Honggao Yan - US grants

Acquisition of DNA Analysis Instrumentation for Molecular Studies Our goal is to establish a common core facility for DNA sequence analysis. We propose to purchase: an Automated DNA Sequencer; a molecular biology CATALYST Labstation; an Oligo Synthesizer; a PhosphorImager workstation; a Fluorimager; a Computer System; a Biomek II robot; a Thermocycler: an Automated Film Developer: a Vacuum Centrifuge: a Photodocumentation Station; and two Ultra Cold Freezers (-80 and -30). The participating faculty address a wide range of research related to biodiversity and biotechnology. Their efforts share a common theme of DNA sequence analysis and fall into two major areas: 1) Molecular genetic approaches to study gene expression, RNA processing, and molecular phylogeny, and 2) Molecular approaches in the study of Population Genetics, Ecology, and Evolution. In the area of Molecular genetic approaches to study gene expression. RNA processing. and molecular phylogeny, faculty research and research training include: Whalen's molecular genetic analyses of interactions between bacterial pathogens and plants; Marquez-Magana's investigations of the molecular mechanisms that regulate flagellin gene expression in Bacillus subtilis; and Davis's functional significance and regulation of spliced leader RNA trans-splicing in metazoa, the evolution of parasitism in flatworms, and the molecular phylogeny of flatworms and early metazoa. The focus of Ramirez' s research is to clone the RECI gene and further characterize the RPD3 (a.k.a. REC3) genes of Saccharomyces cerevisiae which are implicated in the repair of damaged DNA and in mitotic recombination. Goldman focuses on a structural unit of the eukaryotic chromosome, the chromatin domain or loop, and its potential role as a functional unit in the regulation of gene expression in mammalian X- chromosome inactivation and genomic imprinting. Perara investigates molecular approaches to the problem of protein transport across cell ular membranes, with the primary focus on a systematic comparison of the molecular mechanisms of secretory protein transport across the endoplasmic reticulum (ER) membrane and the bacterial periplasmic membrane. In the area of Molecular approaches in the study of Population Genetics. Ecology and Evolution, faculty research and research training include: Parker/Cullings' studies of plant community and evolutionary ecology, dynamics of plant recruitment, life history evolution and community turnover rates; Smith/Bayliss' investigations of evolutionary ecology and conservation biology with research focus on the role of transition zones between rain forest and savannah in speciation and their role as related to conservation of rain forest species; Arp's studies of physiological adaptation to environment, sulfide tolerance and detoxification in mudflat invertebrates, ecology and physiology of hydrothermal vent organisms; Randall's investigations of evolution of communication and social organization in desert rodents; and Routman' s use of modem molecular genetics to examine population structure, biogeography and intraspecific phylogeny, and the genetic architecture of complex phenotypes. Larson's research is in population biology of nearshore teleost fishes, including their behavior, feeding ecology, life histories, and population structure. Desjardin focuses on taxonomy and phylogeny of fleshy fungi, primarily Agaricales, using traditional and modern aspects of systematics, including cladistic and morphometric analysis, utilizing morphological, ecological, physiological, genetic and molecular data. Hollibaugh addresses physiological ecology of bacterial communities in geochemical cycles and foodwebs. Orrego employs the techniques of molecular genetics for the study of genetic variation in natural populations as displayed by sequences obtained via long distance PCR methods. Our proposed instrumentation will increase the amount and efficiency of our research an d training efforts and enable us to train a greater number of students. The requested equipment will provide the highest resolution available for the analysis of DNA/RNA using PCR amplification, DNA sequencing, Denaturing Gradient Gel Electrophoresis (DGGE), Single Strand Conformation Polymorphisms (SSCP), and a variety of other DNA/RNA detection and analytical methods. With the increased throughput capacity for large numbers of samples that can be processed rapidly and efficiently with longer sequence reading (>700 bp), we will be able to do new high resolution analysis on much greater numbers of samples more rapidly than in the past. For example, the ABI 377 Sequencer provides new longer gel formats that allow one to read 700 bases/run and 36 lanes/run that can be completed in 8 hour (or overnight) runs and 300-500 bp runs in 2.25 hours, allowing 3 runs in an eight hour period. Several faculty (Davis, Hollibaugh, and Parker) will collectively sequence in excess of 10,000 samples/year which represents one run of 36 samples each day for a year. While this alone clearly justifies our need, the acquisition of the proposed instrumentation would also allow us the capacity to triple this number. We will also use the equipment for faculty enhancement short courses; since 1991 we have presented 10 such courses-to over 240 faculty from 35 states.

