Research in the Bothner lab has two main focuses: (1) investigating the role of conformational change in protein function, and (2) application of chemical and systems biology approaches to biological stress response. This research takes us from the atomic scale provided by high resolution structural models of viruses and hydrogenases all the way to the identification of clinical markers for disease. A diverse set of analytical, biophysical, biochemical, and cell biology techniques are employed. The Bothner lab is part of newly established Department of Energy Center studying electron transfer and the Thermal Biology Institute.
(1) Nature has evolved active bio-architectures that have conformational plasticity
and respond to environmental signals. Proteins are naturally dynamic; therefore, knowledge
of the frequency, range, and coordination of motion in large complexes is critical
to understanding function and to the development of bio-inspired nanomaterials. Mechanistic
studies of electron transfer within protein complexes for bioenergy production are
an important area of research. Additional projects in this area include use of adeno
associated virus (AAV) in targeted gene therapy, protein cages from extremophiles,
and the mechanism of small molecule Hepatitis B antivirals.
(2) Cellular response to stress (such as traumatic injury, oxygen, viral infection) involves numerous networks and signaling pathways. We use changes in protein abundance and activity along with metabolomics to elucidate the pathways and networks that control biology. 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. The lab is also developing protein sensors and pushing the advent of real-time metabolomics to facilitate direct biological to digital conversion for personalized healthcare and disease prevention.
Radical AdoMet enzymes utilize a [4Fe-4S] cluster and S-adenosylmethionine (AdoMet) to initiate diverse radical reactions in biology. These enzymes are found in all kingdoms of life and are involved in central biochemical processes including ribonucleotide reduction, glucose metabolism, DNA repair, and cofactor biosynthesis. REU students will be exposed to the fundamentals of biochemical research, as well as to the general principles governing the chemistry of metals in biological systems. One of the potential REU projects in the Broderick lab involves probing the roles of radical AdoMet enzymes in assembly of complex metal clusters in biology. Specifically, we are exploring the roles of two radical AdoMet enzymes, HydE and HydG, and one GTPase, HydF, in assembling the organometallic cluster at the active site of the [FeFe]-hydrogenase. Last summer, REU student Christina Green expressed HydE and HydF in E. coli under different conditions, purified the expressed proteins, and analyzed the proteins using spectroscopic methods as well as elemental analysis. Her work has provided support to the idea that cysteine is the substrate of HydE, and we are currently preparing results for publication (Christina will be a co-author).
Dr. Michael Ceballos in cooperation with faculty at the Universiti Teknologi Mara (UiTM) and UNIMAS (University of Malaysia) offer several projects in biological and chemical sciences. Sample projects include: (a) studying the physical and chemical properties of traditional-use plants among the Bidayuh, Penan, and other tribal communities on the island of Borneo such as identifying, quantifying, and cataloging antioxidant, antimicrobial, and other properties of tribal-use plants in Northern Sarawak and identifying, quantifying, and cataloging different species of bamboo; (b) bioprospecting exotic bacteria and fungi for novel enzymes for use in biofuels production processes; and (c) studying interactions between coral reef species, marine bacteria, algae, and viruses. Both lab work and field work comprise these efforts.
In the Cloninger group, we are using carbohydrate-functionalized dendrimers, which are highly branched macromolecules, to study galectin-3 mediated cancer cellular processes. Galectin-3 is a galactoside-binding protein that is known to play a role in cancer cellular aggregation/tumor formation and metastasis. REU students working on this project will use lactose functionalized dendrimers in a cancer cellular migration "scratch" assay to determine whether glycodendrimers inhibit or enhance galectin-3 mediated cellular migration. This migration assay serves as an in vitro model for cancer cellular metastasis. REU students will learn tissue culture techniques, microscopy, and data analysis. As needed, and as time allows, they will also perform glycodendrimer synthesis.
We are actively developing new cascade reaction pathways to efficiently synthesize complex molecules. These new cascade reaction pathways cause complex skeletal rearrangements that define multiple stereogenic centers in a single operation. New strategies for the synthesis of complex molecules including natural products and pharmaceutical targets will emerge from this research. REU students could have projects in one of three areas as follows: (1) using multiple pericyclic processes in a cascade; (2) using a single enantioselective transition metal catalyst to perform multiple reactions; (3) using synergistic (or cooperative) catalytic cycles to perform unique yet complementary functions. Our aim is to develop truly useful methods that are general in their scope and have good functional group tolerance.
Our research program focuses on the investigations of protein structures and functions and the profiling of metabolites (metabolomics) as potential biomarkers of diseased cells. Our approach uses a variety of techniques, including nuclear magnetic resonance (NMR) and also involves also a variety of other biophysical techniques including mass spectrometry, protein biochemistry, circular dichroism, and in vitro protein assays. REU students are encouraged to engage at all levels of the research including: (i) learning molecular biology skills to clone DNA into bacterial vectors; (ii) overexpressing proteins in in vitro recombinant protein expression systems using E. coli cells; (iii) acquiring 2D and 3D NMR experiments on MSU.s 600 MHz NMR spectrometer; (iv) analyzing multidimensional heteronuclear NMR data using computer software such as Sparky, NMRPipe, and solving protein structures using programs such as CYANA, XPLOR_NIH; (v) and conducting metabolite profiling experiments with associated analyses of 1D and 2D 1H and 13C NMR spectra to identify and quantify various metabolites that may be good biomarkers of distinct cellular states. Structural Biology: Our structural biology and protein structure determination research is focused on understanding the molecular links between protein chemical structures, internal dynamics, and biochemical functions. NMR metabolomics: Metabolite levels are closely related to phenotypes, and the number of cellular metabolites is much lower than the number of genes or proteins in cells. Metabolic changes are regulated by gene expression and are widely influenced by environmental stresses. The goal of this research is to identify small-molecule markers that could be used as diagnostics of cellular responses or diseased states.
