Current Trainees

 
Nicole Chumbler (Mentor: Borden Lacy,Microbiology and Immunology)

We have developed a high throughput screen for small molecule inhibitors of Toxin B (TcdB) from the gram-positive bacterium, Clostridium difficile.  This screen is luminescent, using ATP as a metabolic indicator to measure cell death.  We are currently screening a 160,000 compound library available through the Vanderbilt High Throughput Screening facility.  To date, we have screened 16,000 compounds and identified 176 hits with two common chemical scaffolds. 

We are also investigating the mechanism of the cysteine protease domain in these toxins.  In the field, it is accepted that cysteine protease autoprocessing is required for toxin activity.  We have interesting toxicity assay data that suggests the current dogma is not as straight forward as accepted.  We are investigating this data further with more toxicity and cell rounding assays. 

Additionally, we are investigating the differences in autoprocessing efficiency and reducing agent requirements between TcdA and TcdB.  TcdA requires 100 times more reducing agent and 1000 times more InsP6 than TcdB in in vitro cleavage assays.  We are continuing to follow up this observation with mutagenesis experiments to find a disulfide bond in TcdA.

 
William Birmingham (Mentor: Brian Bachmann, Biochemistry)

I am working with Bacillus cereus phosphopentomutase (PPM), an enzyme that naturally converts ribose-5-phosphate to ribose-1-phosphate in the biosynthesis of nucleosides.  We are evolving the enzyme to increase activity on a non-natural substrate, dideoxyribose-5-phosphate, to use in a biosynthetic pathway for production of dideoxyinosine, an anti-retroviral drug. 

The crystal structures of B. cereus PPM that have been determined by a collaborating laboratory at Vanderbilt University have been used to identify multiple residues to be targeted for saturation mutagenesis.  Mutant libraries for site directed saturation mutagenesis are currently being created and tested with biochemical assays to identify any improved variants.  Results from these experiments will provide details on the molecular interactions between the substrate and the enzyme.  Error prone PCR will also be utilized following saturation mutagenesis to introduce random mutations in the gene to further promote activity on the non-natural substrate. 

An assay for medium-throughput screening of PPM mutant libraries has already been developed and will significantly reduce the amount of time required to screen the PPM variants, providing a faster method for identifying an improved mutant enzyme.

 
Megan Wadington (Mentor: Richard Armstrong, Biochemistry)

The glutathione transferase (GST) superfamily of proteins plays an important role in the cellular detoxication of endogenous and xenobiotic compounds. However, proteins in the GST family exhibit considerable diversification with regard to chemistry, activity and substrate specificity. Escherichia coli K-12 encodes nine glutathione transferase paralogues, but only two of the gene products (SspA and Gst) have been assigned a function based on experimental evidence. Megan Wadington is currently working on a project that aims at describing both functionally and structurally the other seven GST paralogues (YliJ, YncG, YfcG, YfcF, YghU, YibF and the membrane bound YecN). To this end, she has employed a variety of approaches with the aim of elucidating the function(s) of the proteins in this important superfamily. These approaches include analysis of each paralogues, (1) genome context, (2) functional and structural properties, (3) phenotypic response in knock out strains and (4) interaction with protein partners.  Specifically, her investigation focuses on two E. coli GST paralogues, YghU and YfcG.

Megan Wadington Publications
Wadington, M. C.; Ladner, J. E.; Stourman, N. V.; Harp, J. M.; Armstrong, R. N., Analysis of the Structure and Function of YfcG from Escherichia coli Reveals an Efficient and Unique Disulfide Bond Reductase. Biochemistry 2009, 48 (28), 6559-6561.

 

Dawn Mackley (Mentor: Jeffrey Johnston, Chemistry)

Currently in the Johnston group I am working on the developing and application of a novel amide bond forming reaction involving an umpolung coupling of α-bromo nitroalkanes and activated amines. While traditional amide bond couplings rely on the condensation of activated carboxylic acids and amines, this reaction employs α-bromo nitroalkanes as nucleophilic carboxylic acid surrogates, coupling them to electrophilic N-haloamines.  One of the major advantages presented by this reaction is that there is no opportunity for epimerization at the α-carbon, a problem common in condensative peptide coupling. 

