2-STATE THERMAL UNFOLDING OF A LONG DIMERIC COILED-COIL - THE ACANTHAMOEBA MYOSIN-II ROD

Acanthamoeba myosin II rod is a long alpha-helical coiled-coil with a flexible hinge containing a helix-breaking proline. The thermal stabil ity of the complete rod domain of myosin II (residues 849-1509), a mut ant in which the hinge proline was replaced by alanine (P398A), and a mutant with the whole hinge region deleted (Delta(384-408)) was studie d in 0.6 and 2.2 M KCI, pH 7.5. In analytical ultracentrifugation stud ies, the purified myosin II rods sedimented as monodisperse dimers wit h sedimentation coefficients S-20,S-w=3.8 S (wild-type, M-r=149 000) a nd 3.6 S (P398A and Delta(384-408)) Circular dichroism (CD) and differ ential scanning calorimetry (DSC) showed that the thermal unfolding of the myosin II rod is reversible and highly cooperative. The unfolding of the rod is coupled to a dissociation of the chains, as shown by HP LC gel filtration at high temperatures and by the concentration depend ence of the transition temperature. The CD and DSC data are consistent with a two-state mechanism (T-m similar to 40 degrees C, Delta H simi lar to 400 kcal/mol) in which the dimeric rod unfolds with concomitant formation of two unfolded monomers. We found no evidence for independ ent unfolding of the two rod domains that are separated by the hinge r egion. The only difference observed in the unfolding of the mutant rod s from that of the wild type was a similar to 2 degrees C increase in the thermal stability of the hinge-deletion mutant. Thus: the mechanis m of unfolding the Acanthamoeba myosin II rod is different from those of skeletal muscle myosin rod and tropomyosin, for which non-two-state thermal transitions have been observed. The cooperative unfolding of the entire coiled-coil rod of Acanthamoeba myosin II may underlie the previously reported regulatory coupling between its N-terminal head an d C-terminal tail.