Guanylate kinase (GK) plays an essential role in the cGMP cycle and may be involved in guanine nucleotide-mediated signal transduction pathways. The long-term goal of the project is to establish quantitative structure- function relationships for yeast GK that will account for its catalytic mechanism and nucleotide specificity, using a combination of kinetic and thermodynamic methods, site-directed mutagenesis, and biophysical methods. The specific aims of the project include: (1) To determine the kinetic pathway and energetics of the GK-catalyzed reaction by steady- state and transient kinetics and thermodynamic measurements. The goal is to obtain a complete free energy profile which will be the basis for dissecting the contributions of individual amino acid residues to catalysis and substrate specificity. (2) To evaluate the roles of the active site residues in catalysis and nucleotide specificity by site- directed mutagenesis. The contributions of these residues to each step of catalysis will be quantitated by evaluating the effects of mutations on the reaction profile as described in Specific Aim 1. Furthermore, how the amino acid residues interact with the substrates will be probed by examining the kinetics and/or stereochemistry of substrate analogues. (3) To assess the effects of mutations on the structure and stability by biophysical methods. The goal is not only to define the roles of the amino acid residues in structure and conformational stability but also to provide the essential structural information for quantitative interpretation of the results obtained in Specific Aim 2. (4) To identify the amino acid residues in close proximity to the bound ATP by NMR and dock the nucleotide into the crystal structure of yeast GK by computer graphics with the distance constraints from the NMR data. (5) To engineer new substrate specificity. The initial goal is to redesign the GMP site by mutagenesis so that the enzyme catalyzes phosphoryl transfer from ATP specifically to IMP, XMP, or AMP. The ATP site will also be modified at the late stage of the project so that it is specific for GTP, ITP, or XTP.

structural biology; proteins; biomedical resource; bacteria;

Training in the use of DMX electronics.

Folate cofactors are essential for the growth of all organisms. While man and animals are able to take folates from foods, most microorganisms have to synthesize folates de novo. Thus, enzymes involved in folate biosynthesis are targets of antimicrobial agents. 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) catalyzes the first reaction in the folate biosynthesis pathway. The long-term goals of the project is to elucidate the structure and mechanism of HPPK and to design inhibitors for the enzyme. HPPK was expressed in E. coli at high level. MALDI-MS analysis was used to determine the molecular weight of the recombinant HPPK. The result showed that the N-terminal methionine was removed in vivo.

The goal of the proposed multidisciplinary research is to elucidate the structure-function relationships of 6-hydroxymethyl-7,8- dihydropterin pyrophosphokinase (HPPK), the first enzyme in the folate biosynthesis pathway. Our system is recombinant E. coli HPPK because the E. coli isozyme is the best model enzyme for studying the mechanisms of enzymatic pyrophosphoryl transfer. The results will provide the detailed information on the structure of HPPK and how it catalyzes the transfer of pyrophosphate. The proposed research is of fundamental importance to enzymology because very little is known about the structures and mechanisms of any pyrophosphokinases. Furthermore, the structural and mechanistic information that will be obtained from the proposed research will be essential for design of inhibitors for HPPK. Since most microorganisms must synthesize folate de novo but man and animals cannot and don't need to, HPPK inhibitors may become new antimicrobial agents. This is of great biomedical significance especially in light of rapidly increasing antibiotic resistance in recent years that has rendered the current antibiotics ineffective for treating many microbial infections and caused a worldwide health care crisis. In Specific Aim l, the kinetic pathway and energetics of the HPPK-catalyzed reaction will be determined by steady-state kinetic, transient kinetic, thermodynamic and stereochemical analyses. In Specific Aim 2, site-directed mutagenesis, in conjunction with biochemical and biophysical characterizations of the mutants, will be used to identify the active-site residues and to elucidate their roles in substrate binding and catalysis. In Specific Aim 3, biophysical methods will be used to assess the effects of mutations on the structure and stability of HPPK at both free and substrate-bound states. In Specific Aim 4, the solution structure of the free HPPK will be determined by double-and triple-resonance multidimensional NMR. In specific Aim 5, NMR will be used to identify the active-site residues and to determine the structures of HPPK in complex with substrate analogues.

