RESUMO
Ligands interacting with Mycobacterium tuberculosis recombinant proteins were identified through use of the ability of Cibacron Blue F3GA dye to interact with nucleoside/nucleotide binding proteins, and the effects of these ligands on crystallization were examined. Co-crystallization with ligands enhanced crystallization and enabled X-ray diffraction data to be collected to a resolution of atleast 2.7 Å for 5 of 10 proteins tested. Additionally, clues about individual proteins' functions were obtained from their interactions with each of a panel of ligands.
Assuntos
Cromatografia de Afinidade/métodos , Cristalografia por Raios X/métodos , Nucleotídeos/química , Triazinas/química , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Eletroforese em Gel de Poliacrilamida , Escherichia coli/química , Escherichia coli/genética , Ligantes , Mycobacterium tuberculosis/química , Mycobacterium tuberculosis/genética , NADP Trans-Hidrogenases/química , NADP Trans-Hidrogenases/genética , Nucleotídeos/genética , Mapeamento de Interação de Proteínas/métodos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
We show that Cibacron Blue F3GA dye resin chromatography can be used to identify ligands that specifically interact with proteins from Mycobacterium tuberculosis, and that the identification of these ligands can facilitate structure determination by enhancing the quality of crystals. Four native Mtb proteins of the aldehyde dehydrogenase (ALDH) family were previously shown to be specifically eluted from a Cibacron Blue F3GA dye resin with nucleosides. In this study we characterized the nucleoside-binding specificity of one of these ALDH isozymes (recombinant Mtb Rv0223c) and compared these biochemical results with co-crystallization experiments with different Rv0223c-nucleoside pairings. We found that the strongly interacting ligands (NAD and NADH) aided formation of high-quality crystals, permitting solution of the first Mtb ALDH (Rv0223c) structure. Other nucleoside ligands (AMP, FAD, adenosine, GTP and NADP) exhibited weaker binding to Rv0223c, and produced co-crystals diffracting to lower resolution. Difference electron density maps based on crystals of Rv0223c with various nucleoside ligands show most share the binding site where the natural ligand NAD binds. From the high degree of similarity of sequence and structure compared to human mitochondrial ALDH-2 (BLAST Z-score = 53.5 and RMSD = 1.5 A), Rv0223c appears to belong to the ALDH-2 class. An altered oligomerization domain in the Rv0223c structure seems to keep this protein as monomer whereas native human ALDH-2 is a multimer.
Assuntos
Aldeído Desidrogenase/isolamento & purificação , Proteínas de Bactérias/isolamento & purificação , Proteínas Mitocondriais/isolamento & purificação , Mycobacterium tuberculosis/enzimologia , Nucleosídeos/química , Sefarose/análogos & derivados , Aldeído Desidrogenase/biossíntese , Aldeído Desidrogenase/genética , Aldeído-Desidrogenase Mitocondrial , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Humanos , Isoenzimas/biossíntese , Isoenzimas/genética , Isoenzimas/isolamento & purificação , Proteínas Mitocondriais/biossíntese , Proteínas Mitocondriais/genética , Nucleosídeos/metabolismo , Ligação Proteica , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Sefarose/químicaRESUMO
Sucrose is the cornerstone of higher plant metabolism. Produced by photosynthesis, sucrose is the main substrate for respiration and biosynthesis. The emerging idea is that sucrose may act as regulator of its own metabolism, characterized in particular by a permanent process of degradation and formation. This sucrose turnover may control several important physiological functions. Of particular concern is an energy dependent cycle involving the hexokinase. This report presents an experimental approach to define quantitatively physiological states of suspension-cultured plant cells wih reference to their sucrose content and respiration rate. Sucrose depletion of normal cells incubated in a medium devoid of sugar is measured in vivo using 13C and respiration is simultaneously recorded. Results obtained with sucrose-storing cells and Arabidopsis thaliana show that respiration rate is closely linked to the available sucrose. Sucrose-depleted cells offer a stable model to study the bioenergetics of the process.
Assuntos
Acer/metabolismo , Arabidopsis/metabolismo , Respiração Celular/fisiologia , Sacarose/metabolismo , Acer/citologia , Arabidopsis/citologia , Células Cultivadas , Modelos Biológicos , Ressonância Magnética Nuclear BiomolecularRESUMO
Proteomic methods are required for the study of many different aspects of plant function. Important issues in proteomics include the molecular complexity of proteins, given that there are hundreds of thousands of chemically and physically distinct proteins in plants, and the context of protein functions with respect to both genomes and the environment. Available genomic and gene sequences greatly simplify the identification of proteins using improved techniques of mass spectrometry. This improved capability has led to much discussion on proteomes, and some experimentation using proteomic methodologies aimed at modest numbers of proteins. The scale of proteomics is open, for the number of proteins and genes considered at any one time is as dependent on the nature of the scientific question posed as on technical resources and capabilities. We know just enough about plant proteomes to imagine the breathtaking scope of our ignorance. There are tremendous opportunities for new molecular biologists to define the nature of the protein machines that transduce genetic and environmental information, and transform simple energy and matter, to give plants.