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Biomedical subjects

T J Boggon

Publications and source records attributed to T J Boggon.

12 recordsLinked to original sources
PubMed2001

G-protein signaling through tubby proteins.

Dysfunction of the tubby protein results in maturity-onset obesity in mice. Tubby has been implicated as a transcription regulator, but details of the molecular mechanism underlying its function remain unclear. Here we show that tubby functions in signal transduction from heterotrimeric GTP-binding protein (G protein)-coupled receptors. Tubby localizes to the plasma membrane by binding phosphatidylinositol 4,5-bisphosphate through its carboxyl terminal "tubby domain." X-ray crystallography reveals the atomic-level basis of this interaction and implicates tubby domains as phosphorylated-phosphatidyl- inositol binding factors. Receptor-mediated activation of G protein alphaq (Galphaq) releases tubby from the plasma membrane through the action of phospholipase C-beta, triggering translocation of tubby to the cell nucleus. The localization of tubby-like protein 3 (TULP3) is similarly regulated. These data suggest that tubby proteins function as membrane-bound transcription regulators that translocate to the nucleus in response to phosphoinositide hydrolysis, providing a direct link between G-protein signaling and the regulation of gene expression.

Active Transport, Cell Nucleus↗
PubMed2000

Synchrotron X-ray reciprocal-space mapping, topography and diffraction resolution studies of macromolecular crystal quality.

A comprehensive study of microgravity and ground-grown chicken egg-white lysozyme crystals is presented using synchrotron X-ray reciprocal-space mapping, topography techniques and diffraction resolution. Microgravity crystals displayed reduced intrinsic mosaicities on average, but no differences in terms of strain over their ground-grown counterparts. Topographic analysis revealed that in the microgravity case the majority of the crystal was contributing to the peak of the reflection at the appropriate Bragg angle. In the ground-control case only a small volume of the crystal contributed to the intensity at the diffraction peak. The techniques prove to be highly complementary, with the reciprocal-space mapping providing a quantitative measure of the crystal mosaicity and strain (or variation in lattice spacing) and the topography providing a qualitative overall assessment of the crystal in terms of its X-ray diffraction properties. Structural data collection was also carried out at the synchrotron.

Crystallography, X-Ray↗
PubMed1999

Implication of tubby proteins as transcription factors by structure-based functional analysis.

Tubby-like proteins (TULPs) are found in a broad range of multicellular organisms. In mammals, genetic mutation of tubby or other TULPs can result in one or more of three disease phenotypes: obesity (from which the name "tubby" is derived), retinal degeneration, and hearing loss. These disease phenotypes indicate a vital role for tubby proteins; however, no biochemical function has yet been ascribed to any member of this protein family. A structure-directed approach was employed to investigate the biological function of these proteins. The crystal structure of the core domain from mouse tubby was determined at a resolution of 1.9 angstroms. From primarily structural clues, experiments were devised, the results of which suggest that TULPs are a unique family of bipartite transcription factors.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Purification, crystallization and initial X-ray analysis of the C1 subunit of the astaxanthin protein, V600, of the chondrophore Velella velella.

The subunit C1 of the carotenoid-binding protein, V600, of the chondrophore Velella velella has been purified and crystallized. The crystals, which were grown by the vapour-diffusion method from ammonium sulfate as the major precipitant, diffract beyond 3 A and show little radiation damage over long periods (greater than 100 h) on a Cu Kalpha rotating-anode X-ray source. The space group of the crystals is P212121 with cell dimensions a = 42.0, b = 80.9, c = 110. 6 A.

Animals↗
PubMed1999

Bound-solvent structures for microgravity-, ground control-, gel- and microbatch-grown hen egg-white lysozyme crystals at 1.8 A resolution.

A number of methods can be used to improve the stability of the protein crystal-growth environment, including growth in microgravity without an air-liquid phase boundary, growth in gels and growth under oil ('microbatch'). In this study, X-ray data has been collected from and structures refined for crystals of hen egg-white lysozyme (HEWL) grown using four different methods, liquid-liquid dialysis on Earth and in microgravity using the European Space Agency's (ESA) Advanced Protein Crystallization Facility (APCF) on board the NASA Space Shuttle Life and Microgravity Spacelab (LMS) mission (STS-78), crystallization in agarose gel using a tube liquid-gel diffusion method and crystallization in microbatch under oil. A comparison of the overall quality of the X-ray data, the protein structures and especially the bound-water structures has been carried out at 1.8 A. The lysozyme protein structures corresponding to these four different crystallization methods remain similar. A small improvement in the bound-solvent structure is seen in lysozyme crystals grown in microgravity by liquid-liquid dialysis, which has a more stable fluid physics state in microgravity, and is consistent with a better formed protein crystal in microgravity.

Amino Acid Sequence↗
PubMed1997

CCD video observation of microgravity crystallization of lysozyme and correlation with accelerometer data.