Previously, I have worked on developing a method for the enantioselective synthesis of phenyl glycine amino acids, through the enantioselective aza-Henry reaction of bromonitromethane and Boc-protected aryl imines.  The resulting α-bromonitro adduct can then be coupled to a variety of amines, including amino acids and peptides, with conservation of stereochemistry at the α-carbon of the phenyl glycine.  In the past year, I have developed a novel, enantioselective addition of bromonitromethane to N-TMS-protected imines.  The resulting adducts can then be trapped with a wide variety of acid chloride or chloroformate reagents, allowing for the incorporation of a number of different protecting groups into the α-bromo nitro phenylglycine surrogates.  I have extended the scope of protecting groups which can be used in this chemistry to include those which are commonly used in peptide synthesis, such as the FMOC, Cbz, and Bz protecting groups, among others.  I am currently working to extend the scope of this reaction in regards to the type of arylglycines that can be synthesized using this method.  In the future, I hope to be able to use this reaction in the total synthesis of an arylglycine-rich natural product.

 
Kellen Harkness (Mentor: David Cliffel, Chemistry)

In the past year, I have continued to develop the use of ion mobility-mass spectrometry (IM-MS) for the characterization of thiolate ligands conjugated to gold nanoparticles (AuNPs). These AuNPs are being utilized as a supramolecular tool for use in vivo and ex vivo due to their ease of control, versatile surface chemistry, and biocompatible nature. Understanding the results of experiments with AuNPs in biological systems requires a thorough understanding of the AuNP surface, an understanding which has been elusive for years due to complications with spectroscopic techniques for these unique nanomaterials. The technique I am developing circumvents these complications through the use of fragmentation and mass analysis. This approach yields important information regarding the surface chemistry of the AuNP, as validated for nanoparticles with surface properties ranging from hydrophobic to hydrophilic, including those which evidence microphase separation. The relative abundances of ligands have been measured for these AuNPs with good accuracy, proving to be superior to current spectroscopic methods in many cases. The use of the strategy which I have developed has already proven instrumental in several manuscripts which are currently in preparation or under consideration for publication.

 
Adam Ketron (Mentor: Neil Osheroff, Biochemistry)

DNA topoisomerases are ubiquitously expressed essential enzymes that are responsible for modulating DNA topology and resolving such issues as DNA over- and underwinding, knotting, and catenation. Specifically, type II topoisomerase (topo II) acts by passing a double-stranded segment of DNA through a transient double-stranded break generated by the enzyme in a separate DNA segment. An important intermediate in this catalytic cycle is the topo II-DNA cleavage complex in which the newly generated termini of the cleaved DNA are covalently linked to the enzyme. Despite being critical for topo II activity, this complex represents a potential danger to the cell because collision with nucleic acid tracking machinery results in a permanent double-stranded break. Under normal conditions, equilibrium levels of cleavage complexes are tolerated by cells. However, in the presence of compounds known as topo II poisons, enzyme-mediated DNA cleavage is stabilized, leading to an accumulation of genetic damage. Many topo II poisons are effective anticancer agents. My current research seeks to investigate the interaction between the DNA intercalative topo II poison amsacrine and the enzyme-DNA complex. I have determined several relationships between the molecular structure of amsacrine, topo II poison activity, and DNA intercalation. Future work will include more extensive study of amsacrine-stabilized topo II-mediated DNA cleavage, as well as a mechanistic study of topo II-mediated DNA ligation in the absence of cleavage.

