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Physico-Chemical Methods in Biotechnology and Material Science Speakers and Conference Topics: Ryszard W. Adamiak, Institute of Bioorganic Chemistry, Professor, Head of the Laboratory of Structural Chemistry of Nucleic Acids. Molecular modeling is the ultimate step in several of our research projects, all based on joint applications of the total synthesis of regioselectively modified (stable isotopes, fluorophores) RNA, thermodynamics, multinuclear NMR spectroscopy, laser spectrofluorimetry and protein interactions. Within the models of the bulge loop- and apical loop-modified HIV-1 TAR RNA structures the importance of considering the long distance non-covalent interactions in solution (using the Particle Mesh Ewald method) becomes evident. "In aqua" simulation of molecular dynamics, magnesium binding protocols all require extensive use of super computing. Paul Agris, North Carolina State University, Professor, Department of Biochemistry. Our research program involves the study of functional and structural genomics, the structure-function relationships for nucleic acids in biology and in biomedical science. Using such techniques as diverse as bioorganic synthesis, biochemistry and molecular biology, spectroscopy and NMR leading to the molecular modeling, we determine structure-function relationships for the unique chemistries of modified RNAs. Recently, we have determined that specific RNA modifications are critical to translation and have designed functioning analogs to tRNA as biological tools and potential pharmaceutical targets. Stefan Franzen, North Carolina State University, Assistant Professor, Department of Chemistry. Oxidants from metabolic activity, ionizing radiation, and genotoxic chemicals are responsible for DNA damage (1-4) and ultimately such damage results in mutagenesis, carcinogenesis, and aging. Our efforts involve the investigation of damaged purines, more specifically, oxo-Guanine and oxo-Adenine. The study of such altered bases is of interest since DNA polymerase, the enzyme that has a DNA repair function, allows DNA synthesis to proceed past these lesions. These lesions result in the possibility of mismatched base pairs and DNA conformational changes. Since structure is indicative of function, conformational studies on relevant oligomers will be investigated. In order to perform a thorough investigation of structure and conformation, a spectral library of NMR, FT-IR, and Raman data for relevant oligomers will be produced. We will couple an artificial neural network to molecular spectroscopy resulting in a robust classification method for damaged versus normal DNA. Hanna Gracz, North Carolina State University, Laboratory Supervisor of the Nuclear Magnetic Resonance Facility. The ferritin IRE mRNA translation regulatory element in animal mRNA was studied by molecular modeling (using MC-SYM and DOCKING) and by NMR spectroscopy. Co(III)hexammine was used to model hydrated Mg2+. All IREs bind IRPs (Iron Regulatory Proteins). A G-C base pair, conserved in ferritin IREs, spans an internal loop/bulge in the middle of an A-helix an combined with a dynamic G-U base pair, formed a pocket suitable for Co(III)hexammine binding. Based on effects of Co(III)hexammine and Mg2+ on the 1H-NMR spectrum and results of automatic docking into the IRE model, the IRE bound Co(III)hexammine at the pocket in the major groove; Mg2+ binds to the IRE at the same site based on NMR data, analogy to Co(III)hexammine and on the Mg2+ inhibition of Cu(phen)2 cleavage at the site. Distortion of the IRE helix by the internal loop/bulge near a conserved unpaired C required for IRP binding and adjacent to an IRP crosslinking site, suggests a role for the pocket and HL/IL interactions in ferritin IRE/IRP. Keith Gubbins, North Carolina State University, W.H. Clark Distinguished University Professor, Department of Chemical Engineering. Our research program is aimed at understanding, at the molecular level, the behavior of nano-dimensional fluids and solids, and the influence of surface forces on such materials. Nano-porous materials (solid materials having pores of nanometer dimension), such as zeolites, activated carbons, silicas, etc., play a prominent role in chemical processing, particularly in separations and as catalysts and catalyst supports. Fluids confined in such porous materials possess many novel properties that can form the basis of future technologies, involving energy storage, novel reactions and separations, fabrication of small devices of molecular dimensions, etc. The underlying theme of our work is to develop molecular models which accurately describe the materials and systems of interest, and to use rigorous methods or statistical mechanics to determine the detailed properties and behavior of the system. Comparisons with experiment are used to check the models, but the ultimate goal is to use the simulations to carry out experiments that cannot be undertaken in the laboratory. Experimental studies complement the simulation work, and comparison of the two frequently leads to important