Lysozyme has been crystallized using the ESA Advanced Protein Crystallization Facility onboard the NASA Space Shuttle Orbiter during the IML-2 mission. CCD video monitoring was used to follow the crystallization process and evaluate the growth rate. During the mission some tetragonal crystals were observed moving over distances of up to 200 micrometers. This was correlated with microgravity disturbances caused by firings of vernier jets on the Orbiter. Growth-rate measurement of a stationary crystal (which had nucleated on the growth reactor wall) showed spurts and lulls correlated with an onboard activity: astronaut exercise. The stepped growth rates may be responsible for the residual mosaic block structure seen in crystal mosaicity and topography measurements.

Acceleration↗
PubMed1997

Partial improvement of crystal quality for microgravity-grown apocrustacyanin C1.

The protein apocrustacyanin C(1) has been crystallized by vapour diffusion in both microgravity (the NASA space shuttle USML-2 mission) and on the ground. Rocking width measurements were made on the crystals at the ESRF Swiss-Norwegian beamline using a high-resolution psi-circle diffractometer from the University of Karlsruhe. Crystal perfection was then evaluated, from comparison of the reflection rocking curves from a total of five crystals (three grown in microgravity and two earth controls), and by plotting mosaicity versus reflection signal/noise. Comparison was then made with previous measurements of almost 'perfect' lysozyme crystals grown aboard IML-2 and Spacehab-I and reported by Snell et al. [Snell, Weisgerber, Helliwell, Weckert, Hölzer & Schroer (1995). Acta Cryst. D51, 1099-1102]. Overall, the best diffraction-quality apocrustacyanin C(1) crystal was microgravity grown, but one earth-grown crystal was as good as one of the other microgravity-grown crystals. The remaining two crystals (one from microgravity and one from earth) were poorer than the other three and of fairly equal quality. Crystal movement during growth in microgravity, resulting from the use of vapour-diffusion geometry, may be the cause of not realising the 'theoretical' limit of perfect protein crystal quality.

Journal Article↗
PubMed1997

Partial improvement of crystal quality for microgravity-grown apocrustacyanin C1.

The protein apocrustacyanin C1 has been crystallized by vapour diffusion in both microgravity (the NASA space shuttle USML-2 mission) and on the ground. Rocking width measurements were made on the crystals at the ESRF Swiss-Norwegian beamline using a high-resolution psi-circle diffractometer from the University of Karlsruhe. Crystal perfection was then evaluated, from comparison of the reflection rocking curves from a total of five crystals (three grown in microgravity and two earth controls), and by plotting mosaicity versus reflection signal/noise. Comparison was then made with previous measurements of almost 'perfect' lysozyme crystals grown aboard IML-2 and Spacehab-1 and reported by Snell et al. [Snell, Weisgerber, Helliwell, Weckert, Holzer & Schroer (1995). Acta Cryst. D51, 1099-1102]. Overall, the best diffraction-quality apocrustacyanin C1 crystal was microgravity grown, but one earth-grown crystal was as good as one of the other microgravity-grown crystals. The remaining two crystals (one from microgravity and one from earth) were poorer than the other three and of fairly equal quality. Crystal movement during growth in microgravity, resulting from the use of vapour-diffusion geometry, may be the cause of not realising the 'theoretical' limit of perfect protein crystal quality.

Carrier Proteins↗
PubMed1996

Lysozyme crystal growth kinetics monitored using a Mach-Zehnder interferometer.

A Mach-Zehnder interferometer has been developed for the monitoring of the kinetics of the diffusion process in protein crystal growth. This device can be used in conjunction with the ESA Advanced Protein Crystallization Facility (APCF), which allows experiments under microgravity conditions (e.g. on board the NASA Space Shuttle). Experimental trials on the ground have been carried out with the interferometer using the engineering model of the APCF and a protein dialysis reactor. Chicken egg-white lysozyme crystal growth, as a test, has thereby been monitored directly. The changes of concentration in the solution over time have been determined via the refractive index measurements made and subsequently correlated with visual monitoring of crystal growth in a repeat experiment.

Journal Article↗
PubMed1996

The crystal structure analysis of d(CGCGAASSCGCG)2, a synthetic DNA dodecamer duplex containing four 4'-thio-2'-deoxythymidine nucleotides.

The crystal structure refinement of the synthetic dodecamer d(CGCGAASSCGCG), where S = 4'-thio-2'-deoxythymidine, has converged at R=0.201 for 2605 reflections with F > 2sigma(F) in the resolution range 8.0-2.4 A for a model consisting of the dodecamer duplex and 66 water molecules. A comparison of its structure with that of the native dodecamer d(CGCGAATTCGCG) has revealed that the major differences between the two structures is a change in the conformation of the sugar-phosphate backbone in the regions at and adjacent to the positions of the modified nucleosides. Examination of the fine structural parameters for each of the structures reveals that the thiosugars adopt a C3'-exo conformation in d(CGCGAASSCGCG), rather than the approximate C1'-exo conformation found for the analogous sugars in the structure of d(CGCGAATTCGCG). The observed differences in structure between the two duplexes may help to explain the enhanced resistance to nuclease digestion of synthetic oligonucleotides containing 4'-thio-2'-deoxynucleotides.

Base Sequence↗