Current limits of structural biology: The transient interaction between cytochrome c 6 and photosystem I.

Trimeric photosystem I from the cyanobacterium Thermosynechococcus elongatus (TePSI) is an intrinsic membrane protein, which converts solar energy into electrical energy by oxidizing the soluble redox mediator cytochrome c 6 (Cyt c 6 ) and reducing ferredoxin. Here, we use cryo-electron microscopy and small angle neutron scattering (SANS) to characterize the transient binding of Cyt c 6 to TePSI. The structure of TePSI cross-linked to Cyt c 6 was solved at a resolution of 2.9 Å and shows additional cofactors as well as side chain density for 84% of the peptide chain of subunit PsaK, revealing a hydrophobic, membrane intrinsic loop that enables binding of associated proteins. Due to the poor binding specificity, Cyt c 6 could not be localized with certainty in our cryo-EM analysis. SANS measurements confirm that Cyt c 6 does not bind to TePSI at protein concentrations comparable to those for cross-linking. However, SANS data indicate a complex formation between TePSI and the non-native mitochondrial cytochrome from horse heart (Cyt c HH ). Our study pinpoints the difficulty of identifying very small binding partners (less than 5% of the overall size) in EM structures when binding affinities are poor. We relate our results to well resolved co-structures with known binding affinities and recommend confirmatory methods for complexes with K M values higher than 20 µM.

Antioxidant effect of functionalized alkyl-organotellurides: a study in vitro.

We evaluated the in vitro antioxidant effect of alkyl-organotellurides A-D on lipid peroxidation and protein carbonylation in rat liver homogenates. The thiol oxidase and thiol peroxidase-like activities of compounds were investigated. delta-Aminolevulinic acid dehydratase (delta-ALA-D) activity was determined in rat liver homogenates. Compounds A-D protected against lipid peroxidation induced by Fe(2+)/EDTA and sodium nitroprusside (SNP). According to the confidence limits of the IC(50) values of compounds A-D, the IC(50) values for organotellurides followed the order: C (0.30 microM) < or = B (0.40 microM) < D (0.68 microM) < A (2.90 microM), for Fe(2+)/EDTA, and B (0.21 microM) < or = C (0.33 microM) < or = D (0.43 microM) < A (1.21 microM) for SNP-induced lipid peroxidation. Compounds A-D reduced protein carbonyl content to control levels. The results demonstrated an inverse correlation between thiol oxidase and delta-ALA-D activities. This study supports an antioxidant effect of organotellurides A-D on rat liver.

Transformation of 2,2'-dichlorodiisopropyl ether in mixed and pure culture.

An aerobic enrichment culture derived from a groundwater contaminated with organic and chloroorganic compounds was adapted to the transformation of 2,2'-dichlorodiisopropyl ether (DDE) in a continuous fixed-bed bioreactor. Continuous DDE removal efficiencies over 90% were achieved with a model water containing 3.3 mM methanol as co-substrate at DDE loading rates of up to 150 micromol l(-1) day(-1) with a hydraulic retention time of 24 h. In batch cultures, a stoichiometric release of 2 micromol chloride per micromol DDE transformed was observed. From the mixed culture, a strain was isolated that is able to grow on DDE as the sole energy and carbon source, tolerating DDE concentrations of up to 1 mM. Based on 16S rRNA sequencing, the strain is affiliated with the genus Rhodococcus.

Detection of small-molecule enzyme inhibitors with peptides isolated from phage-displayed combinatorial peptide libraries.

BACKGROUND: The rapidly expanding list of pharmacologically important targets has highlighted the need for ways to discover new inhibitors that are independent of functional assays. We have utilized peptides to detect inhibitors of protein function. We hypothesized that most peptide ligands identified by phage display would bind to regions of biological interaction in target proteins and that these peptides could be used as sensitive probes for detecting low molecular weight inhibitors that bind to these sites. RESULTS: We selected a broad range of enzymes as targets for phage display and isolated a series of peptides that bound specifically to each target. Peptide ligands for each target contained similar amino acid sequences and competition analysis indicated that they bound one or two sites per target. Of 17 peptides tested, 13 were found to be specific inhibitors of enzyme function. Finally, we used two peptides specific for Haemophilus influenzae tyrosyl-tRNA synthetase to show that a simple binding assay can be used to detect small-molecule inhibitors with potencies in the micromolar to nanomolar range. CONCLUSIONS: Peptidic surrogate ligands identified using phage display are preferentially targeted to a limited number of sites that inhibit enzyme function. These peptides can be utilized in a binding assay as a rapid and sensitive method to detect small-molecule inhibitors of target protein function. The binding assay can be used with a variety of detection systems and is readily adaptable to automation, making this platform ideal for high-throughput screening of compound libraries for drug discovery.

