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A multinuclear NMR analysis and molecular refinement study of a 5-pyrimidine-GA-purine-3' sequence molecular model reveals the presence of a B-type right-handed helix, with the constituent bases possessing routine anti-glycosidic torsional angles. Type I mismatched bases possess edge-to-face hydrogen bonds while type II base pairs have face-to-face hydrogen bonds. The flanking sequences determine the conformation with 5'-pyrimidine-GA-purine-3' sequences which select type I conformation and the 5'-purine-GA-pyrimidine-3' sequences having type II conformation.

The complimentarity of acetylcholinesterase and butyrylcholinesterase was examined. In addition, residues that are responsible for the substrate and inhibitor specificity of the two esterases from mouse were assigned. Esterase chimeras and site-specific mutants were prepared to enable characterization. Residues F295 and F297 were found to determine acyl pocket specificity. Moreover, this finding suggests that modification of phenylalanines and adjacent residues can alter acyl ester specificity.

A combination of magic-angle spinning (MAS) and solid state NMR (SSNMR) studies which provide structural information on human cytochrome P450 (CYP) 3A4 is described. The results help to understand ligand-specific binding events, allosteric regulation, and the impact of membrane interactions on CYP3A4 activity.

Study is presented in which the folding transition state of a protein is characterized with an interesting and unusual topology, the C-terminal domain of ribosomal protein L9 (CTL9). Results indicate that the folding of CTL9 is broadly consistent with the nucleation-condensation model of protein folding.

A model of the unfolded state of the N-terminal domain of ribosomal protein L9 (NTL9) in the absence of denaturant was generated by substitution of an alanine for phenylalanine 5 located in the core of this protein. Studies showed that the physiologically relevant unfolded state of wild-type NTL9 was more compact than the CD spectrum of the variant F5A since the mutation should reduce the level of hydrophobic clustering in the unfolded state in the absence of denaturant.

Amide H/D exchange studies and thermodynamic measurements are combined to probe pH dependent structure in the unfolded state of the small, mixed alpha-beta protein (C-terminal domain of the ribosomal protein L9) CTL9. The studies revealed that unfolded state of CTL9 contains significant interactions and are hydrophobic in nature that lead to compaction but are not sufficient to confer significant protection to hydrogen bonds.

Research demonstrates that the catalysis of RNase HII of Methanococcus jannaschii is mediated by one metal binding to the enzyme and concomittant substrate binding positions the metal ion. Amino acid residue Asp149 is required for the enzyme activity. Data show that metal binding occurs on three sites and the fourth site is involved with the substrate interaction.

The C-terminal domain of ribosomal protein L9 (CTL9) is a 92-residue α-β protein which contains an unusual three-stranded mixed parallel and antiparallel β-sheet. The protein folds in a two-state fashion, and the folding rate is slow. It is thought that the slow folding may be caused by the necessity of forming this unusual β-sheet architecture in the transition state for folding. This hypothesis makes CTL9 an interesting target for folding studies. The transition state for the folding of CTL9 was characterized by Φ-value analysis. The folding of a set of hydrophobic core mutants was analyzed together with a set of truncation mutants. The results revealed a few positions with high 1-values (≥0.5), notably, V131, L133, H134, V137, and L141. All of these residues were found in the β-hairpin region, indicating that the formation of this structure is likely to be the rate-limiting step in the folding of CTL9. One face of the β-hairpin docks against the N-terminal helix. Analysis of truncation mutants of this helix confirmed its importance in folding. Mutations at other sites in the protein gave small Φ-values, despite the fact that some of them had major effects on stability. The analysis indicates that formation of the antiparallel hairpin is critical and its interactions with the first helix are also important. Thus, the slow folding is not a consequence of the need to fully form the unusual three-stranded β-sheet in the transition state. Analysis of the urea dependence of the folding rates indicates that mutations modulate the unfolded state. The folding of CTL9 is broadly consistent with the nucleation-condensation model of protein folding. [ABSTRACT FROM AUTHOR]

The conventional protein kinase C isoform, PKCβII, is a signaling kinase activated during the hyperglycemic state and has been associated with the development of microvascular abnormalities associated with diabetes. PKCflII, therefore, has been identified as a therapeutic target where inhibitors of its kinase activity are being pursued for treatment of microvascular-related diabetic complications. In this report, we describe the crystal structure of the catalytic domain of PKCβII complexed with an inhibitor at 2.6 Å resolution. The kinase domain of PKCβII was cleaved and purified from full-length PKCβII expressed in baculovirus-infected insect cells. The overall kinase domain structure follows the classical bilobal fold and is in its fully activated conformation with three well-defined phosphorylated residues: Thr-500, Thr-641, and Ser-660. Different from the crystal structures of nonconventional PKC isoforms, the C-terminus of the PKCβII catalytic domain is almost fully ordered and features a novel α helix in the turn motif. An ATP-competitive inhibitor, 2-methyl-1H-indol-3-yl-BIM-1, was crystallized with the PKCβII catalytic domain as a dimer of two enzyme-inhibitor complexes. The bound inhibitor adopts a nonplanar conformation in the ATP-binding site, with the kinase domain taking on an intermediate, open conformation. This PKCβII-inhibitor complex represents the first structural description of any conventional PKC kinase domain. Given the pathogenic role of PKCβII in the development of diabetic complications, this structure can serve as a template for the rational design of inhibitors as potential therapeutic agents. [ABSTRACT FROM AUTHOR]

Investigates the influence of cytochrome P450 (CYP) in converting cyano group of pinacidil to the corresponding amide. Incubation of pinacidil with CYP; Results of kinetics of pinacidil amide formation by CYP; Mediation of YCP3A4 reaction.

Reports on the types of posttranslational modifications found in vivo on the HMG-I and HMG-Y proteins isolated from two human breast epithelial cell lines, MCF-7 and MCF-7/PKC-alpha, that represent different stages of neoplastic progression. Inability of MCF-7 to form tumors when injected into nude mice; Malignant and highly metastasic MCF-7/PKC-alpha.