Current methods of determining the rotational diffusion tensors of proteins in solution by NMR spectroscopy exclusively utilize relaxation rate constants for backbone amide 15N spins. However, the distributions of orientations of N-H bond vectors are not isotropic in many proteins, and correlations between bond vector orientations reduce the accuracy and precision of rotational diffusion tensors extracted from 15N spin relaxation data. The inclusion of both 13C alpha and 15N spin relaxation rate constants increases the robustness of the diffusion tensor analysis because the orientations of the C alpha-H alpha bond vectors differ from the orientations of the N-H bond vectors. Theoretical and experimental results for calbindin D9k, granulocyte colony stimulating factor, and ubiquitin, three proteins with different distributions of N-H and C alpha-H alpha bond vectors, are used to illustrate the advantages of the simultaneous utilization of 13C alpha and 15N relaxation data.

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Ca(2+)-dependent protein kinases (CDPK) have a calmodulin-like domain (CaM-LD) tethered to the C-terminal end of the kinase. Activation is proposed to involve intramolecular binding of the CaM-LD to a junction sequence that connects the CaM-LD to the kinase domain. Consistent with this model, a truncated CDPK (DeltaNC) in which the CaM-LD has been deleted can be activated in a bimolecular interaction with an isolated CaM-LD or calmodulin, similar to the activation of a calmodulin-dependent protein kinase (CaMK) by calmodulin. Here we provide genetic evidence that this bimolecular activation requires a nine-residue binding segment from F436 to I444 (numbers correspond to CPK-1 accession number L14771). Two mutations at either end of this core segment (F436/A and VI444/AA) severely disrupted bimolecular activation, whereas flanking mutations had only minor effects. Intramolecular activation of a full-length kinase was also disrupted by a VI444/AA mutation, but surprisingly not by a F436/A mutation (at the N-terminal end of the binding site). Interestingly, intramolecular but not bimolecular activation was disrupted by insertion mutations placed immediately downstream of I444. To show that mutant enzymes were not misfolded, latent kinase activity was stimulated through binding of an antijunction antibody. Results here support a model of intramolecular activation in which the tether (A445 to G455) that connects the CaM-LD to the kinase provides an important structural constraint and is not just a simple flexible connection.