:Naphthalenesulfonates; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry
D002413:Cations, Divalent; D005456:Fluorescent Dyes; D015335:Molecular Probes; D018994:Myosin Light Chains; D009282:Naphthalenesulfonates; D011994:Recombinant Proteins; C006421:1,5-I-AEDANS; D014364:Tryptophan