We view organisms as dynamic systems composed of networks that are highly interwoven and span molecular classes. We are investigating cellular response to stresses such as hemorrhagic shock, oxidative damage, and viral infection. In addition to transcriptomics, proteomics, and metabolomics approaches, activity based profiling is being used in our studies. 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. The chemical probes are synthesized in the Chemistry and Biochemistry Department at MSU as part of a Cell Signaling and Network Analysis program. The combination of ABPP and metabolomics is a novel approach to enhance systems biological studies. The significance of this work is twofold; fundamental aspects of biochemistry are being elucidated, and biomarkers are being tested.

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).

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).

One of the major "bottlenecks" in bioethanol and biodiesel production is the deconstruction of potential high yield feedstocks that do not compete with food crops. Attempts to construct "artificial cellulosomes" that mimic the natural biomolecular complexes with which certain bacteria and fungi are endowed have not been successful in producing a high efficiency feedstock-to-fuel process that can make bioethanol cost-competitive with fossil fuels. Other attempts at enzyme-mediated feedstock deconstruction also suffer from the effects of feedstock pre-treatment (i.e., acid washes, high-temperature steam explosion), which tend to reduce enzymatic efficiency or completely denature the enzymes (e.g., cellulases). Recently, a group at NASA Ames Research Center constructed a first-generation enzyme sequestration platform (ESP) developed from a hyperthermophilic archaeal chaperonin system. In initial trials with Avicel®, this platform system showed a ~2-fold increase in hydrolytic efficiency. Part of this project was transferred to the Ceballos Lab (UM NARL) for further development. REU students working with Ceballos will learn how to construct fusion proteins, develop "clones", express and purify proteins, and perform assays that assess complex carbohydrate reduction efficiency. They will be trained on FPLC, HPLC, and other state-of-the-art instrumentation. The goal of the project is to determine the most efficient enzyme complements to use with the ESP to achieve maximal deconstruction efficiency on various feedstocks.

Dr. Chattipakorn (MD/PhD), the Director of the Cardiac Electrophysiology Research and Training Center in the Department of Physiology at Chiang Mai University in Thailand, has an established research program in cardiac electrophysiology that can easily welcome to REU students per summer. Projects such as the following study of mitochondria are available, as are other projects in the CERT center including projects that focus on intracellular functions of neuronal cells. The function of cardiac mitochondria is significantly altered when stresses such as heart attack and oxidative stress occur. Cardiac mitochondria have been isolated and will be studied under various stress conditions, and the protective effects of several herbs and medicinal agents on the mitochondria will be studied by REU students.

The widespread use of antibacterial agents has had a dramatic beneficial effect on human health in the last century. However, since pathogens are increasingly resistant to current antibacterial agents, new antibacterials are urgently needed. We have developed a dendrimer with quaternary ammonium terminal groups that are capped by a long alkyl chain. Although the mechanism of action of these compounds against bacteria isn't fully understood, they do have nanomolar EC50 values with E. coli. Moreover, E. coli are unable to develop resistance to the new dendrimers. This project was developed by an REU student. In future years, REU students will determine the EC50 values for antibacterial dendrimers against additional gram negative and gram positive strains of bacteria.

Our studies of protein structures and metabolomics biomedical research uses a variety of techniques including nuclear magnetic resonance (NMR) mass spectrometry, protein biochemistry, circular dichroism, and in vitro protein assays. REU students will be encouraged to engage at all levels, including learning molecular biology skills to clone cDNA and overexpress proteins in in vitro recombinant protein expression systems, running 2D and 3D NMR experiments, analyzing multidimensional heteronuclear NMR data, and conducting metabolite profiling experiments with the associated chenomxTM 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. Proteins for our structure/function studies include: (1) crenarchaeal viral proteins from Sulfolobus spindle shaped viruses; (2) proteins involved in heme acquisition and Staphylococcus aureus pathogen growth and survival; (3) leaf senescence and nitrogen remobilization in plants; (4) protein dynamics studies of functionally altered mutants of the tryptophan repressor protein (TrpR). Projects studying metabolic changes, which are regulated by gene expression and are widely influenced by environmental stresses, include: (1) metabolic profiling of microbial biofilms involved in chronic wounds; (2) profiling of metabolites in Sulfolobus strains that are resistant or very susceptible to crenarchaeal viral infections compared to wild-type organisms; (3) Metabolite profiling of E. coli strains that have been engineered via gene knock-outs followed by adaptive evolution.