new insights. Cynthia Hemmenway, North Carolina State University, Assistant Professor, Department of Biochemistry. The potato virus X (PVX) system is an excellent model for understanding general mechanisms of RNA replication and potentially for designing novel antiviral strategies. Genetic and biochemical approaches are being used to determine the components and reactions required for RNA synthesis. We have developed a tobacco protoplast replication system for in vivo analyses of the cis-acting elements and structures that affect PVX RNA synthesis. To study the biochemical mechanisms and components required for RNA replication, we have also developed a soluble, template-dependent PVX replicas extract from infected tobacco plants. Collectively, these in vivo and in vitro approaches will enable us to understand how RNA/RNA, RNA/ protein, and protein/protein interactions mediate replication of plus-strand RNA viruses. Stefan Jurga, Adam Mickiewicz University, Dean, Professor. Alkylammonium layer-structure compounds CnH2n+1NH3Cl can serve as model bilayer systems for description of structure and molecular dynamics in many aliphatic hydrocarbon lipids, which are of great importance in biology and chemistry. In solid state the alkylammonium compounds crystallize in layers of n-alkylammonium moieties alternating with layers of the counterions, whereas when solved in water they form a variety of lyotropic liquid-crystalline states. Thus a general knowledge on motional and conformational behavior of their hydrocarbon chains and of the polar head groups in the solid, liquid crystal and liquid state is fundamental for understanding the physical properties of chemically and biologically important lipid systems. Wlodzimierz Krzyzosiak, Institute of Bioorganic Chemistry, Professor, Head of the Laboratory of Cancer Genetics. The genes responsible for several hereditary neurological diseases, including Fragile X syndrome, myotonic dystrophy, Huntington disease and several spinocerebellar ataxias, were shown to harbor unstable trinucleotide repeats. Our research in this field is focused on finding the role of RNA level effects in cytopathogenesis as well as on establishing rational basis for RNA-directed gene therapies of these diseases by analyzing RNA structures formed by normal and expanded trinucleotide repeats within the context of their host mRNAs. Human FMR-1 mRNA fragments containing CGG repeats and AGG interruptions show high propensity to form different stable conformers under conditions of structure probing. We have developed two alternative approaches designed to obtain structural information specific for individual conformers. These approaches are included in a new, extended protocol for RNA structure probing in solution using a biochemical approach. Andrzej Legocki, Institute of Bioorganic Chemistry, Professor, Head of the Laboratory of Plant Pathology. Intracellular pathogenesis-related proteins of the PR10 class have been shown to be ubiquitous in the plant kingdom. They are composed of 153-160 amino acids, reveal resistance to protease treatment, lack a signal peptide, and accumulate around the sites of pathogen invasion or wounding. It is suggested that PR10 play an important role in plant development and defense. We have detected in the yellow lupine genome two subfamilies of PR10 genes of which individual members are expressed differentially during the course of plant response to pathogenic or symbiotic microorganisms. Recombinant lupine PR10 proteins where overexpressed in E. coli and crystallized. The structure which has been solved with 2 Å resolution revealed a highly conserved glycine-rich motif in the vicinity of a putative nucleotide binding domain which might be related to PR10 function. Steve Lommel, North Carolina State University, Professor, Department of Plant Pathology. Our laboratory utilizes the well-characterized red clover necrotic mosaic virus (RCNMV) system to study the process of plant virus disease induction. The mechanisms of plant viral movement out of initially infected cells into the surrounding cells, and from leaf to leaf throughout the host, are not understood. We are conducting molecular, biochemical, and cell biology experiments to elucidate the mechanisms of movement and identify host factors that the virus must interact with to facilitate movement, systemic infection and disease. We have identified a novel bipartite RNA regulator of subgenomic RNA synthesis in RCNMV. We hypothesize that this regulator acts to sterically inhibit the procession of RNA polymers on the viral template. This results in a premature termination event, generating a minus strand template for subgenomic RNA (sgRNA) synthesis. We believe this is the first report of RNA-mediated transcriptional regulation in an RNA virus. Based on our current data, we have a working model for the mechanism of sgRNA synthesis in RCNMV that is now being tested by molecular, genetic, and physical experimentation. Wieslaw Kazimierski, Glaxo Wellcome. It has recently been shown (Bioorg. Medchem. Lett. 1998, 8, 3637-3642) that several heterocyclic scaffolds