Progress 12/01/04 to 12/30/09
Outputs
OUTPUTS: Administrative Termination - PI is no longer here to submit reports. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Administrative Termination - PI is no longer here to submit reports.
Publications
- No publications reported this period
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: We have continued to use dye-ligand chromatography in laboratory experiments to identify specific protein-nucleoside ligand couples and understanding the basis for functional molecular interactions. A particular emphasis in 2008 was placed on a set of recombinant proteins from Mycobacterium tuberculosis that had been produced by Drs. Chang-Yub Kim and Thomas Terwilliger of Los Alamos National Laboratory in a structural genomics initiative. This set of proteins was of interest in the context of structural genomics because of unanswered questions about their ligand-based functions. We first screened dozens of recombinant proteins and found that approximately half bound tightly to Cibacron Blue F3GA, which is consistent with our experience with native protein mixtures. However, all but one of the proteins that did bind the dye-ligand was resistant to displacement by purine mononucleotides and nucleosides, and by pyridine dinucleotides - in contrast to our prior data from experiments with native protein mixtures. Further experiments were conducted on recombinant proteins targeted as a consequence of prior ligand-protein identifications made on native Mycobacterium tuberculosis lysates, with confirmation of pyridine dinucleotide binding. Comparative analysis of the amino acid sequences of proteins we identified in the aldehyde dehydrogenase family was made to determine the degree of sequence similarities within the different Mycobacterium tuberculosis members, versus the similarity of each tuberculosis protein to members found in Homo sapiens. The methodology and early results of this project were disseminated at a Beilstein Institut Symposium on Systems Chemistry in May, in an invited paper entitled "High-Throughput Analysis of Nucleoside- and Nucleotide-binding by Proteins", with further dissemination via the website http://www.beilstein-institut.de/index.phpid=201&L=3. PARTICIPANTS: Drs. Chang-Yub Kim and Thomas Terwilliger of Los Alamos National Laboratory collaborated in this project, as structural biologists interested in proteins for which we identified ligands, and as recombinant protein expression experts able to generate proteins suitable for ligand analysis. The collaboration provides opportunities for undergraduate and post-graduate students to participate in this project, including via programs offered by Los Alamos National Laboratory. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Our project has enhanced efforts to determine the structures of proteins in action, which provides key insights into the function of genes. This project also is valuable in development of drugs that target proteins. Protein ligands we identify are used by structural biologists to improve protein crystal production, which speeds up molecular structure determination of proteins involved in cell function and disease.
Publications
- No publications reported this period
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Progress 01/01/07 to 12/31/07
Outputs
The specificity of interactions between nucleotides and proteins is important in understanding the metabolism of xenobiotics. One long-established example is the critical role of aldehyde dehydrogenases in metabolism of natural and foreign toxic chemicals in humans. Our identification of an aldehyde dehydrogenase encoded by gene Rv0223c in the human pathogen Mycobacterium tuberculosis, reported last year, has been followed, first, by analysis of its structure in collaboration with Chang-Yub Kim and Thomas Terwilliger at Los Alamos National Laboratory - LANL, New Mexico. The Rv0223c gene product has close structural similarity to human ALDH2, which raises the possibility that this protein plays a role in pathogenicity and drug resistance, given the evidence in humans. Second, we have identified 3 more nucleotide-binding proteins with putative aldehyde dehydrogenase activity, and these have been selected for cloning and expression by our collaborators at LANL, so that we
may determine specificity of nucleotide binding by the pure recombinant protein, and guide structure determination of protein-ligand complexes. This evidence for expression of multiple aldehyde dehydrogenases in M.tuberculosis, which also show homology to human aldehyde dehydrogenases, provides a discrete opportunity for structural genomic analysis, incorporating our ligand-binding data. A second well established high-profile role of nucleotide-protein interactions is found in important drugs that are variants of naturally occurring nucleotides or nucleosides. A number of candidate drug-compounds against M.tuberculosis have been identified in screens, and we have tested purine candidates developed by Southern Research Institute, Alabama, in our protein-ligand screen. Using crude M.tuberculosis lysate, we have identified on aldehyde dehydrogenase isozyme that binds a methylated adenosine analog, and also a gene product, Rv0577, that was not predicted to bind nucleosides/nucleosides.
The Rv0577 gene was targeted for cloning and expression by LANL, and we have shown that the recombinant protein has a clear preference for binding adenine and methylated adenine nucleosides, but not adenylates. This information is currently being used to guide crystallization efforts at LANL. These results indicate that biochemical analysis of protein-nucleotide interactions provides information useful in targeting potentially important proteins for analysis in structural genomics initiatives and providing insight into the biochemical functions of cellular proteins that may be important for drug development and understanding drug resistance.
Impacts
Our project has enhanced efforts to determine the structures of proteins in action, which provides key insights into the function of genes. This project also is valuable in development of drugs that target proteins.
