2011 Summer Research Projects
Organic Chemistry: Gold-catalyzed Rearrangement of N-Propargyloxypyridines
Carolyn Anderson and Nate Romero
Organic synthesis is a powerful technique that allows access to a wide range of different structural motifs. In this project, we are working to advance a method for the synthesis of N- substituted pyridones; an interesting functional group found in a series of pharmacologically interesting compounds. To date, we have developed a new gold-catalyzed method for accessing this motif by rearranging a related system. The student working in this area will be responsible for preparing and testing additional substrates in the rearrangement and for optimizing this reaction to give the desired products. The student will gain experience with synthetic organic chemistry techniques, including: running reactions, purification, organic spectroscopy, and experimental design.
Organic Chemistry: Mechanism of Benzyloxypyridines to N-Benzyl Pyridone Rearrangement
Carolyn Anderson and Nick Vryhof
In this project, we are working to elucidate the mechanism of an oxygen to nitrogen rearrangement that was discovered in the Anderson labs for the synthesis of N-alkyl pyridones – a functional group that plays an important role in a series of pharmacologically interesting compounds. To date, we have developed an experimental method for accessing these compounds, however, we continue to struggle with exactly how these transformations occur. The student working in this area will be trying to elucidate the mechanism using carefully designed experiments and computational studies. The student will gain experience with techniques, including: running reactions, purification, organic spectroscopy, reaction kinetics, experimental design, and computational methods.
Organic Chemistry: Synthesis of N-Alkyl Pyridone Containing β- and γ- Amino Acids
Carolyn Anderson, Stephen Carnegis and Mitchell Groenenboom
Organic synthesis is a powerful technique that allows access to a wide range of different structural motifs. In this project, we are working to develop a method for the synthesis of N-alkyl pyridone containing β- and γ-amino acids – homologues of the natural amino acids, which contain an interesting functional group found in a series of pharmacologically active compounds. To date, we have discovered an important intermediate in route to these species and have begun to optimize its synthesis. The students working in this area will continue to seek conditions for the preparation of this intermediate and its conversion into the desired amino acids. The students will gain experience with synthetic organic chemistry techniques, including: running reactions, purification, organic spectroscopy, and experimental design.
Characterization of Splice Variants from Zebrafish
Eric Arnoys and Eric Prins
We will finish the initial characterization of the expression, cellular localization, and physical properties of three splicing variants of a potential splicing factor from zebrafish. The student will quantitate mRNA levels from adult tissue, examine expression of fluorescently tagged proteins in cell culture, and purify recombinant proteins for binding studies.
Ligand Binding of Mutant Proteins
Eric Arnoys and Kevin Harris
We will examine the physical properties of mutant proteins in vivo and in vitro. The student will purify the proteins with affinity chromatography and then perform competition studies of binding to immobilized ligands. Thermal stability +/- ligand will be examined with DSC. The student will use affinity co-precipitations to compare binding to known protein partners. We also hope to begin examination of protein stability with fluorine NMR.
Watching Membrane Proteins in Real Time
Eric Arnoys and Jacob Artz
We will characterize the behavior of several membrane-bound proteins in living cells with state-or-the-art techniques to examine their cellular localization, mobility, and interactions with other proteins. Protein targets will be tagged with flouorescent proteins so that they can be viewed in living systems. We will also examine what effect extracellular signals have on the protein's behavior.
Expanding the Scope of Tyrosine-Cysteine Crosslinked Proteins in Biology
Dave Benson, Anand Divakaran, Brandon Burkhart and Elizabeth Porter
Proteins that contain a covalent bond between a set of tyrosine and cysteine residues could be much more wide-spread than currently thought. We are studying one protein known to contain a tyrosine-cysteine crosslink that provides the first committed step in mammalian cysteine metabolism (cysteine dioxygenase). Additional proteins that could but are not currently known to contain tyrosine-cysteine crosslinks are being examined. Included in these target proteins are mammalian hemoglobins, an E2 ubiquitin ligase (UBE 2G2), and an orphaned protein from an obligate anaerobe (BF 4112). A variety of oxidation chemistries will be performed with these proteins followed by proteomic analysis. The proteomic analysis will use our newly acquired MALDI-TOF mass spectrometer and 500 MHz NMR spectrometer to determine when and the extent to which a tyrosine-cysteine crosslink has formed.
