The embryonic and neonatal homodimeric isoforms of myosin heavy chain are coexpressed during muscle development but do not form a hybrid. The proposed study seeks to ascertain if thermodynamic or biological factors determine subunit association. Isolated rod segments will be denatured, renatured and assayed for formation of the heterodimer. The high degree of sequence homology between the rod subunit isoforms necessitates application of new methods to detect the hybrid. These include use of a thiol- blocking/disulfide-forming method coupled with non-reducing SDS-PAGE, a resonance energy transfer procedure, and an immobilized subunit hybridization technique. If hybrids are not detected, the rod isoforms will be expressed in E. coli and subjected to site specific mutagenesis in regions where the sequences are known to differ. The assay will be used to determine the effects of each mutation in the homodimer on heterodimer formation. If hybrids are detected, similar mutagenesis studies will be done to determine those features of the sequence upon which association depends. Understanding these structural features of myosin dimerization is important in describing how myofibrils assemble and function in the contractile process.