The incidence of Type 2 Diabetes (T2D) is high and is growing rapidly. The correlation with obesity is low, however, where 24% of T2Ds are normal weight and 8% of obese people have T2D. As many as half of the people with T2D may be undiagnosed and will only recognize their condition when they have developed serious side effects (loss of vision, kidney function and/or circulation in the feet that often leads to amputation). Metabolic Syndrome is a precursor condition that carries high risk of developing T2Ds. We have recently found that newly diagnosed T2Ds carry previously unknown lipid compounds on plasma albumin. We are in the process of purifying large enough amounts of these compounds for chemical identification, and we will be pursing studies of their metabolism. We have developed a spectrophotometric and antibody binding assay for the occupation of lipid binding sites on plasma albumin that appears to correlate with severity of T2D in a limited number of samples. The project that would be carried out by REU students would be to monitor the occupation of the lipid binding sites in albumin in larger numbers of plasmas from Metabolic Syndrome and T2D patients to help validate the correlation. We also work on mechanisms of Alzheimer's Disease, characterizing new food crops with improved nutritional content, and developing improved proteomic analysis method-- that could offer research opportunities, if students are interested in these areas.
Fluorescence is far and away the most important imaging method targeting problems in modern chemical and structural biology. While the high specificity achieved by targeted probe labeling and background free-detection has produced an increasingly powerful suite of fluorescence imaging tools, a variety of biologically relevant molecules such as signaling neurotransmitters, metabolites, and low molecular weight drugs are difficult to characterize with fluorescent techniques. To image such systems, we are developing label-free chemical imaging methods that are both easy to use and minimize cost. REU students involved with this project will design and construct a label-free imaging system from the ground up, developing a compact ultrafast laser and a complete home-built nonlinear microscope. Our ultimate goal is to develop a robust combination of imaging hardware and data analysis software to enable routine and bench-top stimulated Raman imaging.
Our research activities focus on microbial ecophysiology: the study of the physiology of microorganisms with respect to their habitat. We are interested in how the activity of the “uncultured majority” – the large number of microbes that evades cultivation under laboratory conditions – impacts humans and the environment on a micron to global scale. We believe that only by gaining an understanding of microbes directly in their habitats researchers will be able to elucidate the mechanisms of microbial interactions with the biotic and abiotic world. To accomplish these goals, we apply an integrative approach that bridges the two extremes of the microbial scale bar: the individual cell and the whole community.
Very broadly, the research questions we address are: (i) who is doing what (linking identity and physiology of a cells); (ii) what are the abiotic and biotic factors controlling microbial activity; (iii) how does this activity affect the environment and humans; (iv) what are the limits to microbial metabolism; and (v) how can we discover novel structures, functions, and biotechnological potential within uncharted branches of the tree of life?
In order to address previously unrecognized physiologies and cellular interactions of uncultured microbes, we employ a unique combination of single cell and meta-genomics (as hypotheses generator), bioorthogonal compound screening (to identify parameters driving microbial activity), and targeted stable isotope probing (to identify specific growth-sustaining substrates).
We currently work in three systems: geothermal springs in Yellowstone National Park, sediments from the Guaymas deep-sea basin, and a New England salt marsh. We are particularly interested in revealing the physiology, biogeochemical impact, and ecology of only very recently discovered archaea in these habitats.
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 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 seventeen of these viral proteins; in each case the structures have provided significant functional insight. Third, we are working to elucidate the structure and function of the major components of the prokaryotic adaptive immune system known as CRISPR/Cas in S solfataricus. 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, and asymmetric catalysis using chiral amide complexes of the group(III) metals.
Chemodivergent transformations are those in which two or more functional groups could feasibly react with a given reagent, but minor modifications to the specific conditions (e.g., changing the catalyst) leads to selectivity for one or the other functional group. We are interested in using ligands on transition metal catalysts to control the chemoselectivity of reactions such as cross-couplings. In particular, we aim to develop catalytic systems that can switch their chemoselectivity on the fly based on an external stimulus. REU students working in this research area could do any of the following: study the inherent chemoselectivity of transition metal-catalyzed reactions, synthesize and test novel organic ligands, and/or investigate the use of external stimuli to modify the behavior of catalysts.
The design, snthesis, and characterization of porous adsorbent materials based on carbon is the focus of our research. We use zeolites as templates, which are carbonized in their porous cavities. Lightweight, conductive porous materials are important for ion storage in electrochemical energy storage devices (such as batteries), for gaseous fuel storage, for fluid separation, and for catalysis. REU students in the Stadie group will have opportunities to synthesize and study zeolite-templated carbon materials for a variety of applications.
Solid oxide fuel cells (SOFCs) are solid-state electrochemical devices capable of converting a wide variety of fuels including methane, butane, bio-gas, and syn-gas into electricity and products. In order to produce adequate electrical power, however, SOFCs typically operate at temperatures of 700.C and higher. These conditions . high temperatures, strong redox environments and applied electric potentials . can rapidly accelerate electrode and electrolyte degradation. Participants in this project will use newly developed optical techniques to examine the kinetics and thermodynamics associated with anode degradation in functioning SOFCs. During the course of the REU project, student participants will develop expertise in surface spectroscopy, electrochemical measurements, gas handling manifolds, and high temperature materials synthesis and processing.