TRYPTOPHAN PHOTOPHYSICS IN RABBIT SKELETAL MYOSIN ROD

The fibrous region of myosin (myosin rod) is an alpha-helical, two-str anded coiled-coil made up of identical chains of nearly 1000 residues. Myosin from rabbit skeletal muscle has two tryptophans per chain loca ted at identical hydrophobic d sites in the heptad repeat that forms t he basis for hydrophobic dimerization. The fluorescence excitation and emission spectra of rod in high salt buffer (where the rod exists as a coiled-coil monomer) at 20 degrees C are red- and blue-shifted, resp ectively, from the comparable spectra of N-acetyl-tryptophanamide or L -tryptophan. These spectral shifts, as well as red-shifts in the emiss ion spectra induced by excitation on the red edge of the absorption or by increases in temperature, indicate that (on average) the tryptopha ns are partially exposed to aqueous solvent yet in contact with the pr otein matrix. The tryptophan intensity decays show an unusual bimodal distribution; the major species has a discrete lifetime of about 5.2 n s while the minor species exhibits a complex decay with a broad (3.4 n s full width at half maximum) Gaussian distribution of lifetimes cente red around 1.3 ns. The long lifetime species has a blue-shifted excita tion and red-shifted emission characteristic of the indole chromophore in a polar (probably aqueous) environment while the short lifetime sp ecies has the spectral parameters characteristic of indole in a non-po lar environment. Although assignment of these lifetime species to part icular tryptophans in the rod is problematic, this study indicates tha t the coiled-coil interface presents a complex heterogeneous environme nt that may undergo rapid conformational mobility.