NMR methods have been developed that facilitate the resonance assignment for proteins that can be isotopically enriched with carbon-13 and nitrogen-15 for the study of unlabeled ligands complexed with the uniformly labeled protein. Application of these new methods to the protein calmodulin, in the absence and presence of an unlabeled peptide fragment of skeletal muscle myosin light chain kinase, reveal pronounced structural differences in calmodulin. In the presence of calcium and in the absence of peptide, calmodulin consists of two globular domains, connected by a flexible linker. Nitrogen-15 relaxation studies indicate that the two globular domains reorient nearly isotopically, in contrast to what would be expected for the structure observed in the crystalline state where the two domains are connected by a long `-helix (the so-called """"""""central helix""""""""). In the presence of peptide, the relative orientation of the two calmodulin domains is well determined. The peptide-protein complex adopts an approximately ellipsoidal shape, with the peptide in an `-helical conformation clamped in between the two domains. The structures calculated to date are based on only a fraction of the spectral information available from the 3D and 4D NMR spectra. Work is currently in progress to refine the structure by adding more NOE and J-coupling constraints. spectra using pulsed field gradients.
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