Biomedical subjects

Xinghua Shi

Publications and source records attributed to Xinghua Shi.

4 recordsLinked to original sources

PubMed2006

The National Microbial Pathogen Database Resource (NMPDR): a genomics platform based on subsystem annotation.

The National Microbial Pathogen Data Resource (NMPDR) (http://www.nmpdr.org) is a National Institute of Allergy and Infections Disease (NIAID)-funded Bioinformatics Resource Center that supports research in selected Category B pathogens. NMPDR contains the complete genomes of approximately 50 strains of pathogenic bacteria that are the focus of our curators, as well as >400 other genomes that provide a broad context for comparative analysis across the three phylogenetic Domains. NMPDR integrates complete, public genomes with expertly curated biological subsystems to provide the most consistent genome annotations. Subsystems are sets of functional roles related by a biologically meaningful organizing principle, which are built over large collections of genomes; they provide researchers with consistent functional assignments in a biologically structured context. Investigators can browse subsystems and reactions to develop accurate reconstructions of the metabolic networks of any sequenced organism. NMPDR provides a comprehensive bioinformatics platform, with tools and viewers for genome analysis. Results of precomputed gene clustering analyses can be retrieved in tabular or graphic format with one-click tools. NMPDR tools include Signature Genes, which finds the set of genes in common or that differentiates two groups of organisms. Essentiality data collated from genome-wide studies have been curated. Drug target identification and high-throughput, in silico, compound screening are in development.

Bacteria↗

PubMed2006

Antibody evolution constrains conformational heterogeneity by tailoring protein dynamics.

The evolution of proteins with novel function is thought to start from precursor proteins that are conformationally heterogeneous. The corresponding genes may be duplicated and then mutated to select and optimize a specific conformation. However, testing this idea has been difficult because of the challenge of quantifying protein flexibility and conformational heterogeneity as a function of evolution. Here, we report the characterization of protein heterogeneity and dynamics as a function of evolution for the antifluorescein antibody 4-4-20. Using nonlinear laser spectroscopy, surface plasmon resonance, and molecular dynamics simulations, we demonstrate that evolution localized the Ab-combining site from a heterogeneous ensemble of conformations to a single conformation by introducing mutations that act cooperatively and over significant distances to rigidify the protein. This study demonstrates how protein dynamics may be tailored by evolution and has important implications for our understanding of how novel protein functions are evolved.

Animals↗

PubMed2005

Green fluorescent protein variants as ratiometric dual emission pH sensors. 3. Temperature dependence of proton transfer.

In parts 1 and 2 of this series [Hanson, G. T., McAnaney, T. B., Park, E. S., Rendell, M. E. P., Yarbrough, D. K., Chu, S. Y., Xi, L. X., Boxer, S. G., Montrose, M. H., and Remington, S. J. (2002) Biochemistry 41, 15477-15488; McAnaney, T. B., Park, E. S., Hanson, G. T., Remington, S. J., and Boxer, S. G. (2002) Biochemistry 41, 15489-15494], we described the structure, excited-state dynamics, and applications of pH-sensitive, ratiometric dual emission green fluorescent protein (deGFP) variants with fluorescence emission that is modulated between blue (lambda(max) approximately equal 465 nm) and green (lambda(max) approximately equal 515 nm) depending on the pH of the bulk solvent. In this paper, we consider the energetic origin of the dual emission properties of these GFP variants by examining the temperature dependence of the steady-state absorption and fluorescence emission. In most cases, the quantum yield of the green emission decreased as the temperature was lowered, indicating that the excited-state proton transfer (ESPT) which produces the green emitting form is an activated process. The activation energies of ESPT, determined by modeling the quantum yields of both blue and green emissions between 260 and 298 K in the context of a simple photocycle, were found to be larger at low pH than at high pH. These results indicate that the ratiometric dual emission properties of deGFP mutants are due to this pH-sensitive ESPT rate, combined with a modulation of the ground-state neutral and anionic chromophore populations with pH. The time-resolved fluorescence of one of the deGFP mutants was studied in detail. The time-resolved emission spectra of this mutant are the first ultrafast spectra obtained for a GFP. These spectra demonstrate that the rising kinetics for green emission, considered a hallmark of ESPT, is the sum of the contribution from both the neutral and intermediate anionic forms of the chromophore at the probe wavelength and may not be observed in all mutants that undergo ESPT, depending on the relative contributions of the two forms.

Amino Acid Substitution↗

PubMed2005

Protonation, photobleaching, and photoactivation of yellow fluorescent protein (YFP 10C): a unifying mechanism.

Yellow fluorescent protein (YFP 10C) is widely used as a probe in biology, but its complex photochemistry gives rise to unusual behavior that requires fuller definition. Here we characterize the kinetics of protonation and reversible bleaching over time scales of picoseconds to hours. Stopped-flow and pressure-jump techniques showed that protonation of the fluorescent YFP(-) anion state is two-step with a slow transition that accounts for blinking of 527 nm emission at the single molecule level on the seconds time scale. Femtosecond spectroscopy revealed that the protonated excited-state (YFPH*) decayed predominantly by a radiationless mechanism, but emission at 460 nm was detected within the first picosecond. Limited excited-state proton transfer leads to 527 nm emission characteristic of the YFP(-*) anion. Prolonged continuous wave illumination at the peak of YFP(-) absorbance (514 nm) yields, irreversibly, a weakly fluorescent product that absorbs at 390 nm. This "photobleaching" process also gives a different species (YFPHrb) that absorbs at 350/430 nm and spontaneously regenerates YFP(-) in the dark on the time scale of hours but can be photoactivated by UV light to regenerate YFP(-) within seconds, via a ground-state protonated intermediate. Using a pulsed laser for photobleaching resulted in decarboxylation of YFP as indicated by the mass spectrum. These observations are accounted for in a unifying kinetic scheme.

Amino Acid Sequence↗