Two-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 stability of the complete rod domain of myosin II (residues 849-1509), a mutant in which the hinge proline was replaced by alanine (P398A), and a mutant with the whole hinge region deleted (delta(384-408)) was studied in 0.6 and 2.2 M KCl, pH 7.5. In analytical ultracentrifugation studies, the purified myosin II rods sedimented as monodisperse dimers with sedimentation coefficients s(20,w) = 3.8 S (wild-type, Mr = 149,000) and 3.6 S (P398A and delta(384-408)). Circular dichroism (CD) and differential 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 HPLC gel filtration at high temperatures and by the concentration dependence of the transition temperature. The CD and DSC data are consistent with a two-state mechanism (Tm approximately 40 degrees C, deltaH approximately 400 kcal/mol) in which the dimeric rod unfolds with concomitant formation of two unfolded monomers. We found no evidence for independent unfolding of the two rod domains that are separated by the hinge region. The only difference observed in the unfolding of the mutant rods from that of the wild type was a approximately 2 degrees C increase in the thermal stability of the hinge-deletion mutant. Thus, the mechanism 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 and C-terminal tail.
Zolkiewski
M
M
Laboratory of Biochemistry, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892, USA. z5m@cu.nih.gov
Redowicz
M J
MJ
Korn
E D
ED
Hammer
J A
JA
Ginsburg
A
A
D000048:Acanthamoeba / Q000737:chemistry*; D000818:Animals; D002152:Calorimetry, Differential Scanning; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D009218:Myosins / Q000737:chemistry*; D017510:Protein Folding; D014461:Ultracentrifugation
D009218:Myosins