Identifying Oxidizable Tyrosines
Dave Benson and Ryan Martinie
Tyrosine oxidation is used in a positive (water oxidation in photosythesis) and negative (protein oxidation and destruction) in biology. We are collaborating with Roger DeKock to identify tyrosine residues with the correct geometric constraints to more readily be oxidized. While in the DeKock lab, this student will determine the how the xyz position of an aspartate (conjugate base) oxygen to a tyrosine phenolic oxygen affects the reduction potential of the tyrosyl radical (oxidized tyrosine). xyz positions with reduction potentials that provide tyrosine oxidation at neutral pH will be searched for in the protein databank in the Benson lab. Any candidate proteins will be expressed and examined in the Benson lab.
Formation of Tyrosine-Cysteine Crosslinks
Roger DeKock and Ryan Martinie
This research will investigate the ability of tyrosine and cysteine sidechains of proteins to crosslink through an arylalkylthioether bond (abbreviated as Tyr-Cys). Tyr-Cys crosslinks participate in oxidation catalysis and are formed by two proton-coupled oxidations. Metal-oxygen chemistry and the correct sidechain geometries are necessary for Tyr-Cys crosslink formation. The experimental aspects of this research will occur in the Benson laboratory. The research on the DeKock side of the project will focus on employing computational chemistry to examine the reaction energetics and mechanism.
Cooperativity in Hydrogen Bonding
Roger DeKock and John Strikwerda
For several years we have been completing theoretical studies that relate to cooperativity in hydrogen bonding. For example, we examine molecular cubes with the formula NH3(H2O)7. We believe that these small cubes can serve as prototypes for cooperativity in hydrogen bonded networks in real chemical systems, such as ammonia, NH3, in water. We have completed similar studies on the thirty nine cubes of HF(H2O)7. Additional studies on these systems need to be completed. Those additional studies would be the focus of the summer, 2011 work.
Understanding the motives and barriers to two-way faculty/student feedback in science, math, and engineering
Herb Fynewever, Isaac Bokma and Luke Breems
Usually when students are learning science, math, and engineering they reveal their thinking to the instructor and the instructor uses this information to give feedback to the students. Often, however, this two-way communication is limited to formal assessments such as quizzes and exams, which happen after most of the learning has happened. Research has shown that there are many effective ways to better integrate this communication into the learning process (e.g. interactive classroom delivery and activity) realizing significant gains in student learning. Further research is needed to determine what the barriers are to two-way communication and to devise strategies to remedy these barriers. In this research, students will analyze data from classroom observations and interviews with faculty and students to detect and classify these barriers.
Activation of Glucose Uptake by Cell Stress in Muscle and Fat Cells
Larry Louters and Jared Scripture
A family of membrane imbedded proteins, known as GluT, transport glucose into cells. It has been shown that one member of this family, GluT1, can increase its transport activity very quickly when cells are stressed. However, it is not known if this transporter is activated by stress in cells, such as muscle and fat, which also contain GluT4. This project will measure glucose uptake in muscle and fat tissue culture cells when the cells are stimulated by both insulin and cell stress to see which fraction of uptake in each case can be attributed to transport via GluT1.
Effects of Berberine on Glucose Uptake
Larry Louters and David Kuipers
Berberine is an alkaloid isolated from several herbs and has a long history of use in Chinese medicine. Recently it has been shown that berberine is effective in the treatment of type 2 diabetes. We will investigate the effects of berberine on glucose uptake in tissue culture cells that are either insulin-sensitive or insensitive. This should help us determine if berberine modulates the activity of the GluT1 or GluT4 transporter.
Effects of Membrane Cholesterol on the Transport Activity of GluT1
Larry Louters and Daniel Oram
We will both increase and decrease the content of cholesterol in the cell membrane to see if that alters either basal level glucose uptake or the ability to activate glucose uptake by cell stress. The content of cholesterol will be altered using cholesterol binding drugs.
Cloning PCP Monooxygenase from Mycobacterium Chlorophenolicum
Darla McCarthy and Anna Casto
Pentachlorophenol (PCP) is a fungicide that was used extensively in the past as a wood preservative. Due to overuse and poor disposal practices, PCP is now considered one of the most potent and persistent environmental pollutants. Interestingly, some soil microbes have evolved the ability to metabolize PCP, obtaining energy and carbon for growth in the process. The first step in metabolism of PCP by Mycobacterium chlorophenolicum, a gram-positive bacterium, is catalyzed by an enzyme called PCP monooxygenase, which has been shown to be a member of the cytochrome P450 family of enzymes. We are using degenerate PCR to amplify cytochrome P450 gene(s) from M. chlorophenolicum, with the goal of identifying, cloning, and expressing the protein so that we can better understand its function.
Lasers in Chemistry
Mark Muyskens, Benjamin Markosky and Eric Yu
The summer project will apply laser techniques to the study of several possible chemical systems. The techniques are cavity ring down spectroscopy and ultraviolet laser photochemistry; the systems are liquid-phase fluorescent dyes in solvents and gas-phase fluorinated acetylacetones focusing on the photoelimination process. The work may also involve using computational chemistry to study related systems that complement the experimental work.
