PROTON NMR-SPECTROSCOPY OF MYOSIN SUBFRAGMENT-1 ISOENZYMES

High-resolution proton NMR spectroscopy was used to study the solution structures of the subfragment 1 (S1) isoenzymes (containing either the A1 or A2 light chains) from rabbit skeletal muscle myosin and to investigate their interaction with actin. Superimposed upon broad components, the narrow signals of the S1 spectra were unexpectedly sharp, indicating that domains of varying sidechain mobility occur in the conformation adopted in solution. Peptide amide exchange studies showed that the S1 structure accommodates fluctuations of sufficient amplitude to allow most of the peptide groups to come into contact with the solvent on the time scale of the 1H-NMR experiment. The overall impression is that S1 is a molecule possessing backbone motility as well as domains of different sidechain mobility. Close comparison of the S1(A1) and S1(A2) spectra indicate that the N-terminal 41 residues of the A1 light chain, rich in Lys, Pro and Ala, display a high degree of segmental mobility. The difference spectrum [S1(A1)-S1(A2)] obtained closely resembles the spectral simulation of the 41-residue segment. Upon addition of actin, many of the narrow S1 resonances decreased in intensity or progressively disappeared altogether, indicative of intermediate-slow exchange conditions consistent with the recognized high affinity between the 2 proteins. These changes are interpreted as an overall modulation in the observed and hence more mobile regions of S1. The differences noted between S1(A1) and S1(A2) largely disappeared in their complexes with actin indicating a marked reduction in the segmental mobility of the N-terminal region of the light chain in S1(A1).