 
Andrew Morin (Mentor: Jens Meiler, Chemistry)

Vancomycin is a small-molecule beta-lactam glycopeptide antibiotic which binds and sequesters the free D-Alanine-D-Alanine C-terminus of a key gram-positive bacterial cell wall component, thereby inhibiting proper cell wall biosynthesis and consequently rendering the bacteria vulnerable to osmotic lysis. Although vancomycin is often considered an "antibiotic of last resort", whose use is highly controlled, bacterial resistance to vancomycin and other beta-lactam antibiotics has already become widespread. While a small number of next-generation antibiotics capable of treating these resistant strains are either currently available or in the development pipeline, the pace of new therapeutic development over the last several decades, and into the foreseeable future, cannot not keeping pace with the rate of emergence of resistant pathogens. The most common mechanism of acquired resistance observed in pathogenic bacterial strains is through the substitution of a -D-Lactate in place of the -D-Ala at the free C-terminus of the bacterial peptide. This single replacement of the C-terminal amino linkage by an ester linkage of the lactate, destroying a single hydrogen bond between vancomycin and the peptide, is enough to destroy binding of vancomycin to its bacterial peptide ligand and render the bacteria resistant. The objective of Andrew's research is to develop and validate computational methods for designing novel protein therapeutics. Because the molecular and structural bases for both vancomycin binding and resistance have previously been well classified, the re-design of a protein-binding pocket to bind the resistant D-Ala-D-Lac peptide motif seems an ideal proof-of-concept experiment.

 
Sean DeGuire (Mentor: Gary Sulikowski, Chemistry)

I am currently working on the total synthesis of a natural bicyclo[1.1.0]butane (1)derivative of lenolenic acid.  The acid was isolated as the methyl ester in studies on the function of an unusual catalase-like enzyme found in cyanobacteria Anabanae sp. strain PCC 7120. It is thought that this molecule may be an unstable product of the enzyme that under normal cellular conditions is degraded into a variety of fatty acid derivatives of unknown function in the bacterium. 

This compound is the only natural product isolated to date to contain a biclcyclo[1.1.0]butane. The instability of the structure of the compound from both bicyclobutane as well as the vinyl epoxide (which must be formed stereoselectively) makes the synthesis particularly difficult. While previous work on bicyclobutane structures has demonstrated the ability to have substitution at any point on the rings, no bicyclobutane has been synthesized that contains nearly as large or as functionalized substituents as this natural product. Synthesis of this molecule will also aid in studies to determine the role that this molecule plays in the cyanobacterium; theoretically it is an unstable precursor to a variety of fatty acid derivatives. Analysis of the degradation of the molecule in water may provide insight into the mechanism of formation of the variety of diols, triols, epoxides and cyclopropane compounds that have been isolated as secondary products of the catalase-like enzyme.

I have demonstrated a reaction to form a bicyclo[1.1.0]butane ring in a model system that should be applicable to the synthesis of the compound as well as its methyl ester and am currently working on conditions to apply this model reaction in a cascade reaction to achieve the synthesis of the natural product in both racemic and enantioselective syntheses. 

I am also beginning work on studying the mechanism of action of the potent selective apoptosis inducer, Apoptolidin A by the development of natural product based fluorescent and photoaffinity probes.  Identification of a cellular target of this compound and characterization of its mechanism for selective cytotoxicity could lead to the development of new cancer therapeutics. 

 
Rachel Crowder (Mentor:  Walter Chazin, Biochemistry)

Maintenance of the cellular proteome is defined by processes that 1) refold proteins or 2) discard proteins that are beyond repair or are no longer necessary for cell function.  The first function is facilitated via chaperone proteins, while the latter is often mediated through ubiquitination—a process in which proteins are tagged with chains of the 8.5 kD protein, ubiquitin, and targeted for destruction by the 26S proteasome.  Proper protein-triage decisions are of utmost importance, and malfunctions in either process can lead to disease, including many neurodegenerative diseases and some cancers. 

The ultimate goal of my research is to understand the triage process mediated through a central co-chaperone/E3 ubiquitin ligase—CHIP.  More specifically, I am interested in studying the biochemical and biophysical properties of the transient complex that forms during the transfer of ubiquitin from an E2 conjugating enzyme to a targeted substrate bound to CHIP.  Understanding the structural and chemical interactions of the this complex will give further insight into the properties that regulate the transfer of ubiquitin to a specific substrate that subsequently regulate the turnover of the cell proteome.  This information is essential not only to facilitate the understanding of the preservation of the cell proteome, but also in recognizing the molecular basis for systematic malfunctions that lead to the development of many different neurodegenerative disorders and cancers.  Although work on this project has only recently begun, preliminary preparations show promise for future experimentation.