Protein shells that sequester enzymatic reactions are found in diverse organisms and may provide blueprints for design of functional biomaterials. An ongoing funded project in the lab involves the design and development of a new class of bio-inspired materials utilizing the directed confinement of enzymes within viral protein cage assemblies. While the encapsulated enzymes retain their native catalytic activity the protein cage can be separately optimized as a container that can shield the enzymatic cargo from its environment, enhance stability, and modulate enzymatic activity. The bioactive materials that will result from this research have applications in energy conversion and medicine and will allow biocatalysts to be used in contexts very different from their evolved cellular role. One project that is suitable for REU students utilizes the bacteriophage P22 capsid, which uses a scaffold protein (SP) to direct the assembly of its coat protein (CP) into icosahedral capsids. By creating a genetic fusion of a desired cargo protein with a modified SP, we have demonstrated the co-assembly of SP-fusions and CP into a stable "nano-reactor" in which the cargo is sequestered inside the engineered capsid. These functionalized capsids self-assemble when expressed in E. coli and encapsulate up to 300 copies of the SP-fusion protein within the capsid. Using this approach, the tools of molecular biology and genetic engineering can be used to harness a wealth of biological catalysts, packaged into stabilizing nano-reactors.

The incidence of Type 2 Diabetes (T2D) is high and is growing rapidly. The correlation with obesity is low, where 22% 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.

Cellular DNA absorbs UV sunlight, strongly giving rise to excited electronic states that occasionally decay to mutagenic photoproducts. These photoproducts can lead to skin cancer and are responsible for the familiar tanning response of skin exposed to UV light. The most abundant photoproducts are the cyclobutane pyrimidine dimers (CPDs) formed when two consecutive pyrimidine bases are covalently joined. Our femtosecond laser experiments indicate that thymine-thymine dimers are formed more rapidly than the motions that can alter the local DNA conformation. This suggests that CPDs form when adjacent pyrimidine bases are favorably aligned for reaction at the instant of photoexcitation. DNA-binding proteins that can strongly bend or distort DNA are hypothesized to strongly modulate the probability of CPD formation. In this project, REU students will use gel electrophoresis and two-dimensional fluorescence imaging techniques to determine CPD yields site-specifically along nucleosomal DNAs of known sequence. In eukaryotes, DNA is compacted into chromatin in the cell nucleus. The elementary building block of chromatin is the nucleosome, which consists of ~147 base pairs of DNA, corresponding to 1.75 superhelical turns, wrapped around a protein "ball". By UV irradiating the nucleosomes for varying lengths of time, it will be possible to study dimer formation kinetics. Our ultimate goal is to obtain a fully molecular description of DNA photodamage by comparing results from time-resolved laser spectroscopy, molecular dynamics simulations, and site-specific measurements of photoproduct yields in DNA-protein complexes.

Acetylcholine (ACh) is a neurotransmitter implicated in complex behaviors such as learning, memory, and locomotion. We have previously shown that neurochemically distinct GABAergic subtypes respond to cholinergic modulation in a cell type-specific manner. However, it is not known whether GABAergic neurons that are strongly excited by ACh are specifically targeted by cholinergic afferents. Using existing 3D confocal stacks of fluorescently labeled GABAergic cell types and an antibody to the vesicular acetylcholine transporter, we will examine the spatial distribution of cholinergic terminals with respect to the cell surface of neurochemically identified GABAergic cell types. This project is well suited to undergraduate researchers because it entails image analysis using existing code written in ImageJ and Matlab, is at the interface of projects that involve both neuroscientists and mathematical modelers, sufficient population data is already available, and students gain valuable experience in data analysis by exploring a vast parameter space obtained from the confocal images. The results obtained will provide insight into the role of ACh in control of specific neuronal circuits.

