Research Experience for Undergraduates (REU) Program
Chemical and Systems Biology Approach to Stress Response
Cellular response to stress (such as temperature, oxygen, viral infection) involves numerous networks and signaling pathways. The integration of signals leads to changes in gene and protein regulation. We use changes in protein abundance and activity on a global scale to elucidate the pathways and networks important in Archaea. Activity based protein profiling (ABPP) is a method that has been developed to measure protein activity at the cellular level and is a powerful approach to systems biology. ABPP uses chemically reactive small molecules that specifically target enzyme active site residues. This allows only the catalytically active form of a protein to be identified. The "tagged" proteins are visualized by attaching a fluorescent dye molecule to the probe. Rapid screening of the proteome is accomplished with 1D and 2D gel analysis. Identification of specific proteins that are tagged is then conducted using mass spectrometry based protein identification. Using state-of-the-art mass spectrometry, proteomics, and network analysis approaches, we are elucidating the pathways, proteins, and post-translational regulation events important for cellular survival during stress across all the domains of life. This work has important implications for a wide-range of fields from cancer biology to environmental remediation.
Probing the mechanism of DNA repair by spore photoproduct lyase
Spore photoproduct lyase is an enzyme that catalyzes the repair of UV-induced DNA damage in spore-forming organisms. Such organisms are unusually resistant to UV irradiation, in large part due to the efficient repair of accumulated DNA damage during germination. The UV resistance of spores poses a major threat to human health, as spore-forming organisms such as Clostridium are responsible for numerous diseases (including tetanus and botulism) but are difficult to kill completely due to their UV tolerance. Spore-forming organisms, for example Bacillus anthracis, are also potential agents in bioterrorism. Understanding the molecular mechanism of DNA repair that confers the unusual UV resistance to spores could open up new avenues for killing spores, thus resolving some major threats to human health. Spore photoproduct lyase is the enzyme that repairs spore photoproduct (SP), and is found in Bacillus, Clostridia, and other spore-forming organisms. The REU students involved in this project will utilize an array of biochemical and spectroscopic techniques, as well as chemical synthesis, to probe the mechanism of DNA repair by spore photoproduct lyase. The studies will provide important new insights into the structural and electronic basis for recognition and repair of DNA damage.
The Fluorescence Intensity Changes that Accompany Changes in Protein Structure
The long-range goal of this project is to provide a means for detailed fundamental understanding of the widely exploited phenomenon of fluorescence quenching by electron transfer in any biological setting in terms of structure and dynamics. Using a hybrid quantum mechanical/molecular mechanics procedure we have recently made unprecedented progress in understanding the enigmatic and widely exploited tryptophan fluorescence intensity changes accompanying changes in protein structure. We have extended use of these programs to the study of flavin and dye fluorescence by tryptophan and tyrosine in proteins. We propose to use our programs to investigate the validity of the proposed mechanism by which the ubiquitous enzyme, DNA photolyase, appears to shuttle electrons to its flavin co-factor along a chain of three tryptophans. DNA photolyases (which use blue light as an energy source to repair UV-damaged DNA in a number of organisms) are closely related to cryptochromes (which are ubiquitous blue light receptors used by plants and animals to control behavior, including circadian rhythms and probably directional flight of migratory birds).
Native American Research Laboratories
The Native American Research Laboratories (NARL) at The University of Montana are dedicated to providing "hands-on" research experiences to Native American undergraduate, graduate, and, even, high school students from indigenous communities located throughout the nation. Although NARL is focused on providing Native students with access to modern research instrumentation and training in state-of-the-art research techniques, non-Native and international students also participate in our research groups to facilitate a cross-cultural and highly collaborative research and learning environment. What separates NARL from any other Native-serving institution is this cross-cultural atmosphere and the use of both culturally-relevant faculty role models as well as near-peer mentoring techniques. NARL research projects are currently funded by both the National Science Foundation and the National Aeronautics and Space Administration.
Ongoing projects including the following:
(1) The study of hyperthermophilic organisms from volcanic hot springs: NARL studies "extremophiles", such as hyperthermoacidophilic archaea. Not only do we seek to discover and characterize new species but we also work to elucidate the molecular mechanisms that allow microorganisms to live in the harshest habitats known to support life on Earth. Many of these molecular mechanisms have applications for biotechnology as well as providing the scientific community with information about the extents to which life can exist in extreme environments.
