PubMed Health⌕ Search

Biomedical subjects

Isabel Riba-Garcia

Publications and source records attributed to Isabel Riba-Garcia.

6 recordsLinked to original sources
PubMed2005

Development of a technique to determine bicyclomycin-rho binding and stoichiometry by isothermal titration calorimetry and mass spectrometry.

Bicyclomycin (1) is the only natural product inhibitor of the transcription termination factor rho. Rho is a hexameric helicase that terminates nascent RNA transcripts utilizing ATP hydrolysis and is an essential protein for many bacteria. The paucity of information concerning the 1-rho interaction stems from the weak binding affinity of 1. We report a novel technique using imine formation with rho to enhance the affinity of a bicyclomycin analogue and determine the binding stoichiometry by isothermal titration calorimetry (ITC) and mass spectrometry (MS). Our designed bicyclomycin ligand, 5a-(3-formyl-phenylsulfanyl)-dihydrobicyclomycin (2) (apparent I(50) = 4 muM), inhibits rho an order of magnitude more efficiently than 1 (I(50) = 60 muM). MS shows that 2 selectively forms an imine with K181 in rho. We found that despite the heterogeneity of ATP binding (three tight and three weak) imposed on the rho hexamer, the nearby bicyclomycin binding pocket is not affected, and both 1 and 2 bind with equal affinity to all six subunits.

Adenosine Triphosphatases↗
PubMed2004

Proteomic response to amino acid starvation in Candida albicans and Saccharomyces cerevisiae.

Saccharomyces cerevisiae activates general amino acid control (GCN) in response to amino acid starvation. Some aspects of this response are known to be conserved in other fungi including Candida albicans, the major systemic fungal pathogen of humans. Here, we describe a proteomic comparison of the GCN responses in S. cerevisiae and C. albicans. We have used high-resolution two-dimensional (2-D) gel electrophoresis and peptide mass fingerprinting to develop a 2-D protein map of C. albicans. A total of 391 protein spots, representing 316 open reading frames, were identified. Fifty-five C. albicans and 65 S. cerevisiae proteins were identified that responded reproducibly to 3-aminotriazole (3AT) in a Gcn4p-dependent fashion. The changes in the S. cerevisiae proteome correlated with the response in the S. cerevisiae transcript profile to 3AT treatment (rank correlation coefficient = 0.59; Natarajan et al., Molec. Cell. Biol. 2001, 21, 4347-4368). Significant aspects of the GCN response were conserved in C. albicans and S. cerevisiae. In both fungi, amino acid biosynthetic enzymes on multiple metabolic pathways were induced by 3AT in a Gcn4p-dependent fashion. Carbon metabolism functions were also induced. However, subtle differences were observed between these fungi. For example, purine biosynthetic enzymes were induced in S. cerevisiae, but were not significantly induced in C. albicans. These differences presumably reflect the contrasting niches of these relatively benign and pathogenic yeasts, respectively.

Amino Acids↗
PubMed2003

A systematic approach to modeling, capturing, and disseminating proteomics experimental data.

Both the generation and the analysis of proteome data are becoming increasingly widespread, and the field of proteomics is moving incrementally toward high-throughput approaches. Techniques are also increasing in complexity as the relevant technologies evolve. A standard representation of both the methods used and the data generated in proteomics experiments, analogous to that of the MIAME (minimum information about a microarray experiment) guidelines for transcriptomics, and the associated MAGE (microarray gene expression) object model and XML (extensible markup language) implementation, has yet to emerge. This hinders the handling, exchange, and dissemination of proteomics data. Here, we present a UML (unified modeling language) approach to proteomics experimental data, describe XML and SQL (structured query language) implementations of that model, and discuss capture, storage, and dissemination strategies. These make explicit what data might be most usefully captured about proteomics experiments and provide complementary routes toward the implementation of a proteome repository.

Database Management Systems↗
PubMed2002

Stable isotope labelling in vivo as an aid to protein identification in peptide mass fingerprinting.

Peptide mass fingerprinting (PMF) is a powerful technique for identification of proteins derived from in-gel digests by virtue of their matrix-assisted laser desorption/ionization-time of flight mass spectra. However, there are circumstances where the under-representation of peptides in the mass spectrum and the complexity of the source proteome mean that PMF is inadequate as an identification tool. In this paper, we show that identification is substantially enhanced by inclusion of composition data for a single amino acid. Labelling in vivo with a stable isotope labelled amino acid (in this paper, decadeuterated leucine) identifies the number of such amino acids in each digest fragment, and show a considerable gain in the ability of PMF to identify the parent protein. The method is tolerant to the extent of labelling, and as such, may be applicable to a range of single cell systems.

Deuterium↗
PubMed2002

Dynamics of protein turnover, a missing dimension in proteomics.

Functional genomic experiments frequently involve a comparison of the levels of gene expression between two or more genetic, developmental, or physiological states. Such comparisons can be carried out at either the RNA (transcriptome) or protein (proteome) level, but there is often a lack of congruence between parallel analyses using these two approaches. To fully interpret protein abundance data from proteomic experiments, it is necessary to understand the contributions made by the opposing processes of synthesis and degradation to the transition between the states compared. Thus, there is a need for reliable methods to determine the rates of turnover of individual proteins at amounts comparable to those obtained in proteomic experiments. Here, we show that stable isotope-labeled amino acids can be used to define the rate of breakdown of individual proteins by inspection of mass shifts in tryptic fragments. The approach has been applied to an analysis of abundant proteins in glucose-limited yeast cells grown in aerobic chemostat culture at steady state. The average rate of degradation of 50 proteins was 2.2%/h, although some proteins were turned over at imperceptible rates, and others had degradation rates of almost 10%/h. This range of values suggests that protein turnover is a significant missing dimension in proteomic experiments and needs to be considered when assessing protein abundance data and comparing it to the relative abundance of cognate mRNA species.

Amino Acids↗