Structural and functional studies of chicken myosin heavy chain (myHC): I. Significance of the subfragment 2 region of MyHC in myosin thick filament assembly. II. Dimerization specificity of adult and neonatal MyHC rods

Myosin is a complex hexameric protein which aggregates with other myosin molecules to form thick filaments. Different isoforms of myosin are sequentially expressed in a particular muscle cell type. During periods of developmental transition thick filaments containing one type of myosin isoform are replaced by filaments containing another myosin isoform. This multifaceted process can be better understood by gaining knowledge of myosin assembly and dimerization. The present study was focused on two areas, the first dealing with the assembly of myosin into bipolar thick filaments and the second area examining the dimerization specificity of the rod domain of myosin.;The contribution of the subfragment 2 (S2) subdomain of the myosin rod to assembly of myosin filaments was studied. To achieve this specific aim, a chimeric protein was designed that had a globular protein fused to the N-terminus of the myosin rod. This fusion protein assembled into tapered filaments rather than paracrystals indicating a role for the myosin head in determining the morphology of myosin filaments. However, we could not ascertain the polarity of these filaments. Thus they were not useful in studies designed to determine the role of the S2 region in myosin assembly. However, deletion studies of this protein did show that the C-terminal 100 residues of the myosin rod are required for the aggregation of myosin rod similar to what has been observed for the light meromyosin (LMM) domain.;The dimerization specificity of recombinantly expressed and purified rod domain of adult and neonatal chicken myosin heavy chain (MyHC) was analyzed using metal chelation chromatography. Our results indicated that full-length adult and neonatal rods preferentially formed homodimers. The contribution made towards the dimerization specificity by subdomains of the rod was addressed by making a chimeric protein consisting of the S2 region of the adult isoform and the LMM region of the neonatal isoform. The proportion of heterodimers formed in exchange experiments between the chimera and the neonatal and adult rods rose with an increase in sequence homology between the two exchanging proteins. This suggests that multiple regions of the rod domain of chicken MyHC can contribute towards dimerization specificity.