(2) The study of extreme viruses: NARL also studies thermo-tolerant viruses from hyperthermophilic hosts. Many viruses such as the Sulfolobus Spindle Virus possess unique replication strategies that have not previously been observed in other viruses. By studying these viruses, NARL researches seek to better understand host-virus systems in extreme environments. Virus structure and replication cycle characteristics have also proven to be useful in biotechnological and biomedical applications.
(3) The study of artificial cellulosomes for bioethanol production: In collaboration with NASA Ames Research Center, NARL is studying the ability of macromolecular systems derived from hyperthermophilic archaea to serve as platforms for cellulase binding in an effort to increase the efficiency of lignocellulose biomass deconstruction. This new technology has implications for biofuels and climate change research.
Multivalent Protein-Carbohydrate Interactions
Galectin-3 is a member of the Galectin (galactose-binding) family of lectins. In cancer cells, expression of galectin-3 has been correlated with metastatic potential. Galectin-3 induces homotypic tumor cell aggregation, which results in tumor embolism and increases metastatic potential. Recent research (Prof. Raz, Wayne State Karmanos Cancer Institute) with modified citrus pectin, a water-soluble polysaccharide that is rich in galactose, indicates that carbohydrate arrays can interfere with galectin-3-mediated association of cancer cells. We are synthesizing N-acetyl galactose-functionalized dendrimers (synthetic multivalent frameworks) to study galectin-3/carbohydrate multivalent interactions. REU students will use surface plasmon resonance (SPR) to determine the association constants for binding of Gal-NAc functionalized dendrimers to galectin-3. They’ll tether galectin-3 to the gold chip and obtain k on and k off values using a Biacore SPR instrument.
NMR-based protein structure determination
An essential component of our NMR-based structural biology research is to clone the cDNA, express, and purify large (milligram) quantities of proteins using bacterial recombinant expression systems permitting the isotopic (non-radioactive) 15N and 13C labeling of desired proteins for NMR. Sample preparation is essential to the success of the NMR experiments, and students are trained by making their “own” protein, and become experienced with modern biochemical and analytical research methods. Subsequently, the PI teaches the students how to operate the four-channel DRX600 NMR spectrometer, how to set-up triple resonance 2D and 3D NMR experiments, and how to analyze the complex NMR spectra in order to extract NMR parameters that are needed for protein structure determination by NMR. Hyperthermophilic proteins from archaeal Sulfolobus spindle-shaped viruses are current targets.
Denitrification: Nitrous Oxide Reductase
Denitrification is an intrinsic part of the global nitrogen cycle and is the pathway that balances the cycle, returning fixed nitrogen to the atmosphere. The environmental biology of nitrogen fixation, assimilation, and denitrification substantially impacts agricultural productivity and water quality. Denitrification may release N2O to the atmosphere, thereby contributing to ozone depletion and global warming. Hence there exist clear and direct linkages between basic research on the biology and biochemistry of denitrification and numerous issues of substantial societal interest. Specifically, studies of the structure, mechanism, and metal cluster assembly in nitrous oxide reductase are proposed. In the vast majority of denitrifying organisms nitrous oxide reductase is the terminal enzyme in the denitrification pathway, and nitrous oxide reduction is a major source of ATP under oxygen-limited conditions. The proposed research will directly address questions regarding the structures and reactivities of the novel copper sites in nitrous oxide reductase, and their roles in catalysis.
Genetic Responses to Iron and Oxidative Stress
The research project will focus on probing key biochemical elements of iron and oxidative stress in the model hyperthermophilic archaeon, Sulfolobus solfataricus, from Yellowstone National Park. The mechanism of protection against oxidative stress afforded by the Dps protein from Sulfolobus solfataricus (recently identified Wiedenheft et al PNAS 2005) will be investigated. The inhibition of hydroxyl radical •OH formation by Dps in the presence of the Fenton reagents (Fe(II) + H2O2) will be monitored by EPR. Additionally, experiments will be performed to study the genetic responses in Sulfolobus solfataricus to Fe and oxidative stress. These responses will be investigated using cDNA microarray analysis and mass spectrometry-based proteomics. Using these techniques, we will begin to identify the proteins and mRNA transcripts generated in response to increasing Fe and oxidative stress (H2O2).