DESCRIPTION (provided by applicant): The long-term goal of the project is to determine the structures and molecular mechanisms of catalysis for enzymes in the folate biosynthetic pathway, a proven target pathway for developing antimicrobial agents. The proposed research is to continue our current study on 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK), and to expand our research to include dihydroneopterin aldolase (DHNA). HPPK is an excellent model for studying mechanism of enzymatic pyrophosphoryl transfer. DHNA is a unique aldolase because it requires neither the formation of a Schiff base between the enzyme and its substrate nor metal ions for catalysis, and the enzyme also catalyzes the epimerization of its substrate. The central hypothesis behind the proposed research on HPPK, which is based on the results obtained in the previous funding period, is that HPPK undergoes dramatic conformational changes during its catalytic cycle and the conformational changes play critical roles in its catalysis. Thus, in Specific Aim 1, we will continue our quest for structure determination of HPPK along the catalytic cycle by X-ray crystallography. In Specific Aim 2, we will determine the conformational dynamics of the catalytic loops of HPPK by time-resolved fluorescence energy transfer (FRET) at equilibrium conditions and even as the reaction progresses and the dynamics of its core structure by heteronuclear NMR relaxation at the sub-nanosecond to nanosecond and microsecond to millisecond time scales. Most importantly, in Specific Aim 3, we will correlate the structure and conformational dynamics of HPPK with its catalysis by site-directed mutagenesis, biochemical analysis (particularly transient kinetic analysis), and biophysical methods. The main hypotheses behind the proposed research on DHNA are that (1) the two adlolases from Staphylococcus aureus and E. coil have different binding/catalytic properties and distinct responses to inhibitors and (2) general acid/base catalysis plays a most critical role in the catalytic mechanism of this unique aldolase. Thus, in Specific Aim 4, we will determine the structures of DHNA by X-ray crystallography, particularly the structures of the complexes with neopterin and monopterin, the closest mimics of the Michaelis complexes of DHNA. In Specific Aim 5, we will identify residues involved in general acid/base catalysis in DHNA by a combination of site-directed mutagenesis, transient kinetic pH-rate profile analysis, and NMR spectroscopic titration.

Terpenoids are the most diverse, largest, single family of compounds found in nature. They play vital roles in all living organisms, and have a variety of applications in agriculture, nutrition, and medicine. The building blocks for the biosynthesis of terpenoids are made in nature by one of two biosynthetic pathways. The recently discovered MEP pathway is the pathway for the biosynthesis of the building blocks for terpenoids in most microorganisms, apicoplasts of some protozoa, and plastids of plants. 1-deoxy-D-xylulose 5-phosphate (DXP) reductoisomerase (DXR) catalyzes the biosynthesis of 2-C-methyl-D-erythritol 4-phosphate (MEP), the committing step of the MEP pathway. While there is rich structural information on DXR, the chemical mechanism of the DXR-catalyzed reaction remains unresolved and the transition state of the reaction is unknown. The proposed project is a combined experimental and computational study aimed at addressing these most challenging issues of this important enzyme. It involves the simultaneous measurement of multiple 13C kinetic isotope effects (KIE) on the DXP isomerization by a novel NMR-based method and combined quantum mechanics/molecular mechanics molecular dynamics simulation and 13C KIE computation. Furthermore, the transition state stabilization by DXR will be determined by site-directed mutagenesis and biochemical, X-ray crystallographic, and computational analysis of the site-directed mutant enzymes. The proposed research is not only of fundamental significance for the understanding of DXR catalysis but also valuable for the development of antibiotics, antimalarial drugs, and herbicides targeting DXR. It will also provide an outstanding opportunity for multidisciplinary research training of both graduate and undergraduate students. The results of the proposed research will be incorporated into protein biochemistry and computational chemistry courses taught by the PI and the co-PI at two institutions and will significantly enhance these courses and benefit students taking the courses for many years to come.

