The thermal stability of the myosin II rod has been studied by circular dichroism (CD) and differential scanning calorimetry (DSC). The complete rod domain of myosin II (residues 849-1509) has been expressed in E. coli. At high ionic strength (0.6 M KCl, pH 7.5), the purified myosin II rod is a coiled-coil dimer (about 150,000 MW) of mainly alpha-helical polypeptide chains. CD and DSC experiments show that the thermal unfolding of the myosin II rod is highly cooperative (T about 40 degrees C, deltaH about 400 kcal/mol) and is coupled to a decay of secondary structure. This behavior is similar to that previously found for intact myosin II (M. Zolkiewski, M.J. Redowicz, E.D. Korn, & A. Ginsburg, Arch. Biochem. Biophys. 318, 207-214, 1995), but differs in that the rod unfolding reaction is completely reversible. DSC data are scan-rate dependent because the approach to equilibrium during unfolding of the myosin II rod is slow. The unfolding of the rod is coupled to a dissociation of the two chains, as shown by the lower apparent molecular weight of the protein at high temperatures in HPLC gel filtration and by the concentration dependence of the transition temperature observed in CD. The CD and DSC data for the thermal transition are consistent with a two-state mechanism in which the native dimeric rod unfolds with concomitant formation of two unfolded monomers. This mechanism is significantly different from those for skeletal muscle myosin rod and tropomyosin, for which others have found several partially folded intermediates. However, the two-state unfolding of the myosin II rod is similar to that of small dimeric proteins containing the alpha-helical coiled-coil, leucine zipper motif. Recent studies have shown that a point mutation (Pro to Ala) at the helix-breaking residue Pro 398 of the myosin II rod does not affect the cooperativity or temperature of unfolding. A deletion of rod residues 384-408, which comprise the entire hinge region of the myosin II rod, increases the thermal stability by about 2 degrees C. The hydrodynamic properties of the wild type and mutant myosin II rod proteins are being determined.
