EFFECTS OF IONS AND PH ON THE THERMAL-STABILITY OF THIN AND THICK FILAMENTS OF SKELETAL-MUSCLE - HIGH-SENSITIVITY DIFFERENTIAL SCANNING CALORIMETRIC STUDY

Differential scanning calorimetry (DSC) is unique for studying conformational changes in supramolecular structures because it is immune to interference by the turbidity and other optical artifacts of a sample solution. We have employed DSC to study thermal stability of myosin and actin in their filamentous forms (i.e., thick and thin filaments). The thermal stability of the myosin monomer, as well as polymers, showed remarkable sensitivities to pH and to the ionic strength of the solution. At pH 7.5, the endotherm of myosin filaments was broad and resembled that of the monomer in solution. Reducing the pH to 6.3 split the endotherm of the filament into two major transitions. The first one, with a Tm of 47 °C, a ΔHcal of 805 kcal/mol, and a cooperative ratio (CR) of 0.1, was relatively insensitive to the pH changes whereas the second one which represented approximately 80% of the helical structure was pH sensitive. The second transition released 2.17 H+ per mole at 0.17 M KCl and was defined by a Tm of 53.9 °C, a ΔHcal of 917 kcal/mol, and a CR of 0.35. The major fragment contributing to the splitting of the endotherm was interpreted to be S-2 because the Tm of purified S-2 in a similar medium also shifted from 39.5 °C at pH 7.3 to 49.6 °C at pH 6.0. KCl had similar effects on the shape of the endotherm of the thick filament. A decrease of KCl from 0.2 to 0.1 M enhanced the effect of pH on the second transition. A calculated release of 8.9 K+ per mole was associated with the melting of the major part of the helix. The Tm vs pH curve had an inflection point at pH 6.8 in 0.17 M KCl. These conditions mimicked the physiological conditions. The two major transitions of myosin filaments did not show a strong cooperativity (CR 0.1 and 0.4, respectively), implying that the domains observed in the monomer [Bertazzon, A., & Tsong, T. Y. (1989) Biochemistry 28, 9784–9790] were also present in the filament. In actin, a partial polymerization was observed when the concentration of Ca2+ in the medium was increased (1–10 mM), as assessed by analysis of the DSC endotherms (the CR increased from 0.7 to 1.25). This effect of calcium was not observed in the fully polymerized protein; however, a reduction of the pH from 7.9 to 5.9 increased the Tm from 68.2 to 74.3 °C, with apparently no effect on the cooperative ratio. These results suggest that, in contrast to the myosin filament, the thin filament behaved as a fully cooperative structure. The effect of pH on the thermal stability of F-actin was consistent with the release of 1.4 H+ per mole upon melting. © 1990, American Chemical Society. All rights reserved.