could be used to replace the P1/P2 portion of Amprenavir, resulting in potent HIV-1 inhibitors (e.g. GW 7652X). To explore the potential of these ring systems for the discovery of second-generation inhibitors, we have developed facile synthetic methods leading to mono, di-, and spiro-substituted heterocyclic scaffolds (I-III). When combined with sulfonamide- and indanolamine-derived backbones, these scaffolds yielded novel and potent inhibitors, culminating in the discovery of GW 5950X. This poster will present chemistry developed towards I-III and SAR of two lead series containing these scaffolds. Ryszard Kole, University of North Carolina at Chapel Hill, Professor, Department of Pharmacology. In cells recapitulating ß-thalassemia or cystic fibrosis caused by splicing mutations the antisense oligonucleotides not only inhibited aberrant splicing but also restored correct splicing in a dose dependent and sequence specific fashion, generating correct human ß-globin and CFTR mRNAs and polypeptides. Similarly, antisense oligonucleotides restored a reading frame in the mutated murine dystrophingene in cultured myotubes from mdx mice. Thus, the antisense oligonucleotide treatments provide potential alternatives to gene replacement therapies. Recent results show that modification of splicing by antisense oligonucleotides is also useful as potential anti-cancer therapy. Targeting of the alternatively spliced pre-mRNAs transcribed from genes involved in metastatic progression of cancer cells leads to a change in the ratio of the expressed splice variants with significant effects on the phenotype of the cells. Barbara Ramsay-Shaw, Duke University, Professor, Department of Chemistry. The boranophosphates are isoelectronic and isoionic analogs of the naturally occurring O-phosphate esters. Boranophosphates possess properties that may make them uniquely useful as antisense agents. Boronated DNA dimers are 18-fold more lipophilic than natural nucleotide dimers, and orders of magnitude more resistant to exo and endonucleases. Boronated oligomers form stable duplexes and support RNAse H mediated cleavage of mRNA. Boronated DNA may provide an exceptionally useful addition to the repertoire of nucleic acid mimetics and gene targeting drugs. Edward O. Stejskal, North Carolina State University, Professor Emeritus, Physical and Analytical Chemistry. There has been a growing demand for diffusion measurement by means of pulsed field gradient spin echo (PFGSE) methods. We have recently studied the diffusion of small cations in the presence of large, massively charged anions. We are also using the diffusion of RNA molecules in order to assess whether their samples are monomeric or not. When applied to RNA, this experiment easily distinguishes duplexes RNA's from RNA hairpins, and thus, it can solve one of the perennial problems faced by RNA spectroscopists. Also, the measurement of the diffusion of Xenon-129 absorbed in multiphase polymer systems will make it possible to determine the relationship between the domain sizes and the ability to see the absorbed Xenon as distinguishable lines in the spectrum. Another experiment involves the study of the binding properties and structure of hormone peptides in membrane mimics by Pulsed-Field Gradient Diffusion. One of the main goals of this work is to determine whether the binding of these short peptides to sodium dodecylsulfate (SDS), and dodecylphosphocholine (DPC) micelles and the induced secondary structure upon binding are similar to their binding to lipid bilayers; thus, the validity of the widely held assumption that these micelles are good membrane mimics can be examined. Paul Wollenzien, North Carolina State University, Professor, Department of Biochemistry. Ribosomes perform a universal biochemical process for which the molecular details are not known. It has been proposed for some time that conformational changes in the ribosome orchestrate many important steps in protein synthesis. However, this has been difficult to investigate because of the absence of techniques to determine and monitor higher order structure in large RNAs. We have developed techniques involving crosslinking (UV irradiation and the irradiation with near-UV light of photochemical reagents (psoralens, psoralen derivatives, 4-thiouridine and other photoactive nucleoside derivatives) placed at specific sites in the rRNA in the 30S ribosomal subunit) that allow the determination of intramolecular distances and distance changes in the ribosome. The information allows us to construct and refine a topographical molecular model for the small subunit rRNA. Conformational changes in the 16S rRNA have been detected around the decoding region upon ribosome subunit association, association with initiation factor IF3 and tRNA binding. These changes must be related to the selection of initiator tRNA and the selection and movement of tRNA during the elongation cycle. Recent Meetings There have been no recent meetings on the topic of physico-chemical methods in biotechnology and material science. |
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