A new NMR approach has been developed for the measurement of three-bond J couplings. The approach is applicable to isotopically enriched macromolecules and to concentrated solutions of molecules at natural abundance. The three-bond J couplings between atomic nuclei provide very direct information on the magnitude of the intervening dihedral angles and thereby yield valuable new constraints in the structure determination process of biological macromolecules. The approach has been successfully applied to the measurement of 13C-13C, 13C-15N, 1H-15N, 1H-13C, 1H-113Cd, and HN-Ha J couplings in a range of proteins. It has been demonstrated for the first time that a detailed structural analysis of a detergent-solubilized protein is possible without recourse to perdeuterated detergent. This type of analysis relies on the combined use of 13C- and 15N-edited NMR experiments and pulsed field gradients which make it possible to suppress the intense detergent 1H NMR signals while observing the much weaker protein signals. The approach is demonstrated for the protein calcineurin B, solubilized by a 15 molar excess of the detergent CHAPS. The calcineurin A/B complex remains fully active in the presence of CHAPS and NMR results confirm that the detergent does not significantly alter the protein structure. Resonance assignment of the backbone 1H,13C, and 15N resonances and the secondary structure determination process have been completed. It is evident that the secondary structure of calcineurin B is highly homologous with that of the smaller protein calmodulin.
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