High-resolution differential scanning calorimetric study of myosin, functional domains, and supramolecular structures

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Abstract
High-resolution differential scanning calorimetry (DSC) has been employed to study the thermal stability of myosin, its major constitutive fragments (S-1, light chains, and rod), and also reconstituted thick filaments. The thermal denaturation of soluble myosin was complex and was characterized by a multistep endothermic process for the temperature range from 41 to 60.degree. C. The shape of the endotherm was highly dependent on the pH and the ionic strength of the solution, although the .DELTA.Hcal (calorimetric enthalpy) of denaturation (1715 .+-. 75 kcal/mol) was insensitive to these changes for the solvent conditions used in this study. This value also agrees, with experimental error, with the sum of the denaturation enthalpies obtained for isolated fragments (1724 .+-. 79 kcal/mol). In identical conditions of ionic strength, pH, and heating rate, the computer-calculated differential endotherms of domains belonging to S-1 and light chains were superimposable with those of the isolated fragments. Their responses to changes in the solvent condition were also similar. We suggest that the observd functional independence of the major domains in myosin reflects also the independence of their structural stability. The thermal unfolding of the isolated rod was multiphasic and readily reversible (95%). It occurred between 41 and 60.degree. C, and an .DELTA.Hcal of 1058 .+-. 59 kcal/mol. The melting of S-1 showed a single peak at 46.3 .+-. 0.1.degree. C with an .DELTA.Hcal of 255 .+-. 12 kcal/mol. Light chains melted at 51.0 .+-. 0.2.degree. C with an .DELTA.Hcal of 85 .+-. 15 kcal/mol. Despite the reversibility of the rod, the thermal denaturation of myosin was irreversible. When the ionic strength of the solution was gradually reduced to obtain synthetic filaments, the transition temperatures of domains converged, and the shape of the endotherm appeared sharp, with little effect on the overall enthalpy of denaturation (.DELTA.Hcal of 1679 .+-. 98 kcal/mol). The peak of the overall transition of myosin filaments was 45.degree. C in 0.2 M KCl and increased to 49.degree. C in 0.1 M KCl. Electron micrographs showed different morphologies for filaments grown in an imidazole buffer and that grown in a phosphate buffer although the endotherms of these samples appeared similar. pH had a strong effect on the endotherms of these filaments. Lowering the pH from 7.0 to 6.7 in 0.1 M KCl increased the Tm of the main peak by 1.4.degree. C, resulting in the separation of the endotherm into two distinct transitions. These two transitions were broad, and their .DELTA.HvH values were much smaller than the .DELTA.Hcal. Analysis suggests that the structural domains that constitute a myosin molecule also exist in these filaments and that the stability of these structural domains is equally sensitive to modulation by the ionic strength and the pH of the solution, consistent with the possibility of local structure events in the molecular mechanisms of muscle contraction.