Although proteins populate large structural ensembles, X-ray diffraction data are traditionally interpreted using a single model. To search for evidence of alternate conformers, we developed a program, Ringer, which systematically samples electron density around the dihedral angles of protein side chains. In a diverse set of 402 structures, Ringer identified weak, nonrandom electron-density features that suggest of the presence of hidden, lowly populated conformations for >18% of uniquely modeled residues. Although these peaks occur at electron-density levels traditionally regarded as noise, statistically significant (P < 10 25 ) enrichment of peaks at successive rotameric v angles validates the assignment of these features as unmodeled conformations. Weak electron density corresponding to alternate rotamers also was detected in an accurate electron density map free of model bias. Ringer analysis of the high-resolution structures of free and peptide-bound calmodulin identified shifts in ensembles and connected the alternate conformations to ligand recognition. These results show that the signal in high-resolution electron density maps extends below the traditional 1 r cutoff, and crystalline proteins are more polymorphic than current crystallographic models. Ringer provides an objective, systematic method to identify previously undiscovered alternate conformations that can mediate protein folding and function.

Flavoprotein reductases play a key role in electron transfer in many physiological processes. We have isolated a cDNA with strong sequence similarities to cytochrome P-450 reductase and nitric-oxide synthase. The cDNA encodes a protein of 597 amino acid residues with a predicted molecular mass of 67 kDa. Northern blot analysis identified a predicted transcript of 3.0 kilobase pairs as well as a larger transcript at 6.0 kilobase pairs, and the gene was mapped to chromosome 9q34.3 by fluorescence in situ hybridization analysis. The amino acid sequence of the protein contained distinct FMN-, FAD-, and NADPH-binding domains, and in order to establish whether the protein contained these cofactors, the coding sequence was expressed in insect cells and purified. Recombinant protein bound FMN, FAD, and NADPH cofactors and exhibited a UV-visible spectrum with absorbance maxima at 380, 460, and 626 nm. The purified enzyme reduced cytochrome c, with apparent K m and k cat values of 21 M and 1.3 s ؊1 , respectively, and metabolized the one-electron acceptors doxorubicin, menadione, and potassium ferricyanide. Immunoblot analysis of fractionated MCF7 cells with antibodies to recombinant NR1 showed that the enzyme is cytoplasmic and highly expressed in a panel of human cancer cell lines, thus indicating that this novel reductase may play a role in the metabolic activation of bioreductive anticancer drugs and other chemicals activated by one-electron reduction.Flavin-containing enzymes catalyze a broad spectrum of biochemical reactions ranging from oxidase, dehydrogenase, and mono-oxygenase reactions. Most flavoproteins contain either FMN or FAD as prosthetic groups; however, a small number of enzymes contain both cofactors. In mammalian systems, NADPH cytochrome P-450 oxidoreductase (cytochrome P-450 reductase) was the first such enzyme isolated (1, 2), followed by several other dual flavin enzymes including nitric-oxide synthases (NOS) 1 in higher organisms (3, 4), and CYP102 (5) and sulfite reductase (6) in bacteria. More recently, the cDNA sequence encoding a putative FMN-and FAD-binding enzyme, methionine synthase reductase, has been described (7). Cytochrome P-450 reductase, the most extensively characterized of these enzymes (8 -10), is found in the endoplasmic reticulum of most eukaryote cells and is an integral component of the monooxygenase system transferring electrons from NADPH to cytochromes P-450 via FMN and FAD co-factors. Cytochrome P-450 reductase may also donate electrons to heme oxygenase (11), cytochrome b 5 (12), and the fatty acid elongation system (13), and can reduce cytochrome c (14). Both the crystal and NMR structure of the FMN domain of human cytochrome P-450 reductase (15, 16) and the crystal structure of the NH 2 -terminally truncated form of the rat enzyme (17) have been resolved, providing high resolution structural information on this enzyme class. The amino-terminal region of cytochrome P-450 reductase bears striking amino acid homology with FMN-containing flavodoxins, while the carboxyl-term...

Xenobiotic metabolizing enzymes in the olfactory epithelium have been suggested to catalyse inactivation and facilitate elimination of odorants. We report here the molecular cloning and functional characterization of a human olfactory UDP-glucuronosyltransferase (UGT). The cloned protein is composed of 527 amino acids with an identity of 87% with a rat olfactory UGT and of 43-62% with other human UGT isoforms. Based on the sequence homology, it has been designated hUGT2A1. The gene was mapped to chromosome 4q13 by fluorescence in situ hybridization. The expression appeared to be specific for the olfactory tissue. The substrate specificity of this isoform was assessed using Chinese hamster V79 cells stably transfected with the isolated cDNA. The expressed enzyme showed a broad substrate spectrum including a range of phenolic compounds as well as aliphatic and monoterpenoid alcohols, among them many odorants. Furthermore, some steroids, especially androgens, some drugs and carcinogens were conjugated. The results support a role of the enzyme in olfactory perception and in protection of the neural system against airborne hazardous chemicals.

Insulin has been previously shown to regulate the expression of the hepatic glycogen-targeting subunit, G L , of protein phosphatase 1 (PP1) and is believed to control the activity of the PP1-G L complex by modulation of the level of phosphorylase a, which allosterically inhibits the activity of PP1-G L . These mechanisms contribute to the ability of insulin to increase hepatic glycogen synthesis. Human G L shows >88% amino acid identity to its rat and mouse homologs, with complete conservation of the phosphorylase a binding site. G L is highly expressed in the liver and present at appreciable levels in heart tissue of all three species. Surprisingly, G L is highly expressed in human skeletal muscle while only being detected at very low levels in rat, mouse, and rabbit skeletal muscle. The amino acid sequence of G L predicted from the cDNA is identical in human liver and skeletal muscle and encoded by a gene on chromosome 8 at p23.1. The species-specific difference in the level of expression of G L mRNA and protein in skeletal muscle has important implications for understanding the mechanisms by which insulin regulates glycogen synthesis in human skeletal muscle and for questions regarding whether rodents are appropriate models for this purpose. Diabetes 51:591-598, 2002