Complexes of smooth muscle tropomyosin with F-actin studied by differential scanning calorimetry

Differential scanning calorimetry (DSC) and light scattering were used to a nalyze the interaction of duck gizzard tropomyosin (tropomyosin) with rabbi t skeletal-muscle F-actin. In the absence of F-actin, tropomyosin, represen ted mainly by heterodimers, unfolds at 41 degrees C with a sharp thermal tr ansition. Interaction of tropomyosin heterodimers with F-actin causes a 2-6 degrees C shift in the tropomyosin thermal transition to higher temperatur e, depending on the tropomyosin/actin molar ratio and protein concentration . A pronounced shift of the tropomyosin thermal transition was observed onl y for tropomyosin heterodimers, and not for homodimers. The most pronounced effect was observed after complete saturation of F-actin with tropomyosin molecules, at tropomyosin/actin molar ratios > 1 : 7. Under these condition s, two well-separated peaks of tropomyosin were observed on the thermogram besides the peak of F-actin, the peak characteristic of free tropomyosin he terodimer, and the peak with a maximum at 45-47 degrees C corresponding to tropomyosin bound to F-actin. By measuring the temperature-dependence of li ght scattering, we found that thermal unfolding of tropomyosin is accompani ed by its dissociation from F-actin. Thermal unfolding of tropomyosin is al most completely reversible, whereas F-actin denatures irreversibly. The add ition of tropomyosin has no effect on thermal unfolding of F-actin, which d enatures with a maximum at 64 degrees C in the absence and at 78 degrees C in the presence of a twofold molar excess of phalloidin. After the F-actin- tropomyosin complex had been heated to 90 degrees C and then cooled (i.e. a fter complete irreversible denaturation of F-actin), only the peak characte ristic of free tropomyosin was observed on the thermogram during reheating, whereas the thermal transitions of F-actin and actin-bound tropomyosin com pletely disappeared. Therefore, the DSC method allows changes in thermal un folding of tropomyosin resulting from its interaction with F-actin to be pr obed very precisely.