The interaction of apocalmodulin (apoCaM) with a peptide (Neurop) based on the primary sequence of the calmodulin-binding domain of neuromodulin has been studied by nuclear magnetic resonance (NMR) methods. The NMR spectra of both apocalmodulin and its 1:1 complex with the Neurop peptide have been assigned by triple resonance and nuclear Overhauser effect-(NOE-) based strategies. ApoCaM displays many of the same basic structural features as calcium-saturated calmodulin. Analysis of observed chemical shifts and patterns of NOEs on the main chain indicates extensive and regular secondary structure throughout the N-terminal domain. In contrast, the helices of the C-terminal domain are somewhat irregular and are dynamically averaged. The EF-hands are intact in the N-terminal domain with the loops forming a short antiparallel beta sheet. Under low-salt conditions, two helix-loop-helix EF-hand motifs are present in the C-terminal domain of apoCaM but do not show interstrand NOEs. The spectral perturbations of apoCaM upon complexation with the Neurop peptide are relatively small with the larger chemical shift perturbations occurring in the C-terminal domain. The general secondary structure and tertiary organization appears to remain roughly the same as in free apoCaM. Stoichiometric titration of the apoCaM.Neurop complex with calcium indicates that the C-terminal domain EF-hands have a higher affinity for calcium than N-terminal domain EF-hands. Thus, this complex offers a unique opportunity to examine the structural and energetic consequences of calcium-dependent and calcium-independent binding of peptide to calmodulin.

Selective incorporation of 13C into the methyl groups of protein side chains is described as a means for simplifying the measurement and interpretation of 13C relaxation parameters. High incorporation (> 90%) is accomplished by using pyruvate (3-13C, 99%) as the sole carbon source in the growth media for protein overexpression in E. coli. This improved labeling scheme increases the sensitivity of the relaxation experiments by approximately fivefold when compared to randomly fractionally 13C-labeled protein, allowing high-quality measurements on relatively dilute (< 1 mM) protein samples at a relatively low cost.

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 15N-1H correlation techniques were used to quantitate amide proton exchange rates of both 15N-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.(ABSTRACT TRUNCATED AT 250 WORDS)