Characterizing the Binding Interaction of Insulin with G-Quadruplex DNA: From Single Molecule to Bulk Measurements
Kumar Sinniah and Christine Timmer
G-quadruplexes are noncanonical DNA structures formed from guanine-rich DNA sequences in the presence of monovalent cations such as potassium or sodium ions. These structures are of significant interest due to their possible role in biological processes. Since the human genome contains hundreds of thousands of sequences that have the potential to form DNA quadruplexes, proteins that bind to G-quadruplex DNA may provide a clue to the role of G-quadruplex DNA in biology. This summer we will examine in detail the interactions between the protein insulin and G-quadruplex DNA. Insulin is known to bind with high selectivity to G-quadruplex DNA that contains the sequence of the insulin-linked polymorphic region (ILPR) of the human insulin promoter region. We plan to quantify the strength and specificity of the insulin-quadruplex DNA interaction. The biophysical properties of G-quadruplex and the quadruplex binding proteins such as insulin are essential for understanding the biology of these important biomolecular interactions.
Characterizing Riboflavin Conjugated Nanoparticles for Targeted Drug Delivery in Cancer Therapy
Kumar Sinniah and Amanda Witte
The past summer, we developed a screening protocol for riboflavin mimics for their potential binding activity towards chicken riboflavin binding protein (RfBP). By using two complementary and label-free techniques, we obtained binding constants for a number of riboflavin analogs. The small molecule riboflavin analogs are a novel class of dual acting riboflavin antagonists that target the riboflavin receptor for cellular uptake and display multifunctional activities upon cellular entry. This summer, we plan to characterize three riboflavin conjugated nanoparticles that have been used by our collaborators (a biomedical science team) at the University of Michigan for targeted drug delivery to cancer cells in-vitro. Their work suggests a general route for the selective delivery of anticancer drug molecules to the cancer cells that overexpress riboflavin receptors. Our studies will focus on the riboflavin conjugated nanoparticle binding to RfBP. Specific questions we plan to address are: monovalent binding, steric effects relative to riboflavin and clustering of the RfBPs. This project is a joint collaboration between Calvin College and the Biomedical Sciences program at the University of Michigan.
Studies into the Role of Noncovalent Interactions on Redox Potential and Aggregation
Chad Tatko, Ian Robertson and Gillian Morris
This project will explore the role that aromatic interaction play in perturbing noncovalent interactions. Principle activities will include the synthesis, characterization and analysis of peptides. Analysis will predominantly include potientiometric measurements, NMR, fluorescence and UV/Vis.
Protofibrils: Nucleation, Structure and Therapeutic Binding
Chad Tatko, Ian Robertson and Gillian Morris
Students are exploring model peptides (typically 2/3 stranded beta-hairpins) for the role of oxidative damage as a means for protofibril nucleation. By taking advantage of L-Dopa type residues covalent strategies for macrocyclization are being explored. Additionally, a protofibril replica is being used to determine the role of aromatic networks in thermodynamic stability and Alzheimer's therapeutic binding.
NMR Investigations Into the Nature of Tyr-Cys Crosslinks
Chad Tatko and Alexandra Batt
This project will use multidimensional NMR techniques to explore the formation and occurrence of Tyr-Cys crosslinks in protein structures. This position will work closely with the Benson lab to effect the expression of protein material and purify it to a sufficient quality for NMR investigations.
Exploring the Genetic/Protein Diversity in Brackish and Estuary Flows
Chad Tatko and Rachael Machiele
The essence of this project has been to explore the hypothesis that anthropomorphic changes to the natural waterflow in the Southern Florida watershed has resulted in a decline in genetic diversity of the biotic communities. While this has been studied in avian models there does not seem to be any investigations into the microscopic genetic diversity.
Nanomolecular Building Projects
Doug VanderGriend, Emily Golz, Shelby Lofthus and Liz Vincent
Understanding and controlling the synthesis of supramolecules is a key goal of nanotechnology. The students working on this project will use UV-vis spectroscopy and mass spectrometry to investigate the solution chemistry of transition metal cations, which typically bond to 4-6 ligand molecules in various geometries. These different building blocks can be used to make nanocontainers, nanomachines, and thermochromic solutions. A significant element of the research involves collaboration with a graduate lab at Indiana University, and with an industrial lab at Pleotint, LLC.
I anticipate that the students will not only get ample hands on experience with making up solutions and mastering several analytical devices, but also have a chance to interact in a collaborative effort with other research labs. It is ideal for first or second year students.