Tropomyosin stability: assignment of thermally induced conformational transitions to separate regions of the molecule.
Tropomyosin was prepared from rabbit skeletal muscle and studied in a differential scanning calorimeter. The characteristics of the observed endotherms were studied as a function of p]H, salt concentration, oxidation state of Cys-190, and concentration of the divalent metal ions Ca2+ and Mg2+. The large shifts observed for the Tm values of the components of the endotherms with changing pH and salt concentration are consistent with electrostatic effects being an important determinant of the structural stability of tropomyosin. For reduced tropomyosin or tropomyosin blocked with N-ethylmaleimide, two endotherms were observed with Tm values of 41.5 and 52.5 degrees C at neutral pH in a low-salt buffer. For tropomyosin containing a disulfide link at Cys-190, two endotherms were observed with Tm values of 32 and 52 degrees C under the same conditions. The endotherm at 52 degrees C contains contributions from conformational transitions in two independent structural regions. An analysis of the heat-capacity profiles for the two large cyanogen bromide peptides, CN1A and CN1B, enabled the assignment of two components of the endotherms to structural transitions in the C-terminal region which includes Cys-190 and in the N-terminal region. Calcium and magnesium ions in the 1-10 mM range increased the stability of several of the regions of the structure, presumably by binding to localized areas of excess negative charge. Unfolding of tropomyosin in the 20-70 degrees C range is a multistep process and occurs with an average enthalpy of 4 cal g-1.
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002152:Calorimetry, Differential Scanning; D003488:Cyanogen Bromide; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000494:pharmacology; D010446:Peptide Fragments / Q000032:analysis; D011487:Protein Conformation / Q000187:drug effects; D011817:Rabbits; D014335:Tropomyosin / Q000032:analysis*
D010446:Peptide Fragments; D014335:Tropomyosin; D008274:Magnesium; D003488:Cyanogen Bromide; D002118:Calcium