NMR structure of the N-terminal domain of the replication initiator protein DnaA

DnaA is an essential component in the initiation of bacterial chromosomal replication. DnaA binds to a series of 9 base pair repeats leading to oligomerization, recruitment of the DnaBC helicase, and the assembly of the replication fork machinery. The structure of the N-terminal domain (residues 1-100) of DnaA from Mycoplasma genitalium was determined by NMR spectroscopy. The backbone r.m.s.d. for the first 86 residues was 0.6 +/- 0.2 Angstrom based on 742 NOE, 50 hydrogen bond, 46 backbone angle, and 88 residual dipolar coupling restraints. Ultracentrifugation studies revealed that the domain is monomeric in solution. Features on the protein surface include a hydrophobic cleft flanked by several negative residues on one side, and positive residues on the other. A negatively charged ridge is present on the opposite face of the protein. These surfaces may be important sites of interaction with other proteins involved in the replication process. Together, the structure and NMR assignments should facilitate the design of new experiments to probe the protein-protein interactions essential for the initiation of DNA replication

ACCESS: Design and Sub-System Performance

Establishing improved spectrophotometric standards is important for a broad range of missions and is relevant to many astrophysical problems. ACCESS, "Absolute Color Calibration Experiment for Standard Stars", is a series of rocket-borne sub-orbital missions and ground-based experiments designed to enable improvements in the precision of the astrophysical flux scale through the transfer of absolute laboratory detector standards from the National Institute of Standards and Technology (NIST) to a network of stellar standards with a calibration accuracy of 1% and a spectral resolving power of 500 across the 0.35 -1.7 micrometer bandpass

Metabolomics of Oxidative Stress in Recent Studies of Endogenous and Exogenously Administered Intermediate Metabolites

Aerobic metabolism occurs in a background of oxygen radicals and reactive oxygen species (ROS) that originate from the incomplete reduction of molecular oxygen in electron transfer reactions. The essential role of aerobic metabolism, the generation and consumption of ATP and other high energy phosphates, sustains a balance of approximately 3000 essential human metabolites that serve not only as nutrients, but also as antioxidants, neurotransmitters, osmolytes, and participants in ligand-based and other cellular signaling. In hypoxia, ischemia, and oxidative stress, where pathological circumstances cause oxygen radicals to form at a rate greater than is possible for their consumption, changes in the composition of metabolite ensembles, or metabolomes, can be associated with physiological changes. Metabolomics and metabonomics are a scientific disciplines that focuse on quantifying dynamic metabolome responses, using multivariate analytical approaches derived from methods within genomics, a discipline that consolidated innovative analysis techniques for situations where the number of biomarkers (metabolites in our case) greatly exceeds the number of subjects. This review focuses on the behavior of cytosolic, mitochondrial, and redox metabolites in ameliorating or exacerbating oxidative stress. After reviewing work regarding a small number of metabolites—pyruvate, ethyl pyruvate, and fructose-1,6-bisphosphate—whose exogenous administration was found to ameliorate oxidative stress, a subsequent section reviews basic multivariate statistical methods common in metabolomics research, and their application in human and preclinical studies emphasizing oxidative stress. Particular attention is paid to new NMR spectroscopy methods in metabolomics and metabonomics. Because complex relationships connect oxidative stress to so many physiological processes, studies from different disciplines were reviewed. All, however, shared the common goal of ultimately developing “omics”-based, diagnostic tests to help influence therapies

Structural Studies of MJ1529, an O6-methylguanine-DNA Methyltransferase

The structure of an O{sub 6}-methylguanine methyltransferase from the thermophile Methanococcus jannaschii has been determined using multinuclear multidimensional NMR spectroscopy. The structure is similar to homologues from other organisms that have been determined by crystallography, with some variation in the N-terminal domain. The C-terminal domain is more highly conserved in both sequence and structure. Regions of the protein show broadening reflecting conformational flexibility that is likely related to function

