A capillary-based microfluidic device enables primary high-throughput room-temperature crystallographic screening.

A novel capillary-based microfluidic strategy to accelerate the process of small-molecule-compound screening by room-temperature X-ray crystallography using protein crystals is reported. The ultra-thin microfluidic devices are composed of a UV-curable polymer, patterned by cleanroom photolithography, and have nine capillary channels per chip. The chip was designed for ease of sample manipulation, sample stability and minimal X-ray background.

Spectral X-Ray Diffraction using a 6 Megapixel Photon Counting Array Detector.

Pixel-array array detectors allow single-photon counting to be performed on a massively parallel scale, with several million counting circuits and detectors in the array. Because the number of photoelectrons produced at the detector surface depends on the photon energy, these detectors offer the possibility of spectral imaging. In this work, a statistical model of the instrument response is used to calibrate the detector on a per-pixel basis.

Linear fitting of multi-threshold counting data with a pixel-array detector for spectral X-ray imaging.

Experiments and modeling are described to perform spectral fitting of multi-threshold counting measurements on a pixel-array detector. An analytical model was developed for describing the probability density function of detected voltage in X-ray photon-counting arrays, utilizing fractional photon counting to account for edge/corner effects from voltage plumes that spread across multiple pixels. Each pixel was mathematically calibrated by fitting the detected voltage distributions to the model at both 13.

Towards protein-crystal centering using second-harmonic generation (SHG) microscopy.

The potential of second-harmonic generation (SHG) microscopy for automated crystal centering to guide synchrotron X-ray diffraction of protein crystals was explored. These studies included (i) comparison of microcrystal positions in cryoloops as determined by SHG imaging and by X-ray diffraction rastering and (ii) X-ray structure determinations of selected proteins to investigate the potential for laser-induced damage from SHG imaging. In studies using β2 adrenergic receptor membrane-protein crystals prepared in lipidic mesophase, the crystal locations identified by SHG images obtained in transmission mode were found to correlate well with the crystal locations identified by raster scanning using an X-ray minibeam.

Global radiation damage: temperature dependence, time dependence and how to outrun it.

A series of studies that provide a consistent and illuminating picture of global radiation damage to protein crystals, especially at temperatures above ∼200 K, are described. The radiation sensitivity shows a transition near 200 K, above which it appears to be limited by solvent-coupled diffusive processes. Consistent with this interpretation, a component of global damage proceeds on timescales of several minutes at 180 K, decreasing to seconds near room temperature.

Global radiation damage at 300 and 260 K with dose rates approaching 1 MGy s⁻¹.

Global radiation damage to 19 thaumatin crystals has been measured using dose rates from 3 to 680 kGy s⁻¹. At room temperature damage per unit dose appears to be roughly independent of dose rate, suggesting that the timescales for important damage processes are less than ∼1 s. However, at T = 260 K approximately half of the global damage manifested at dose rates of ∼10 kGy s⁻¹ can be outrun by collecting data at 680 kGy s⁻¹.

The Clp chaperones and proteases of the human malaria parasite Plasmodium falciparum.

The Clp chaperones and proteases play an important role in protein homeostasis in the cell. They are highly conserved across prokaryotes and found also in the mitochondria of eukaryotes and the chloroplasts of plants. They function mainly in the disaggregation, unfolding and degradation of native as well as misfolded proteins.

Focusing, collimation and flux throughput at the IMCA-CAT bending-magnet beamline at the Advanced Photon Source.

The IMCA-CAT bending-magnet beamline was upgraded with a collimating mirror in order to achieve the energy resolution required to conduct high-quality multi- and single-wavelength anomalous diffraction (MAD/SAD) experiments without sacrificing beamline flux throughput. Following the upgrade, the bending-magnet beamline achieves a flux of 8 x 10(11) photons s(-1) at 1 A wavelength, at a beamline aperture of 1.5 mrad (horizontal) x 86 microrad (vertical), with energy resolution (limited mostly by the intrinsic resolution of the monochromator optics) deltaE/E = 1.

Genome-scale protein expression and structural biology of Plasmodium falciparum and related Apicomplexan organisms.

Parasites from the protozoan phylum Apicomplexa are responsible for diseases, such as malaria, toxoplasmosis and cryptosporidiosis, all of which have significantly higher rates of mortality and morbidity in economically underdeveloped regions of the world. Advances in vaccine development and drug discovery are urgently needed to control these diseases and can be facilitated by production of purified recombinant proteins from Apicomplexan genomes and determination of their 3D structures. To date, both heterologous expression and crystallization of Apicomplexan proteins have seen only limited success.

