Biomedical subjects

Russell A Judge

Publications and source records attributed to Russell A Judge.

7 recordsLinked to original sources

PubMed2006

Kinase drug discovery by affinity selection/mass spectrometry (ASMS): application to DNA damage checkpoint kinase Chk1.

Kinase enzymes are involved in a vast array of biological processes associated with human disease; therefore, selective kinase inhibition by small molecules and therapeutic antibodies is an area of intense study. The authors show that drug candidates with immediate value for biological preclinical evaluation can be identified directly through ultra-efficient affinity screening of kinase enzymes and random compound mixtures. The screening process comprises sampling and trapping equilibrium binding between candidate ligands and protein in solution, followed by removal of unbound ligands via 3 rounds of ultrafiltration and direct identification of bound ligands by mass spectrometry. Evaluation of significant peaks is facilitated by automated integration and collation of the mass spectral data and import into custom software for analysis. One Chk1-selective ligand found by using this process is presented in detail. The compound is potent in both enzymatic and Chk1-dependent cellular assays, and specific contacts in the Chk1 active site are shown by X-ray crystallography.

Amino Acid Sequence↗

PubMed2006

Optimizing crystal volume for neutron diffraction: D-xylose isomerase.

Neutron diffraction is uniquely sensitive to hydrogen positions and protonation state. In that context structural information from neutron data is complementary to that provided through X-ray diffraction. However, there are practical obstacles to overcome in fully exploiting the potential of neutron diffraction, i.e. low flux and weak scattering. Several approaches are available to overcome these obstacles and we have investigated the simplest: increasing the diffracting volume of the crystals. Volume is a quantifiable metric that is well suited for experimental design and optimization techniques. By using response surface methods we have optimized the xylose isomerase crystal volume, enabling neutron diffraction while we determined the crystallization parameters with a minimum of experiments. Our results suggest a systematic means of enabling neutron diffraction studies for a larger number of samples that require information on hydrogen position and/or protonation state.

Aldose-Ketose Isomerases↗

PubMed2006

A quasi-Laue neutron crystallographic study of D-xylose isomerase.

The location of hydrogen atoms in enzyme structures can bring critical understanding of catalytic mechanism. However, whilst it is often difficult to determine the position of hydrogen atoms using X-ray crystallography even with subatomic (<1.0 A) resolution data available, neutron crystallography provides an experimental tool to directly localize hydrogen/deuterium atoms in biological macromolecules at resolution of 1.5-2.0 A. D-Xylose isomerase (D-xylose ketol-isomerase, EC 5.3.1.5) is a 43 kDa enzyme that catalyses the first reaction in the catabolism of D-xylose. Linearization and isomerization of D-xylose at the active site of D-xylose isomerase rely upon a complex hydrogen transfer. Neutron quasi-Laue data at 2.2 A resolution were collected at room temperature on a partially deuterated Streptomyces rubiginosus D-xylose isomerase crystal using the LADI instrument at ILL with the objective to provide insight into the enzymatic mechanism. The neutron structure shows unambiguously that residue His 53 is doubly protonated at the active site of the enzyme. This suggests that the reaction proceeds through an acid catalyzed opening of the sugar ring, which is in accord with the mechanism suggested by Fenn et al. (Biochemistry 43(21): 6464-6474, 2004). This is the first report of direct observation of double protonation of His 53 and the first validation of the ring opening mechanism at the active site of D-xylose isomerase.

Aldose-Ketose Isomerases↗

PubMed2005

Evaluation of a model for seeded isothermal batch protein crystallization.

Bulk protein crystallization, unlike small molecule crystallization, has found very limited use in biopharmaceutical manufacture. Most work in this area targets obtaining single large crystals for molecular structure determination by crystallography. Design and optimization of bulk crystallization for protein recovery and purification is much less common, and requires a mathematical model for analysis of laboratory data suitable for scale-up purposes. Traditionally, the crystal size distribution and method of moments is used to characterize the crystallization process. A simpler method is presented in this paper that utilizes the desupersaturation curve. The method uses an approach that does not require expensive instrumentation or characterization of the seed crystal size distribution. The method is extended to allow determination of both the mass deposition rate constant and the growth rate order from a single desuperaturation curve. Experimental data for the bulk crystallization of ovalbumin are used to validate the method. The rate constants and rate order obtained using the new method compare well with literature values. Scale-up is illustrated by prediction of the impact of changes in seed mass on protein crystallization. This new method offers a straightforward and low-cost alternative to traditional methods for the analysis and scale-up of protein crystallization data.

Computer Simulation↗

PubMed2005

Extracting trends from two decades of microgravity macromolecular crystallization history.

Since the 1980s hundreds of macromolecular crystal growth experiments have been performed in the reduced acceleration environment of an orbiting spacecraft. Significant enhancements in structural knowledge have resulted from X-ray diffraction of the crystals grown. Similarly, many samples have shown no improvement or degradation in comparison to those grown on the ground. A complex series of interrelated factors affect these experiments and by building a comprehensive archive of the results it was aimed to identify factors that result in success and those that result in failure. Specifically, it was found that dedicated microgravity missions increase the chance of success when compared with those where crystallization took place as a parasitic aspect of the mission. It was also found that the chance of success could not be predicted based on any discernible property of the macromolecule available to us.

Crystallography, X-Ray↗

PubMed2004

An ultraviolet fluorescence-based method for identifying and distinguishing protein crystals.

Intrinsic ultraviolet fluorescence has been investigated as a rapid non-invasive method for identifying and distinguishing protein crystals. An epi-fluorescence microscope, which provides for excitation and viewing of fluorescence from above the sample, and a straight-through geometry, which provides excitation from above and views fluorescence from underneath the sample, were tested with protein and non-protein crystal samples. In both systems the protein crystals were observed to fluoresce brightly, providing a high contrast against background solution fluorescence, thus enabling protein crystals to be identified and distinguished from non-protein crystals.

Aldose-Ketose Isomerases↗

PubMed2002

Seeing the heat -- preliminary studies of cryocrystallography using infrared imaging.

As preparation for an extensive study that aims to image the cryocooling process of macromolecular crystals, the ability to thermally image solid objects and liquids at temperatures far below 273 K is demonstrated. In the case of a large lysozyme crystal (1.0 x 0.7 x 0.2 mm), qualitative measurements show the cooling process to take about 0.6 s with the cooling taking place in a wave starting from the face of the crystal nearest to the origin of the cryostream and ending at the point furthest away from the origin. Annealing of this lysozyme crystal, cooled under good cryoprotectant conditions, shows that cold striations form perpendicular to the cooling stream. These striations become more pronounced after successive annealing. Cryocooling of a non-cryoprotected crystal of glucose isomerase displayed an 'S-shaped' cold front wave traveling across the sample. These preliminary results are qualitative but show the power of infrared imaging as a new tool for fundamental and practical cryocrystallography studies.

Cold Temperature↗