Local conformation of rabbit skeletal myosin rod filaments probed by intrinsic tryptophan fluorescence.
Rabbit skeletal myosin rod contains two tryptophan residues per chain (four per coiled-coil) that are located about 50 and 175 A from the N-terminus of the light meromyosin (LMM) region of rod. We have characterized the local polarity, excited-state photophysics, solvent accessibility, and rotational dynamics of these tryptophans in myosin rod filaments at 125 mM KCl using steady-state and time-resolved fluorescence techniques. The fluorescence decays were described using a complex bimodal distribution with a discrete long-lifetime component of 5.44 ns (amplitude of 0.51) and a Gaussian distribution of short lifetimes with mean of 0.105 ns and width of 2.15 ns (amplitude 0.49). The discrete long-lifetime species was efficiently quenched by the neutral quencher acrylamide with a bimolecular collision constant (kq) of 0.85 x 10(9) M-1 s-1. The emission spectrum, lifetime distribution, and quenching behavior of the tryptophans in myosin rod monomers (at 0.5 M KCl) were quite similar. Time-resolved anisotropy decays of the rod monomers and filaments exhibited nearly identical double-exponential decays to a constant. Each had a fast, subnanosecond component (amplitude 0.07), probably corresponding to fast wobble of the tryptophans on the coiled-coil surface, a slower, approximately 6-ns component (amplitude approximately 0.04), corresponding to an unidentified internal, segmental mode of motion of the coiled-coil, and a constant (r infinity) of 0.15.(ABSTRACT TRUNCATED AT 250 WORDS)
Chang
Y C
YC
Department of Food Science, Rutgers University, New Brunswick, New Jersey.
Ludescher
R D
RD
D020106:Acrylamide; D000178:Acrylamides; D000818:Animals; D005454:Fluorescence Polarization; D009132:Muscles / Q000737:chemistry*; D009218:Myosins / Q000737:chemistry*; D011487:Protein Conformation; D011817:Rabbits; D013997:Time Factors; D014364:Tryptophan / Q000737:chemistry*
D000178:Acrylamides; D020106:Acrylamide; D014364:Tryptophan; D009218:Myosins