The C-Terminal RpoN Domain of sigma54 Forms an unpredicted Helix-Turn-Helix Motif Similar to domains of sigma70

The ''{delta}'' subunit of prokaryotic RNA-polymerase allows gene-specific transcription initiation. Two {sigma} families have been identified, {sigma}{sup 70} and {sigma}{sup 54}, which use distinct mechanisms to initiate transcription and share no detectable sequence homology. Although the {sigma}{sup 70}-type factors have been well characterized structurally by x-ray crystallography, no high-resolution structural information is available for the {sigma}{sup 54}-type factors. Here we present the NMR derived structure of the C-terminal domain of {sigma}{sup 54} from Aquifex aeolicus. This domain (Thr323 to Gly389), which contains the highly conserved RpoN box sequence, consists of a poorly structured N-terminal tail followed by a three-helix bundle, which is surprisingly similar to domains of the {sigma}{sup 70}-type proteins. Residues of the RpoN box, which have previously been shown to be critical for DNA binding, form the second helix of an unpredicted helix-turn-helix motif. This structure's homology with other DNA binding proteins, combined with previous biochemical data, suggest how the C-terminal domain of {sigma}{sup 54} binds to DNA

Entropy drives selective fluorine recognition in the fluoroacetyl–CoA thioesterase from Streptomyces cattleya

Fluorinated small molecules play an important role in the design of bioactive compounds for a broad range of applications. As such, there is strong interest in developing a deeper understanding of how fluorine affects the interaction of these ligands with their targets. Given the small number of fluorinated metabolites identified to date, insights into fluorine recognition have been provided almost entirely by synthetic systems. The fluoroacetyl-CoA thioesterase (FlK) from Streptomyces cattleya thus provides a unique opportunity to study an enzyme-ligand pair that has been evolutionarily optimized for a surprisingly high 106 selectivity for a single fluorine substituent. In these studies, we synthesize a series of analogs of fluoroacetyl-CoA and acetyl-CoA to generate nonhydrolyzable ester, amide, and ketone congeners of the thioester substrate to isolate the role of fluorine molecular recognition in FlK selectivity. Using a combination of thermodynamic, kinetic, and protein NMR experiments, we show that fluorine recognition is entropically driven by the interaction of the fluorine substituent with a key residue, Phe-36, on the lid structure that covers the active site, resulting in an ∼5- to 20-fold difference in binding (KD). Although the magnitude of discrimination is similar to that found in designed synthetic ligand-protein complexes where dipolar interactions control fluorine recognition, these studies show that hydrophobic and solvation effects serve as the major determinant of naturally evolved fluorine selectivity

Selective Chromium(VI) Ligands Identified Using Combinatorial Peptoid Libraries

Hexavalent chromium [Cr(VI)] is a worldwide water contaminant that is currently without cost-effective and efficient remediation strategies. This is in part due to a lack of ligands that can bind it amid an excess of innocuous ions in aqueous solution. We present herein the design and application of a peptoid-based library of ligand candidates for toxic metal ions. A selective screening process was used to identify members of the library that can bind to Cr(VI) species at neutral pH and in the presence of a large excess of spectator ions. There were 11 sequences identified, and their affinities were compared using titrations monitored with UV-vis spectroscopy. To identify the interactions involved in coordination and specificity, we evaluated the effects of sequence substitutions and backbone variation in the highest affinity structure. Additional characterization of the complex formed between this sequence and Cr(VI) was performed using NMR spectroscopy. To evaluate the ability of the developed sequences to remediate contaminated solutions, the structures were synthesized on a solid-phase resin and incubated with environmental water samples that contained simulated levels of chromium contamination. The synthetic structures demonstrated the ability to reduce the amount of toxic chromium to levels within the range of the EPA contamination guidelines. In addition to providing some of the first selective ligands for Cr(VI), these studies highlight the promise of peptoid sequences as easily prepared components of environmental remediation materials