Research Experience for Undergraduates (REU) Program
Activity-Based Protein Profiling of the Viral Infection Process
Metabolic state, differentiation, or infection lead to cells with dramatically different protein profiles. Activity based protein profiling (ABPP) is a method that has been developed to address the activity level of proteins on a global scale and constitutes a new strategy for functional proteomics. ABPP uses chemically reactive small molecules that specifically target 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 proteomics technology in conjunction with ABPP, we are seeking to identify novel cellular proteins that are hijacked by viruses during the infection process. The significance of this work is two fold; the basic biology of viruses will be elucidated and new targets for antiviral agents will be identified.
Iron-Sulfur Clusters in Biological Radical Reactions
The overall objective of this project is to delineate the detailed chemical mechanism of radical generation by the Fe/S-S-adenosylmethionine (the so-called radical SAM) superfamily of enzymes. These enzymes span a remarkably diverse range of reactions and appear to be represented across the phylogenetic kingdom, with hundreds of radical SAM enzymes identified. In humans, radical SAM enzymes are involved in the biosynthesis of lipoic acid, the synthesis of heme, and the biosynthesis of the molybdopterin cofactor, among many other essential functions, some as yet unidentified. Despite the diversity of reactions catalyzed, our overriding hypothesis is that the adenosylmethionine-dependent iron-sulfur enzymes all operate by a common mechanism in which a reduced cluster interacts with S-adenosylmethionine to generate an adenosyl radical intermediate, which is directly involved in catalysis. To investigate this novel chemistry, biochemical, spectroscopic, mechanistic, and structural studies of pyruvate formate-lyase activating enzyme (PFL-AE) will be pursued. In addition we propose to explore the nature of the PFL-AE iron-sulfur cluster in whole cells.
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).
Structure-Function Characteristics of Lentivirus Proteins
Feline Immunodeficiency Virus (FIV) causes fatal disease in domestic cats and apathogenic infections in other feline species. HIV (Human), SIV (Simian), and FIV share similar life cycle characteristics and produce similar proteins during infection and replication. Of particular interest are the viral proteins Reverse Transcriptase (RT) and the envelope glycoprotein, gp120. Although the RT and gp120 literature is extensive, there are fewer studies that compare structure-function relationships between similar proteins from different species-specific lentivirus strains. We hypothesize that three-dimensional structural differences in similar proteins from different species-specific strains of lentiviruses influence the dynamics of RT-DNA and gp120-CD4/CCR4 interactions. REU students may be assigned to work on one of the three components of the investigation: (1) producing protein and determining bioactivity using techniques in molecular biology and biochemistry; (2) elucidating protein structural properties using data from Nuclear Magnetic Resonance Spectroscopy or Fluorescent Spectroscopy experiments; or, (3) comparing three-dimensional structural characteristics between samples using bioinformatics and molecular modeling software. Given that FIV strains were in part isolated from cougars indigenous to the traditional Native American hunting grounds of northwestern Montana, this research project bears a level of cultural relevance.
In addition to the FIV project, projects to study the nutrient flow in the pine forest due to mycorrhizal fungal symbiosis and to identify chemical contaminants from former clandestine methamphetamine lab sites on the Flathead Indian Reservation (by Drs. Leighton and Johnson) are also available to students wishing to perform summer research with SKC faculty.
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.
Our group focuses on solving the three-dimensional structures of large and complex proteins using recently developed, state-of-the-art solution NMR methods. Our research efforts are cross-disciplinary in nature. Most of our projects concentrate on the relationship between structure and function, and are in collaboration with other faculty at MSU. These investigators provide us with technical expertise for the biological components of the research, and permit us to interpret our NMR-based structural and dynamic data in terms of the proteins' biological functions. Thus, our research is devoted to several aspects of NMR spectroscopy and structural biology.
Amine Oxidases and Lysyl Oxidases
Copper-containing amine oxidases and lysyl oxidases are widely distributed in nature and are involved in the metabolism of biogenic primary amines, in the maturation of connective tissue, and numerous other physiological processes. The human vascular adhesion protein (HVAP) is an amine oxidase, and its oxidase activity is directly involved in cellular adhesion. Multiple “lysyl oxidase like” (LOXL) proteins have been recognized in mammals, and these proteins may substitute for lysyl oxidase, but may also have roles in multiple cellular processes, including differentiation, proliferation, and motility. A multidisciplinary approach emphasizing spectroscopy, kinetics, site directed mutagenesis, and crystallography is proposed to elucidate the chemical and biological principles that define key structure-function relationships in amine oxidases and lysyl oxidases. Major goals of this project are to define the active-site structures, catalytic mechanisms, and the molecular bases for substrate specificity and selective inhibition in amine oxidases, including HVAP and LOXLs.
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) + H 2O 2) 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 (H 2O 2).
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 four 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 are the protein/RNA interactions that regulate translation in an iron dependent manner. Second, we are involved in structural studies of crenarchaeal viral proteins. We have now determined structures for eight of these viral proteins; in each case the structures have provided significant functional insight. Third, we are in involved structural studies of antibodies that recognize the CD4 binding site of gp120. We have determined the structure of Fab F105. The structure suggests new strategies to elicit a broadly neutralizing response against HIV. Fourth, we are working towards 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 four areas.
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.
Our research focuses on the development of new synthetic methodologies to efficiently produce biologically important compounds that contain nitrogen and/or phosphorus. Our targets include nucleotides, carbohydrates, alkaloids with possible anti-tumor activity, and cyclic peptides. Our new synthetic methods include a photochemical rearrangement (alkaloids), reactions of pentacovalent phosphorus compounds, and the use of large ring conformational control in addition reactions to form cyclic peptides. We also perform high level ab initio calculations on our systems to try to explain, and eventually predict, the reactivities observed.
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.
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.
In my laboratory, we study the isolation and characterization of many novel bioactive compounds from plant pathogenic fungi and bacteria for the development of new drugs and for agricultural applications. For example, two new compounds have recently been isolated-a novel lactamic acid and achlorinated macrocyclic lactone-that are candidates for use in plant disease control. Comprehensive studies of the genetics, biochemistry, and biology of plant associated microbes are also performed in our group.
Molecular Models for Proteins with Inorganic Active Sites
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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