This project is a study by a combination of modern experimental and computational methodologies on 1-deoxy-D-xylulose 5-phosphate reductoisomerase, the most important enzyme in a pathway for the biosynthesis of the building blocks for terpenoids. This pathway is absent in human and animals but essential for most microorganisms, some protozoa, and plants. The research is not only of fundamental significance but also valuable for the development of antibiotics, antimalarial drugs, and herbicides targeting this enzyme. It will also provide an outstanding opportunity for multidisciplinary research training of both graduate and undergraduate students.

DESCRIPTION (provided by applicant): The long-term goal of the proposed research is to develop diagnostic tools for otitis media (OM) and other pneumococcal infections and research tools for studying pneumococcal pathogenesis. OM is a major public health problem in young children with an enormous economic burden in the US as well as in the world. S. pneumoniae not only is a major bacterial cause of OM but also causes additional life-threatening or invasive infections. Pneumococcal infection is one of the four killer infectious diseases and probably the world's single biggest killer of young children. Although S. pneumoniae is one of the most important human pathogens, the laboratory diagnosis of pneumococcal infections is still dependent on traditional microbiological methods and very few new methods are reliable enough for the laboratory diagnosis of pneumococcal infections. The traditional microbiological methods and tests not only are laborious but also have many limitations. There is an urgent need for new laboratory diagnostic tests for pneumococcal diseases, as the limitations of current diagnostic tests hinder the timely and accurate diagnosis of pneumococcal infections, which in turn negatively affects not only the treatment of the diseases but also the assessment of the effectiveness of control measures. Aptamers are small single-stranded nucleic acids that bind a molecular or cellular target with high affinity and their biochemical properties rival or are superior to those of antibodies in a variety of analytical, diagnostic, and potential therapeutic applications, particularly in ease of production, batch uniformity, shelf life, and cost. In this exploratory two-year grant application, we propose to develop aptamers against two major pneumococcal virulence factors, choline-binding protein A (CbpA, also known as PspC or SpsA) and pneumococcal surface protein A (PspA). Both virulence factors are multidomain multifunction proteins and play major roles in pneumococcal evasion of host immunity and pathogenesis. The molecular bases for their functions are the ability of CbpA to bind human complement factor H (FH) and the ectodomain of pIgR and the ability of PspA to bind apo- and holo-lactoferrin (LF) and to inhibit complement deposition. Specific Aim 1 is to develop aptamers against the virulence factors and Specific Aim 2 is to map where in the virulence factors the aptamers bind and develop aptamer beacons for the detection of the virulence factors. The proposed research will set up the stage for exploring the idea of using aptamers for the development of diagnostic tests for pneumococcal infections and provide research tools for visualizing pneumococcal pathogenesis. Based on the essential roles of pneumococcal virulence factors in pneumococcal pathogenesis and the ability of the developed aptamers to neutralize the two most important virulence factors, the proposed research will also set up the stage for testing the hypothesis of antivirulence strategy as a novel alternative/complementary strategy for combating pneumococcal resistance to classical antibiotics. PUBLIC HEALTH RELEVANCE:Otitis media, or middle ear infection, is a major public health problem in young children both in the US and in the world. Streptococcus pneumoniae not only is the major bacterial cause of otitis media but also causes additional life-threatening or invasive infections. The proposed research will set up the stage for exploring the idea of using aptamers for the development of diagnostic tests for pneumococcal infections and research tools for visualizing pneumococcal pathogenesis and for testing the hypothesis of antivirulence strategy as a novel potential strategy against otitis media and other pneumococcal infections.