Identification of conserved lipid/detergent-binding sites in a high-resolution structure of the membrane protein cytochrome c oxidase.

Well ordered reproducible crystals of cytochrome c oxidase (CcO) from Rhodobacter sphaeroides yield a previously unreported structure at 2.0 A resolution that contains the two catalytic subunits and a number of alkyl chains of lipids and detergents. Comparison with crystal structures of other bacterial and mammalian CcOs reveals that the positions occupied by native membrane lipids and detergent substitutes are highly conserved, along with amino acid residues in their vicinity, suggesting a more prevalent and specific role of lipid in membrane protein structure than often envisioned.

Crystal structure of vancosaminyltransferase GtfD from the vancomycin biosynthetic pathway: interactions with acceptor and nucleotide ligands.

The TDP-vancosaminyltransferase GtfD catalyzes the attachment of L-vancosamine to a monoglucosylated heptapeptide intermediate during the final stage of vancomycin biosynthesis. Glycosyltransferases from this and similar antibiotic pathways are potential tools for the design of new compounds that are effective against vancomycin resistant bacterial strains. We have determined the X-ray crystal structure of GtfD as a complex with TDP and the natural glycopeptide substrate at 2.

Crystal structure of a tetrameric GDP-D-mannose 4,6-dehydratase from a bacterial GDP-D-rhamnose biosynthetic pathway.

d-Rhamnose is a rare 6-deoxy monosaccharide primarily found in the lipopolysaccharide of pathogenic bacteria, where it is involved in host-bacterium interactions and the establishment of infection. The biosynthesis of d-rhamnose proceeds through the conversion of GDP-d-mannose by GDP-d-mannose 4,6-dehydratase (GMD) to GDP-4-keto-6-deoxymannose, which is subsequently reduced to GDP-d-rhamnose by a reductase. We have determined the crystal structure of GMD from Pseudomonas aeruginosa in complex with NADPH and GDP.

Structure of the TDP-epi-vancosaminyltransferase GtfA from the chloroeremomycin biosynthetic pathway.

During the biosynthesis of the vancomycin-class antibiotic chloroeremomycin, TDP-epi-vancosaminyltransferase GtfA catalyzes the attachment of 4-epi-vancosamine from a TDP donor to the beta-OHTyr-6 of the aglycone cosubstrate. Glycosyltransferases from this pathway are potential tools for the combinatorial design of new antibiotics that are effective against vancomycin-resistant bacterial strains. These enzymes are members of the GT-B glycosyltransferase superfamily, which share a homologous bidomain topology.

The structure of mammalian cyclooxygenases.

Cyclooxygenases-1 and -2 (COX-1 and COX-2, also known as prostaglandin H2 synthases-1 and -2) catalyze the committed step in prostaglandin synthesis. COX-1 and -2 are of particular interest because they are the major targets of nonsteroidal antiinflammatory drugs (NSAIDs) including aspirin, ibuprofen, and the new COX-2-selective inhibitors. Inhibition of the COXs with NSAIDs acutely reduces inflammation, pain, and fever, and long-term use of these drugs reduces the incidence of fatal thrombotic events, as well as the development of colon cancer and Alzheimer's disease.

Structure of the MUR1 GDP-mannose 4,6-dehydratase from Arabidopsis thaliana: implications for ligand binding and specificity.

GDP-D-mannose 4,6-dehydratase catalyzes the first step in the de novo synthesis of GDP-L-fucose, the activated form of L-fucose, which is a component of glycoconjugates in plants known to be important to the development and strength of stem tissues. We have determined the three-dimensional structure of the MUR1 dehydratase isoform from Arabidopsis thaliana complexed with its NADPH cofactor as well as with the ligands GDP and GDP-D-rhamnose. MUR1 is a member of the nucleoside-diphosphosugar modifying subclass of the short-chain dehydrogenase/reductase enzyme family, having homologous structures and a conserved catalytic triad of Lys, Tyr, and Ser/Thr residues.

Structure of the UDP-glucosyltransferase GtfB that modifies the heptapeptide aglycone in the biosynthesis of vancomycin group antibiotics.

Background: Members of the vancomycin group of glycopeptide antibiotics have an oxidatively crosslinked heptapeptide scaffold decorated at the hydroxyl groups of 4-OH-Phegly4 or beta-OH-Tyr6 with mono- (residue 6) or disaccharides (residue 4). The disaccharide in vancomycin itself is L-vancosamine-1,2-glucose, and in chloroeremomycin it is L-4-epi-vancosamine-1,2-glucose. The sugars and their substituents play an important role in efficacy, particularly against vancomycin-resistant pathogenic enterococci.

