ENERGETICS OF TARGET PEPTIDE RECOGNITION BY CALMODULIN - A CALORIMETRIC STUDY

Calmodulin is a small protein involved in the regulation of a wide var iety of intracellular processes. The cooperative binding of Ca2+ to ca lmodulin's two Ca2+ binding domains induces conformational changes whi ch allow calmodulin to activate specific target enzymes. The associati on of calmodulin with a peptide corresponding to the calmodulin bindin g site of rabbit smooth muscle myosin light chain kinase (smMLCKp) was studied using isothermal titration microcalorimetry. The dependence o f the binding energetics on temperature, pH, Ca2+ concentration, and N aCl concentration were determined. It is found that the binding of cal modulin to smMLCKp proceeds with negative changes in enthalpy (Delta H ), heat capacity (Delta C-p), and entropy (Delta S) near room temperat ure, indicating that it is an enthalpically driven process that is ent ropically unfavorable. From these results it is concluded that the hyd rophobic effect, an entropic effect which favors the removal of nonpol ar protein groups from water, is not a major driving force in calmodul in-smMLCKp recognition. Although a large number of non-polar side-chai ns are buried upon binding, these stabilize the complex primarily by f orming tightly packed van der Waals interactions with one another. Bin ding at acidic pH was studied in order to assess the contribution of e lectrostatic interactions to binding. It is found that moving to acidi c pH results in a large decrease in the Gibbs free energy of binding b ut no change in the enthalpy, indicating that electrostatic interactio ns contribute only entropically to the binding energetic. The accessib le surface area and atomic packing density of the calmodulin-smMLCKp c rystal structure are analyzed, and the results discussed in relation t o the experimental data. (C) 1997 Academic Press Limited.