Nguyen Huu Xuong

Computer analysis of two-dimensional gels

Abstract New methods and computer programs are described which enable one to analyze autoradiograms produced by two-dimensional gel electrophoresis. These programs are completely automatic with respect to finding spots resolved by such gels and quantitating the radioactivity in them. Semiautomatic programs have also been developed to match the spot patterns of different autoradiograms, and to follow the synthesis of any individual polypeptide through a series of gels.

Crystal structure of a phosphatase-resistant mutant of sporulation response regulator Spo0F from Bacillus subtilis.

BACKGROUND Spo0F, a phosphotransferase containing an aspartyl pocket, is involved in the signaling pathway (phosphorelay) controlling sporulation in Bacillus subtilis. It belongs to the superfamily of bacterial response regulatory proteins, which are activated upon phosphorylation of an invariant aspartate residue. This phosphorylation is carried out in a divalent cation dependent reaction catalyzed by cognate histidine kinases. Knowledge of the Spo0F structure would provide valuable information that would enable the elucidation of its function as a secondary messenger in a system in which a phosphate is donated from Spo0F to Spo0B, the third of four main proteins that constitute the phosphorelay. RESULTS We have determined the crystal structure of a Rap phosphatase resistant mutant, Spo0F Tyr13-->Ser, at 1.9 A resolution. The structure was solved by single isomorphous replacement and anomalous scattering techniques. The overall structural fold is (beta/alpha)5 and contains a central beta sheet. The active site of the molecule is formed by three aspartate residues and a lysine residue which come together at the C terminus of the beta sheet. The active site accommodates a calcium ion. CONCLUSIONS The structural analysis reveals that the overall topology and metal-binding coordination at the active site are similar to those of the bacterial chemotaxis response regulator CheY. Structural differences between Spo0F and CheY in the vicinity of the active site provide an insight into how similar molecular scaffolds can be adapted to perform different biological roles by the alteration of only a few amino acid residues. These differences may contribute to the observed stability of the phosphorylated species of Spo0F, a feature demanded by its role as a secondary messenger within the phosphorelay system which controls sporulation.

Crystallographic structure of Rhodospirillum molischianum ferricytochrome c′ at 2.5 Å resolution

Abstract This paper describes the 2.5 A crystallographic structure determination of ferricytochrome c ′ from the photosynthetic bacterium Rhodospirillum molischianum . The molecule is a symmetric dimer, with each 128-residue polypeptide chain incorporating a covalently bound protoheme IX prosthetic group. The monomer is structurally organized as an array of four nearly parallel α-helices, which pack most closely at one end and thereafter spatially diverge to accommodate the heme prosthetic group. Although local features of the heme attachment pattern resemble those seen in cytochrome c , the heme iron in cytochrome c ′ is pentaco-ordinate with a solvent-exposed histidine residue furnishing the single axial ligand to the heme iron. Subunit association in the dimeric molecule is principally stabilized by helix interactions, which are qualitatively similar to those occurring within each monomer. These interactions result in a dimer geometry that situates the exposed regions of both hemes on the same molecular surface. The structural basis for some of the physiochemical properties cytochrome c ′ are examined and compared to those of other heme proteins of known structure.

Crystallization studies of cAMP-dependent protein kinase. Crystals of catalytic subunit diffract to 3.5 A resolution.

The catalytic subunit of cAMP-dependent protein kinase from porcine heart has been crystallized in several different crystal forms. One of these forms diffracts to 3.5 A resolution. It is in monoclinic space group P2(1) with a = 64.24 A, b = 143.58 A, c = 48.40 A, alpha = gamma = 90 degrees and beta = 106.9 degrees.

Crystallization studies of cAMP-dependent protein kinase: Cocrystals of the catalytic subunit with a 20 amino acid residue peptide inhibitor and MgATP diffract to 3.0 Å resolution

Crystallographic studies of the catalytic subunit of cAMP-dependent protein kinase demonstrate that the presence of a 20 amino acid residue peptide inhibitor and MgATP during crystallization yields crystals with a different space group and, more significantly, makes an important difference in the quality of the resulting crystals. Under identical experimental conditions, the kinase crystallizes in a cubic space group P4(1)32 (a = b = c = 169.24 A), when no substrates or inhibitors are present, and in the hexagonal space group P6(1)22 (or P6(5)22) (a = b = 80.16 A, c = 288.07 A, alpha = beta = 90 degrees, gamma = 120 degrees) when a 20-amino acid residue peptide inhibitor and MgATP are present. Moreover, the hexagonal crystal diffracts to a resolution of 3.0 A, while the cubic crystals diffract to a resolution of 4.0 A.

BEAM-ish: a graphical user interface for the physical characterization of macromolecular crystals

Crystal mosaicity is determined from the measurement of the reflection angular width and can be used as an indicator of crystal perfection. A new method has been developed that combines the use of unfocused synchrotron radiation, super-fine phi slicing and a CCD area detector to simultaneously measure the mosaicity of hundreds of reflections . The X-ray beam characteristics and Lorentz correction are deconvoluted from the resulting reflection widths to calculate the true crystal mosaicity.

A new direct detection camera system for electron microscopy

High resolution electron imaging is very important in nanotechnology and biotechnology fields. For example, Cryogenic Electron-Microscopy is a promising method to obtain 3-D structures of large protein complexes and viruses. We report on the design and measurements of a new CMOS direct-detection camera system for electron imaging. The active pixel sensor array that we report on includes 512 by 550 pixels, each 5 by 5 μm in size, with an ~8 μm epitaxial layer to achieve an effective fill factor of 100%. Spatial resolution of 2.3 μm for a single incident e- has been measured. Electron microscope tests have been performed with 200 and 300 keV beams, and the first recorded Electron Microscope image is presented.

Is Dihydropteridine Reductase an Anomalous Dihydrofolate Reductase, a Flavin-Like Enzyme, or a Short-Chain Dehydrogenase?

Dihydropteridine reductase (DHPR, EC 1.6.99.10) and dihydrofolate reductase (DHFR, EC 1.5.1.3) both employ a reduced dinucleotide cofactor to convert a dihydro pteridine substrate to a tetrahydropteridine product. In the former case the substrate has a quinonoid dihydro structure whereas in the latter the 7,8-dihydro form is the substrate. The quinonoid form resembles a flavin molecule and the enzymatic mechanism of reduction has common features to this latter class of compound. Additionally, DHPR contains a specific Tyr XXX Lys motif in its sequence that allows comparison with a class of short chain dehydrogenases.1 The relationship of DHPR to these differing enzymatic types is illustrated briefly in the following discussion.

A CMOS Sensor for Nano-Imaging

We report on the design and measurements of a new direct electron imaging sensor. The active pixel sensor array consists of 512 by 550 pixels, each 5 by 5 μm in size, with a ∼8 μm epitaxial layer, to achieve an effective fill factor of 100%. Spatial resolution of 2.3 μm for a single incident e-has been measured. Tests have been performed with 200 and 300 keV beams, and an image of a catalase crystal, taken with the sensor, is presented. This novel sensor technology opens the door for studies of nanostructures and biological samples at higher resolution in both space and time.