SUBUNIT INTERACTIONS WITHIN AN EXPRESSED REGULATORY DOMAIN OF CHICKENSKELETAL 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, consist s of two classes of light chains that stabilize an alpha-helical segme nt of the heavy chain. RD from chicken skeletal muscle myosin was prep ared in Escherichia coli by coexpression of a 9-kDa heavy chain fragme nt with the essential light chain. Recombinant regulatory light chain (RLC), wild type or mutant, was added separately to reconstitute the c omplex. The affinity of RD for divalent cations was determined by meas uring the change in fluorescence of a pair of heavy chain tryptophans upon addition of calcium or magnesium. The complex bound divalent cati ons with high affinity, similar to the association constants determine d for native myosin, The intrinsic fluorescence of the tryptophans cou ld be used as a donor to measure the fluorescence resonance energy tra nsfer distance to a single labeled cysteine engineered at position 2 o n RLC, Dansylated Cys(2) could also serve as a donor by preparing RLC with a second cysteine at position 79 which was labeled with an accept or probe. These fluorescence resonance energy transfer distances (24-3 0 Angstrom), together with a previous measurement between Cys(2) and C ys(155) (Wolff-Long, V. L., Tao, T., and Lowey, S. (1995) J. Biol. Che m. 270, 31111-31118) suggest a location for the NH2 terminus of RLC th at appears to preclude a direct interaction between the phosphorylatab le serine and specific residues in the COOH-terminal domain.