Probing myosin light chain 1 structure with monoclonal antibodies.

Five monoclonal antibodies that react with different regions of myosin light chain 1 from human ventricular myocardial muscle were used to obtain information on interactions between the light chain 1 and heavy chains and generally on the tertiary structure of the light chain 1 within the myosin head. We performed Western blot assays of the five antibodies with myosins from different cardiac and skeletal muscles, with different proteolytic fragments of bovine ventricular myosin light chain 1 (LC1) and to different recombinant fragments of human ventricular LC1 and rat fast skeletal light chain LC1/LC3. The five antibodies were mapped in three different regions of the light chain 1: two antibodies mapped within the first eight amino-terminal residues, two between residues 71 and 74, and one between residues 129 and 134. The apparent dissociation constants of the last three antibodies, determined by antibody-antigen equilibria in solution, were lower than when isolated light chains were used as antigens. It is probable that the corresponding amino acids involved in the antibody epitopes were either involved in interactions between the light and heavy myosin subunits, or somehow hindered by the myosin heavy chain bulk. In contrast, the apparent dissociation constants measured for both other antibodies were higher when myosin, rather than isolated light chains, was used as antigen. Thus LC1 fixation to heavy chains within the myosin molecule induced conformation changes at the amino-terminal end of the light chain 1. No difference in the accessibility of this mobile LC1 segment was detected in the presence of actin. Finally, observed differences in epitope accessibility on the light chain LC1 in myosin, as compared with chymotryptic subfragment 1 (SF1), indicated conformational differences between native myosin and extensively studied SF1 molecules.