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, asymmetric synthesis using chiral amido and related complexes of the group(III) metals and stereocontrolled cyclization reactions initiated by stabilized carbocations and free radicals.

We are examining biochemical mechanisms involved in the production of hydrogen and reduced carbon by photosynthetic algae and cyanobacteria. We are using molecular biological, biochemical, and biomimetic approaches to understand the biochemical mechanisms, patterns of gene expression, and factors important for enzyme stability to generate superior biotechnological solutions for the production of biofuels. Algal and cyanobacterial mixed cultures are engineered to maximize biofuel production using site-directed mutagenesis, deletion mutant analysis, global transcriptional analysis, and bioprospecting in extreme environments such as the thermal environments in Yellowstone National Park in Wyoming and the Great Salt Lake in Utah. In a second project, we are examining the structure and mechanism of several novel carboxylating-enzymes. Key carboxylating enzymes are involved in pathways for the metabolism of molecules such as propylene, isopropanol, and acetone that are produced in common anthropogenic activities and in the chemical industry. Recently, we have also begun to examine novel carboxylating enzymes involved in the 3-hydroxypropionate pathway and modified versions of the citric acid cycle that are present in anoxygenic phototrophs and members of the archaea. These studies involve coupling detailed biochemical studies with structural work using x-ray diffraction methods to study the enzyme with bound substrates, products, and inhibitors to elucidate the structural basis for catalysis.

Our research program has four primary components: (1) the synthesis of biologically active natural products and natural product derivatives for potential use as therapeutic agents; (2) the development of novel synthetic methodologies, with an emphasis on catalysis and catalytic-enantioselective reactions; (3) collaborative testing of natural products and derivatives thereof to study biological activity and mode of action; and (4) elucidation of organic and organometallic reaction mechanisms by experimental and computational methods. Two biologically active natural products we are currently investigating are the cytotoxic alkaloid acutumine and welwistatin, an alkaloid with significant potential as a multi-drug resistant (MDR)-reversing therapeutic. We select synthetic targets on the combined basis of interesting biological activity (potency, unique mode of action) and structural complexity. In the course of our studies we attempt to develop general methods for complex-molecule synthesis which are more broadly applicable; typically, these encompass transition-metal and organic small-molecule catalyzed reactions. Other projects currently ongoing in the group are focused on the use of electron-deficient, -acidic metals to promote new modes of chemical reactivity, as well as the use of chiral organic catalysts capable of hydrogen-bond activation in catalytic-enantioselective synthesis. Fundamental to reaction development is a thorough understanding of the operable reaction mechanism; in the area of catalysis we make frequent recourse to experimental (e.g. kinetics, activation parameters, by-product formation) and computational (DFT and ab initio quantum mechanics) methods.

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 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.

Cell membranes keep essential genetic material protected from extra-cellular threats such as variations in pH, osmotic pressure and pathogens. Biochemical studies provide information about membrane composition in different organisms, and structure-function relationships suggest specific roles played by different membrane components in regulating membrane transport and flexibility. Missing from our understanding of membrane properties is quantitative, predictive data about how different membrane components organize and interact cooperatively to provide membranes with the functionality to maintain viable life processes. Members of the Walker Research Group use a suite of thermodynamic and spectroscopic methods to examine molecular structure and organization in model membranes monolayers and bilayers. The stability and miscibility of these membranes are tested as functions of membrane composition and of exposure to different analytes. These analytes include simple surfactants such as those often used to penetrate and break apart cell membranes of potentially hostile microbes.