 
Joseph Manna (Mentor:  Lawrence Marnett, Biochemistry)

Prostaglandin-glycerols are produced from the oxygenation of COX-2 followed by the respective synthases, but whether a hydrolase exists to cleave the prostaglandin-glycerols into free prostaglandin and glycerol remains unknown.  Our lab and others attempted to investigate this by first examining the hydrolases that were known to cleave 2-AG and AEA, respectively, MAGL and FAAH.  However, none of the prostaglandin-glycerols investigated showed any significant degree of hydrolysis in the presence of FAAH or MGL. The hydrolysis of prostaglandin-glycerols, specifically PGE2-G, was our main focus in our studies because we showed that this substrate was metabolized into free prostaglandin and free glycerol.

This hydrolysis of PGE2-G to PGE2 is of particular interest because PGE2 has been shown to have a large array of different physiological responses in cells and tissues and these effects are mediated through PGE2  interaction with distinct PGE2 receptors that are G-protein coupled receptors.  The largest interest in this hydrolysis is focused on the fact that many of the PGE2 receptors are important in the regulation, survival and proliferation of a variety of different cancers; including breast, non-small cell lung and adenocarcinomas.  Investigation on a number of different cancer cell lines has shown that they posses a hydrolase responsible for the conversion of PGE2-G to PGE2. 

Our lab is using newly developed mass spectrometer techniques to assess the state and extend of conversion of these prostoglandins.  We are beginning to identify some of the characteristics of this hydrolase, including that it is a serine hydrolase, and are working to help identify this enzyme.  With identification it may be possible to develop new treatments for some of the cancers that are dependent on PGE2­ for survival.

 
Matthew Bryant (Mentor:  David Wright, Chemistry)

Porphyrins and metalloporphyrins are essential bioinorganic cofactors for life. Heme, an example of a metal porphyrin (Iron Protoporphyrin IX) is the cofactor molecule for the protein hemoglobin which is responsible for binding and transport oxygen in red blood cells.  Metalloporphyrins are versatile molecules that can be used in optical detection, protein purification, electrochemistry, and novel materials.  The Wright lab has always been focused on heme¹s properties and involvement with disease such as malaria and formation of hemozoin. 

Using bioavailable and versatile molecules for bioinorganic chemistry research is my focus for my projects.  Metal porphyrins provide a multifunctional molecule with useful optical properties. These colored materials can be used in a variety of applications for molecular sensing and detection through fluorescence techniques.  The charged metal center can be used for binding to histidine rich proteins, an important biomarker of malaria.  The propionate groups can be functionalized easily to introduce new qualities to the porphyrin molecule.  This approach will span the gap between biochemistry, inorganic chemistry, and materials chemistry.

 
Brittney Allison (Mentor:  Jens Meiler, Chemistry)

Protein family superfolds are of much significance because they show nature’s preference for certain folds.  The Meiler lab has taken a particular interest in the (βα)8 barrel (also known as a TIM-barrel) superfold because of its prevalence among proteins with known crystal structure, and because of its 2-fold symmetry.  This fold is popular among enzymes because the substrate enters one end of the barrel, and the product exits the other end. 

Currently I am studying protein-ligand binding of these (βα)8 barrels, specifically HisF in the histidine biosynthesis pathway and indole-3-glycerol phosphate synthase in the tryptophan biosynthesis pathway.  I am most interested in redesigning the protein-ligand interface of these proteins, using RosettaLigand of the Rosetta modeling suite.  De novo design of protein pocket interfaces has proven to be a difficult challenge, so I hope to achieve success by using proteins with a known binding pocket and crystal structure.  I will first test if I can improve binding of the native substrate, and then test if (with few mutations) I can bind non-native ligands.  The designs with the most favorable energy will be tested experimentally and validated with 2D NMR spectroscopy.  Current results show that RosettaLigand does indeed design sensible mutations within the protein-ligand binding site.  As I continue to work with RosettaLigand developers in my lab, I become closer to designing models that I can test experimentally.

 

 
 






           
           
           

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