Tracking global changes in post-translational protein modifications
Better methods are needed to track protein post-translational modifications as a function of biological stimuli, health and disease. We have been developing and exploiting new global methods of ultra-sensitive differential analysis of the amounts of proteins in stimulated and control samples, using new multicolor fluorescent dyes synthesized in collaboration with the Grieco group. Here, we plan to modify some of the multicolor fluorescent detection dyes to react with sugar groups on glycoproteins (by adding hydrazine or boron-based chemistries) in collaboration with the Cloninger group. Complex mixtures of proteins extracted from control and experimental samples will be labeled with different colored fluorescent reagents, the labeled samples will be mixed and the proteins will be separated on 2D gels. The changes in levels of glycoproteins will be determined by laser scanning of the gels, and the regulated molecules will be isolated from the gels and studied by mass spectrometry. A similar strategy for global differential analysis of nitrosothiols in collaboration with the Singel group will also be performed.
Protein Structure-Function Relationships
The Lawrence laboratory employs X-ray crystallography and other biochemical techniques in the study of structure function relationships in three major areas. First, we are studying the structural and functional basis of iron transport, iron homeostasis and the response to reactive oxygen species. Of particular interest is the protein machinery of the transferrin cycle [(a) transferrin, b) transferrin receptor, c) Steap3, a member of a unique family of ferric reductases and d) Divalent Metal Ion Transporter 1)] as well as proteins such as TMPRSS6 that play a prominent role in regulating levels of hepcidin, the major hormone controlling systemic iron homeostasis. Second, we are involved in structural studies of crenarchaeal viral proteins. We have now determined structures for eleven of these viral proteins; in each case the structures have provided significant functional insight. Third, we are working toward general mechanisms of small molecule delivery across the blood brain barrier. We are following up on our structural studies of transferrin receptor (TfR) by identifying small molecules that bind within an interdomain pocket in this receptor. Projects appropriate for REU students are available in all three areas.
Development of new methods for organic synthesis
Our research spans topics ranging from stereocontrolled total synthesis to asymmetric catalysis and ligand design. Specific areas of current emphasis include the design and synthesis of biologically active heterocycles, asymmetric cycloadditions using chiral cobalt and rhodium catalysts, asymmetric synthesis using chiral amido and related complexes of the group(III) metals and stereocontrolled cyclization reactions initiated by stabilized carbocations and free radicals.
Synthesis of glycosyl ureas and new antibiotics
The growing emergence of antibiotic-resistant bacteria is now recognized as a global health issue. In response to this medical concern, the search for a new class of aminoglycoside antibiotics has been intensified. The research on glycosyl ureas, in which the O- and N-glycosidic bonds are replaced with the urea-glycosidic linkages, has recently emerged. We have recently developed a novel method for the stereoselective synthesis of a- and b-glycosyl ureas via palladium(II)-catalyzed rearrangement of glycal imidates. In our approach, the nature of the palladium-ligand complex controls the selectivity at C(1) position. Since the stereoselective synthesis of N-glycosyl trichloroacetamides is relatively straightforward, an REU student will broaden the substrate scope to other carbohydrate donors besides the glycal donor. Our goal is to explore the possibility of directly converting a-glycosyl trichloroacetamides into the corresponding a-glycosyl ureas.
Mechanism based inhibitors in the study of novel carbolylases
Elucidating the mechanism of two novel carboxylases using a structure-function based approach in which a proposed mechanism for the catalytic activity is derived from a variety of biochemical and structural studies is a long-range goal of this project. The carboxylases currently being investigated are involved in microbial epoxide and ketone metabolism and include a 2-ketopropyl Coenzyme M carboxylase / oxidoreductase and an acetone carbolylase. Elucidating the mechanism requires combining biochemical data with the determination (by X-ray diffraction methods) of a suite of structures representing mechanistically relevant states. We utilize the mechanistic proposals derived from our structural work to design and synthesize mechanism-based inhibitors. We then characterize the kinetics of enzyme inhibition of each inhibitor and determine co-crystal structures, the results of which in sum typically provide significant insights into the enzyme mechanism. Practical summer research experiences within the overall project include for example the design and synthesis of a proposed inhibitor or the detailed kinetic characterization of an inhibitor that has been previously synthesized.
