A comparison of 2H- and 13C-based NMR relaxation methods to characterize the dynamics of methyl groups in proteins is presented. Using human ubiquitin as a model system, the field dependence of carbon and deuterium relaxation parameters has been measured and used to probe the utility of various forms of the model-free formalism in revealing the underlying dynamics. We find that both approaches reveal the same overall dynamical features provided that suitable parametrization and model-free spectral densities are employed. It is found that the original and extended model-free formalisms yield different descriptions of the methyl group dynamics and that the extended version is more appropriate for the analysis of carbon relaxation. Because of the inherent differences in the types of information that 2H and 13C offer, deuterium methods appear to provide robust access to methyl symmetry axis order with the least amount of data, while carbon methods provide more robust access to model-free parameters defining the time scale of methyl rotation and methyl symmetry axis motion.

The majority of known proteins are too large to be comprehensively examined by solution NMR methods, primarily because they tumble too slowly in solution. Here we introduce an approach to making the NMR relaxation properties of large proteins amenable to modern solution NMR techniques. The encapsulation of a protein in a reverse micelle dissolved in a low-viscosity fluid allows it to tumble as fast as a much smaller protein. The approach is demonstrated and validated with the protein ubiquitin encapsulated in reverse micelles prepared in a variety of alkane solvents.

The protein kinase C-related protein kinases (PRKs) have been shown to be under the control of the Rho GTPases and influenced by autophosphorylation. In analyzing the relationship between these inputs, it is shown that activation in vitro and in vivo involves the activation loop phosphorylation of PRK1/2 by 3-phosphoinositide-dependent protein kinase-1 (PDK1). Rho overexpression in cultured cells is shown to increase the activation loop phosphorylation of endogenous PRKs and is demonstrated to influence this process by controlling the ability of PRKs to bind to PDK1. The interaction of PRK1/2 with PDK1 is shown to be dependent upon Rho. Direct demonstration of ternary (Rho⅐PRK⅐PDK1) complex formation in situ is provided by the observation that PDK1 is recruited to RhoB-containing endosomes only if PRK is coexpressed. Furthermore, this in vivo complex is maintained after phosphoinositide 3-kinase inhibition. The control of PRKs by PDK1 thus evidences a novel strategy of substrate-directed control involving GTPases.The protein kinase C-related protein kinases (PRKs) 1 are a subfamily of serine/threonine-specific kinases independently identified by molecular cloning, protein purification, and polymerase chain reaction-based screens for novel PKC isoforms (1-3). Two members of this subfamily have been fully cloned and characterized: PRK1 (also termed protein kinase N) and PRK2. They are activated by fatty acids and phospholipids in vitro, although the in vivo significance of this potential is as yet uncharacterized (4, 5). Two-hybrid screens and affinity chromatography identified the PRKs as potential effectors of Rho family GTPases (6 -8). The novel amino-terminal HR1 domain (9) was subsequently identified as the Rho-interacting region (10, 11), an interaction that is presumed to disrupt the autoinhibitory effect produced by a pseudosubstrate-catalytic domain contact (12). Indeed, the GTPase interaction has been shown to increase modestly the activity of the intact kinase (6 -8). The observed proteolytic activation of these kinases would support the role of an allosteric amino-terminal GTPase interaction in the regulation of activity (13,14). In addition to any allosteric component, Rho GTPases can also be responsible for the location of these kinases, with RhoB causing localization to an endosomal compartment in fibroblasts (15), a translocation event that is associated with the accumulation of a hyperphosphorylated form of the kinase.In addition to GTPases, other PRK interactions have been identified. The adapter protein NCK has been shown to interact with a proline-rich region just N-terminal of the kinase domain of PRK2 (16). A similar region is absent in PRK1, suggesting a specificity in the upstream recruitment of these kinases. A potential role for PRKs in the regulation of the cytoskeleton has been proposed following the demonstrated disruption of fibroblast actin stress fibers by the expression of a catalytically inactive PRK2 (8) and the observed PRK1 interaction with the head domain of intermediate fil...