500 MHz 1H-NMR study of the N-terminal N-trimethylalanine residue of LC-1 and LC-2 light chains m rabbit fast skeletal myosin solutions

High-resolution proton NMR at 500MHz has been used to study the N-trimethyl terminals of LC-1 and LC-2 light chains in rabbit fast skeletal myosin solutions. The observed resonance is a sum of two Lorentzians withΔv = 5 ± 1 Hz, δ = 3.23 ppm and Δv = 12 ± l Hz, δ = 3.22ppm, respectively. By selective proteolytic modifications samples lacking either the N-terminal segment of LC-1 up to Lys-17 (papain modification) or the N-terminal segment of LC-2 up to Arg-7 (trypsin modification) were prepared. From the NMR spectra of the modified samples the narrower -N⁺(CH₃)₃ methyl resonance is shown to originate from LC-1 and the broader from LC-2. Thus in solution LC-1 and LC-2 N-terminals do not behave identically and there exists between both terminals no interaction reflected by linewidth or chemical shift variation when either N-terminal is removed. In intact as well as in modified myosins the number of resonating protons corresponds within experimental error to the expected values. The comparison of the present NMR results with the rates of proteolytic cleavage suggests that in solution these light chains could be folded back along S1 but without impeding the motion of the N-terminal residues, while in filaments the LC-1 and LC-2 light chains would expose their median sensitive part to proteolytic enzymes.