Complexes of smooth muscle tropomyosin with F-actin studied by differential scanning calorimetry.
Differential scanning calorimetry (DSC) and light scattering were used to analyze the interaction of duck gizzard tropomyosin (tropomyosin) with rabbit skeletal-muscle F-actin. In the absence of F-actin, tropomyosin, represented mainly by heterodimers, unfolds at 41 degrees C with a sharp thermal transition. Interaction of tropomyosin heterodimers with F-actin causes a 2-6 degrees C shift in the tropomyosin thermal transition to higher temperature, depending on the tropomyosin/actin molar ratio and protein concentration. A pronounced shift of the tropomyosin thermal transition was observed only 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 conditions, two well-separated peaks of tropomyosin were observed on the thermogram besides the peak of F-actin, the peak characteristic of free tropomyosin heterodimer, and the peak with a maximum at 45-47 degrees C corresponding to tropomyosin bound to F-actin. By measuring the temperature-dependence of light scattering, we found that thermal unfolding of tropomyosin is accompanied by its dissociation from F-actin. Thermal unfolding of tropomyosin is almost completely reversible, whereas F-actin denatures irreversibly. The addition of tropomyosin has no effect on thermal unfolding of F-actin, which denatures 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. after complete irreversible denaturation of F-actin), only the peak characteristic of free tropomyosin was observed on the thermogram during reheating, whereas the thermal transitions of F-actin and actin-bound tropomyosin completely disappeared. Therefore, the DSC method allows changes in thermal unfolding of tropomyosin resulting from its interaction with F-actin to be probed very precisely.
Levitsky
D I
DI
A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Russia.
Rostkova
E V
EV
Orlov
V N
VN
Nikolaeva
O P
OP
Moiseeva
L N
LN
Teplova
M V
MV
Gusev
N B
NB
D000199:Actins / Q000378:metabolism*; D000818:Animals; D002152:Calorimetry, Differential Scanning; D019281:Dimerization; D004372:Ducks; D005895:Gizzard, Avian; D006358:Hot Temperature; D009130:Muscle, Smooth / Q000378:metabolism*; D009391:Nephelometry and Turbidimetry; D010590:Phalloidine / Q000494:pharmacology; D011485:Protein Binding; D017510:Protein Folding; D055503:Protein Multimerization; D011817:Rabbits; D014335:Tropomyosin / Q000378:metabolism*
D000199:Actins; D014335:Tropomyosin; D010590:Phalloidine