DIFFERENTIAL SCANNING CALORIMETRIC STUDIES ON CALMODULIN IN THE PRESENCE OF MONOCATIONS, DICATIONS, AND ETHANOL

Changing the monocations in solution with calmodulin (0.225-0.450 mM at pH = 7.5) alters the broad reversible denaturization observed with differential scanning calorimetry (maxima near 50°C), and an overall destabilization of this protein's tertiary and secondary structure is found in solutions containing ethanol (1.5-3.0 M). With tetraethylammonium ions present (12 mM), the excess heat capacity curve, corrected for intrinsic heat capacity variations, can be fit to a single two-state transition with a maximum near 49°C. With Na+ or K+ present at the same concentration, the maximum is in the 52-54°C range, and with only Li+ (12 mM) present the maximum is near 59°C. Two transitions are needed to fit adequately the corrected excess heat capacity curves with only alkali-metal ions present. With 5.0 mM Mg2+ and 12.5 mM K+ present, the melting region has a maximum near 80°C and can be represented by two transitions with enthalpy changes similar to those observed with only alkali-metal monocations present; similar effects are seen for Sr2+ and Mn2+. Up to a mole ratio of two Ca2+ ions per calmodulin, the excess heat capacity curves are similar in shape and have nearly the same maxima as those found in the presence of monocations, implying that any structural change linked to Ca2+ binding and/or preferential binding of Ca2+ to a native state is associated with substantial occupation of the second, third, and fourth sites. The unusually large slope in the excess heat capacity curves, seen before the transition region with only monocations present, is maintained up to 100°C when calmoldulin's four metal ion binding sites are nearly filled with Ca2+, Cd2+, or Co2+ ions. These results are adequately represented by (1) a sequential melting model involving three states, two being highly structured (native) and one a random coil (denatured), and (2) preferential stabilization of the first two states by metal ion binding to the protein. © 1990 American Chemical Society.