Publications
- No publications reported this period
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Progress 01/01/06 to 12/31/06
Outputs
We are interested in understanding the specificity of interactions between nucleotides and proteins, from the point of view of determining which proteins in cells bind to nucleotides, and the selectivity of binding by which a protein may discriminate between closely related nucleotides or nucleotide analogs. To this end, we have have developed a ligand-protein interaction screen that aims to enhance experimental progress in functional genomics of nucleotide-binding proteins from Mycobacterium tuberculosis, in collaboration with Chang-Yub Kim and Thomas Terwilliger at Los Alamos National Laboratory, New Mexico. The screen can be used to identify protein targets for structural analysis and to identify ligands (natural and xenobiotic) for co-crystallization for structure-function analysis. In this method, proteins (pure or in in crude cytosolic extracts) are bound to a dye affinity resin. About half of proteins, including many nucleotide-binding proteins, bind to this
resin. Proteins that interact with a series of specific ligands are identified by elution with those ligands followed by mass spectrometry-based fingerprinting of tryptic peptides and matching to genomic databases. We have demonstrated several applications of this approach. First, our ligand-binding data, based on elution of total cell extracts bound to the affinity resin, led to thirteen Mycobacterium tuberculosis proteins being targeted by the LANL group for cloning and crystallization. The structure of one of these targeted proteins (Rv0223c, a putative aldehyde dehydrogenase) has been solved, with a bound nucleotide, and multiple crystals with different nucleotides have been generated. Additionally, individual proteins from Mycobacterium tuberculosis were screened for nucleotide-ligand interactions. Conversely protein targets of nucleoside-analog anti-tuberculosis drug candidates were identified. These results indicate that biochemical analysis of protein-nucleotide interactions
provides information useful in both reducing bottlenecks in high-throughput structural genomics initiatives (e.g. by improving crystal quality) and providing insight into the biochemical functions of cellular proteins.
Impacts
Our project has enhanced efforts to determine the structures of proteins in action, which provides key insights into the function of genes. This project also is valuable in development of drugs that target proteins.
Publications
- No publications reported this period
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Progress 01/01/05 to 12/31/05
Outputs
We have studied the oxidation and synthesis of exogenous NADH by isolated, purified potato tuber mitochondria, using fluorescence spectroscopy and other methods of biochemical analysis, in collaboration with R. Douce (Grenoble). We have discovered an important mechanism regulating the redox state and the rate of respiration in plant cells. This mechanisms serves to determine under which conditions cytoplasmic NADH is oxidized by the external NADH-dehydrogenase, on the outer surface of the inner mitochondrial membrane (where no permeability barriers exist between the enzyme and the cytosolic pool of NADH). Most importantly, NADH oxidation by this enzyme is effectively shut of when NADH levels approach micromolar levels, i.e. the concentration of cytoplasmic NADH found in normal, respiring plant cells. This inhibition is imposed by NAD (which is normally a thousand-fold more abundant than NADH in plant cells), which causes the steady-state kinetics of the NADH
dehydrogenase to switch from a simple hyperbolic NADH isotherm to a strongly sigmoidal isotherm. This change in kinetics, which reflects the apparent affinity of NADH for the NADH-dehydrogenase enzyme, indicates that quaternary interactions are taking place between different subunits in or on the membrane, which results in the rate of NADH oxidation rising extremely sharply in response to small increases in NADH at concentrations above a few micromolar. In contrast, when NADH levels change below about one micromolar, this dehydrogenase remains inactive. This regulation of the external NADH dehydrogenase also serves to maintain cytoplasmic redox levels, helping clamp the cytoplasmic [NAD]/[NADH] ratio at about 1000, and limiting energy expenditure via futile cycling of metabolites (notably malate and oxaloacetate) between cytoplasm and mitochondria that could occur by the combined action of dicarboxylate anion carriers, malate dehydrogenase inside and out of the mitochondria, and the
external NADH dehydrogenase. The mechanism of regulation of the external NADH dehydrogenase is evident under conditions of either net import (oxidation) of reducing equivalents (i.e. heterotrophic metabolism; primary mitochondrial function is respiration) or net export of reducing equivalents (most importantly in photosynthetic plant cells where mitochondrial metabolism serves to support photorespiration, rather than oxidative phosphorylation).
Impacts
Our results provide a unifying framework for understanding how plant cells manage oxidations (biological versions of chemical reactions such as combustion) while performing the important functions of respiration and photosynthesis. The mechanisms by which plants perform the reactions of energy transformation, using carbon fuel to perform chemical and mechanical work, are so much more efficient and less destructive than the much cruder versions employed in human machines and processes. Further, the regulation of oxidation of carbohydrates in plant cells is very different than in animals, including the mechanism elucidated in our study, which has implications for understanding how plants respond to environmental stress and pests, and to genetic modifications.
Publications
- No publications reported this period
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