Hydrogen-bond activation for annulation reactions
The specific aim of this project is to develop a general annulation method to form 5- and 7-membered carbocycles in a highly enantioselective manner. Despite the prevalence of 5- and 7-membered rings in a wide variety of biologically significant organic small molecules, methods for their efficient construction remain elusive. A strategically direct method for annulation is the union of an electron-poor oxyallyl cation with a 2- or 4-carbon electron-rich unsaturated component in a formal [2+3] or [4+3] sense. Recent advances (Eric Jacobsen, Harvard University) have demonstrated that chiral thiourea-containing catalysts can promote the formation of electron-poor heteroatom-stabilized carbocations through what is believed to be hydrogen-bond activation. In the present context it is hypothesized that such an activation would allow for the formation of a reactive oxyallyl cation intermediate under mild conditions, whilst controlling its subsequent cycloaddition with good selectivity.
Chemistry of NO-Hemoglobin Interactions
Oxygenation of tissues in higher organisms is regulated through modulation of blood flow via dilation of vessels in the microcirculation in response to ambient oxygen tension. The molecular mechanism by which oxygen is sensed and oxygen-tension signals are transduced to dilate these vessels is a major unanswered question. In collaboration Jonathan Stamler at Duke University Medical School, we have hypothesized that hemoglobin (Hb) in the red blood cells (RBCs) functions as the oxygen sensor, and that the effects of Hb allostery on its chemical interactions with the endogenous vasodilator NO establishes a transduction mechanism for the oxygen-responsive deployment of NO-vasodilatory activity. The pivotal chemical component in this mechanism is the S-nitroso derivative of Hb (SNO-Hb, nitrosated at the thiol of Cys-93 of the b -subunits in human Hb), whose release of NO-bioactivity is coupled to the allosteric transition undergone by Hb in its release of oxygen. Using spectroscopic (UV/Vis and EPR, primarily) methods and chemical analysis, we have identified and characterized the novel chemistry fundamental to the hypothesis, and have elucidated conditions – reflective of the physiological situation – that support such chemistry, as well as conditions that disfavor it and lead to more conventional behavior. Ultimately, we aim to develop integrated, phenomenological models that enable quantitative predictions of the outcome – product distributions and bioactivity – of NO hemoglobin interactions under various, biologically relevant conditions of reagent and effector concentrations, and thus elucidate the complex chemistry underlying RBC-mediated vasodilation.
Isolation of characterization of novel bioactive compounds
In my laboratory, we isolate and characterize many
novel bioactive compounds from endophytic microbes. These microbes have a unique association with higher plants. Some of the products that they make are novel and useful in agriculture, medicine and industry. Recently, we have discovered an endophyte that makes diesel-related hydrocarbons. Others make novel antibiotics, antioxidants, and anticancer agents. Comprehensive studies of the genetics, biochemistry, and biology of plant associated microbes are also performed in our group. Collaborative research arrangements are in place with other institutions such as BYU and Yale universities.
Virtual Chemical Models for Metalloenzymes
The aim of the research project is to develop realistic molecular models for proteins with inorganic active sites, which are implicated in electron-transfer and small molecule activation processes in biology. Stellacyanin and its variants from Cucumis sativus are particularly exciting members of the family of copper-containing proteins, since they have been in the focus of extensive experimental studies with limited computational investigations. They provide a rich experimental database for evaluation, development, and optimization of integrated computational methodologies. The REU student will develop DFT/MO/MM models for wild-type stellacyanin and apply the modeling strategy for variant proteins. Cu site models will be constructed and their structures will be optimized in gas phase and solution by DFT theory. The isolated Cu site models will be embedded into a two layered protein model, where the steric and electronic effects from a 7-10 Å protein environment will be explicitly calculated by MO theory. The latter model will be further extended to a complete protein model, where the steric interactions from the protein secondary structure will be simulated by molecular mechanical methods. The optimized Cu site structures at each level of modeling will be compared to spectroscopic data from the literature.
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