THE ENERGETICS AND DYNAMICS OF MOLECULAR RECOGNITION BY CALMODULIN

Amide hydrogen exchange has been used to examine the structural dynamics and energetics of the interaction of a peptide corresponding to the calmodulin binding domain of smooth muscle myosin light chain kinase with calcium-saturated calmodulin. Heteronuclear NMR N-15-H-1 correlation techniques were used to quantitate amide proton exchange rates of both N-15-labeled and unlabeled amide protons of the smMLCK peptide complexed to calmodulin. Hydrogen exchange slowing factors were determined for 18 of the 19 amide hydrogens and found to span 6 orders of magnitude. The first six residues of the bound peptide were found to have slowing factors near 1 and are considered not to be hydrogen bonded, consistent with the previously reported model for the structure of the peptide. The pattern of hydrogen exchange of hydrogen-bonded amide hydrogens is indicative of end-fraying behavior characteristic of helix-coil transitions, The effective statistical mechanical parameters revealed by the end fraying are consistent with exchange from a highly solvated state. However, the slowing factors of the first hydrogen-bonded amide hydrogens are large, indicating the requirement for a reorganization of the calmodulin-peptide complex before the helix-coil transitions leading to exchange can occur. Taken together, these observations suggest that the collapsed complex reorganizes with an associated free energy change of 5.5 kcal/mol to a more open state where the helical peptide is highly solvated and undergoes helix-coil transitions leading to exchange, The free energy difference between the most and least stable intrahelical amide hydrogen bonds of the bound peptide is estimated to be approximately 2.5 kcal/mol. In reverse, these events are effectively those of the collapse of the initial encounter complex to the final stable complex structure. The results presented demonstrate the potential of hydrogen exchange for studying the dynamics and energetics of interprotein interactions along the free energy path from the initial encounter complex to the final equilibrium structure.