Mutational and X-ray crystallographic analysis of the interaction of dihomo-gamma -linolenic acid with prostaglandin endoperoxide H synthases.

Prostaglandin endoperoxide H synthases-1 and -2 (PGHSs) catalyze the committed step in prostaglandin biosynthesis. Both isozymes can oxygenate a variety of related polyunsaturated fatty acids. We report here the x-ray crystal structure of dihomo-gamma-linolenic acid (DHLA) in the cyclooxygenase site of PGHS-1 and the effects of active site substitutions on the oxygenation of DHLA, and we compare these results to those obtained previously with arachidonic acid (AA).

Prostaglandin endoperoxide H synthase-1: the functions of cyclooxygenase active site residues in the binding, positioning, and oxygenation of arachidonic acid.

Prostaglandin endoperoxide H synthases (PGHSs) catalyze the committed step in the biosynthesis of prostaglandins and thromboxane, the conversion of arachidonic acid, two molecules of O(2), and two electrons to prostaglandin endoperoxide H(2) (PGH(2)). Formation of PGH(2) involves an initial oxygenation of arachidonate to yield PGG(2) catalyzed by the cyclooxygenase activity of the enzyme and then a reduction of the 15-hydroperoxyl group of PGG(2) to form PGH(2) catalyzed by the peroxidase activity. The cyclooxygenase active site is a hydrophobic channel that protrudes from the membrane binding domain into the core of the globular domain of PGHS.

Peroxidase activity in prostaglandin endoperoxide H synthase-1 occurs with a neutral histidine proximal heme ligand.

Prostaglandin endoperoxide H synthases-1 and -2 (PGHS-1 and -2) convert arachidonic acid to prostaglandin H(2) (PGH(2)), the committed step in prostaglandin and thromboxane formation. Interaction of peroxides with the heme sites in PGHSs generates a tyrosyl radical that catalyzes subsequent cyclooxygenase chemistry. To study the peroxidase reaction of ovine oPGHS-1, we combined spectroscopic and directed mutagenesis data with X-ray crystallographic refinement of the heme site.

Crystal structure of SQD1, an enzyme involved in the biosynthesis of the plant sulfolipid headgroup donor UDP-sulfoquinovose.

The SQD1 enzyme is believed to be involved in the biosynthesis of the sulfoquinovosyl headgroup of plant sulfolipids, catalyzing the transfer of SO(3)(-) to UDP-glucose. We have determined the structure of the complex of SQD1 from Arabidopsis thaliana with NAD(+) and the putative substrate UDP-glucose at 1.6-A resolution.

The role of arginine 120 of human prostaglandin endoperoxide H synthase-2 in the interaction with fatty acid substrates and inhibitors.

Arg-120 is located near the mouth of the hydrophobic channel that forms the cyclooxygenase active site of prostaglandin endoperoxide H synthases (PGHSs)-1 and -2. Replacement of Arg-120 of ovine PGHS-1 with a glutamine increases the apparent Km of PGHS-1 for arachidonate by 1,000-fold (Bhattacharyya, D. K.

Allosteric regulation of type I hexokinase: A site-directed mutational study indicating location of the functional glucose 6-phosphate binding site in the N-terminal half of the enzyme.

The Type I isozyme of mammalian hexokinase has evolved by a gene duplication-fusion mechanism, with resulting internal duplication of sequence and ligand binding sites. However, 1:1 binding stoichiometry indicates that only one of these is available for binding the product inhibitor, Glc-6-P; the location of that site, in the N- or C-terminal half, remains under debate. Recent structural studies (Aleshin et al.

Identification and SAR for a selective, nonpeptidyl thrombin inhibitor.

A novel, nonpeptidyl thrombin inhibitor, L-636,619 (1), was identified via topological similarity searching over the Merck Corporate Sample Database. X-ray crystallographic studies determined the geometry for ligand binding to the enzyme. Chemical modification of the P1 and P3 segments of the ligand resulted in enhanced potency and improvement in the chemical stability of the lead.

The structure of mammalian hexokinase-1.

We have determined the structures of the glucose-6-phosphate (G6P)-inhibitable 100,000 Mr Type I hexokinase from rat and the G6P-sensitive 50,000 Mr hexokinase from Schistosoma mansoni at a resolution of 2.8 and 2.6 A respectively.