Drug target validation: lethal infection blocked by inducible peptide.

Genome projects are generating large numbers of potential new targets for drug discovery. One challenge is target validation, proving the usefulness of a specific target in an animal model. In this paper, we demonstrate a new approach to validation and assay development. We selected in vitro specific peptide binders to a potential pathogen target. By inducing the expression of a selected peptide in pathogen cells causing a lethal infection in mice, the animals were rescued. Thus, by combining in vitro selection methods for peptide binders with inducible expression in animals, the target's validity was rigorously tested and demonstrated. This approach to validation can be generalized and has the potential to become a valuable tool in the drug discovery process.

Synthesis and structure-activity relationships of a series of novel thiazoles as inhibitors of aminoacyl-tRNA synthetases.

A series of novel aminoacyl adenylate mimics has been prepared and evaluated for their inhibitory activity against aminoacyl-tRNA synthetases. Several of these thiazole derivatives displayed potent and selective enzyme activity against both Gram-positive and Gram-negative bacteria.

Src homology 2 domain deletion mutants of p60v-src do not phosphorylate cellular proteins of 120-150 kDa.

We have constructed seven deletions in the src homology 2 (SH2) domain of the Rous sarcoma virus src gene and have expressed them and wild-type v-src (wt v-src) in Rat 1 fibroblasts. Transfected cells containing mutant DNAs have reduced focus forming activity when compared to cells containing the wt v-src DNA. In most cases, established cell lines that express these mutants have altered growth properties in soft agar. The src proteins isolated from mutant cell lines have reduced tyrosine kinase activity. We also see differences in the phosphorylation of cellular proteins in vivo. Unlike the wt protein kinase, the SH2 domain mutant kinases do not phosphorylate a set of cellular proteins ranging in size from 120-150 kDa.

Interaction between the urea cycle and the orotate pathway: studies with isolated hepatocytes.

Two enzymes catalyze the synthesis of carbamylphosphate (CP) in the liver. One is intramitochondrial and utilizes ammonia to make CP for ureagenesis; the second is cytoplasmic and utilizes glutamine to produce CP for pyrimidine biosynthesis. The extent to which the metabolic independence of the two pathways is abridged by the use of a common precursor was examined with measurements of the incorporation of [14C]NaHCO3 into orotic acid, uridine nucleotides, and urea in isolated hepatocytes. Pyrimidine synthesis was markedly stimulated by physiological concentrations of ammonia, and the stimulation was antagonized by ornithine. At intracellular concentrations of ornithine and levels of ammonia found in the portal circulation, some 90% of pyrimidine synthesis was ammonia-dependent. When the glutamine-dependent activity was released from feedback inhibition with galactosamine, the ammonia-dependent incorporation still accounted for 2/3 of pyrimidine synthesis. These results do not support the widely held view that the cytoplasmic enzyme is the sole source of CP for pyrimidine biosynthesis in the liver. They suggest instead that the bulk of the CP incorporated into hepatic pyrimidines is of mitochondrial origin. However, an experiment with intact animals failed to provide decisive evidence on this interpretation. Pyrimidine biosynthesis was sharply inhibited by the addition of uridine, but ureagenesis was unaffected. When physiological levels of ammonia were provided, the sensitivity of pyrimidine biosynthesis to uridine was lost. Although inhibition of the ammonia-dependent enzyme by pyrimidines has been observed with cell-free preparations, it was not evident in the intact cell. Thus, to the extent that the CP consumed in pyrimidine biosynthesis is of mitochondrial origin, feedback control of the orotate pathway appears to be thwarted.