(Oral presentation abstracts will be added as they are submitted via online registration - see above link.)

1. Patrick Halvey, "Deficiencies in DNA mismatch repair (MMR) systems occur in hereditary non-polyposis colorectal cancer (HNPCC) and in 10-15% of sporadic colorectal cancers (CRC)." Loss of function mutations in MMR proteins such as MLH1 and MSH2 lead to genetic instability, which contributes to tumor formation. Widespread mutations at the genome level presumably give rise to alterations in protein expression profiles; however, little is known about the effects of MMR deficiency on the proteome. We carried out global shotgun proteomic analysis of four MMR proficient (MMR+) CRC cell lines (HT-29, COLO-205, CaCo2, SW480) and four MMR deficient (MMR-) CRC cell lines ( HCT-116, LS174T, LoVo, RKO). Tryptic peptides from whole cell lysates were separated by isoelectric focusing (IEF) and analyzed by liquid chromatography/tandem mass spectrometry (LC-MS/MS) on a linear ion trap quadrupole (LTQ) mass spectrometer. Resulting spectra were searched against protein databases using the Myrimatch search algorithms and proteins were identified using parsimonious assembly with IDpicker. When data from all cell lines were assembled together, 5047 protein groups were identified (4.8% protein FDR). Statistical analysis of group-wise differences identified a small number of proteins that differentiated MMR+ and MMR- cell lines, with notably increased spectral counts for DNA repair proteins in MMR+ cell lines (MSH2, RAD50B, MRE11 and others). We further examined protein expression levels for genes known to be mutated in these cell lines. These genes include the KRAS, p53, APC, BAX and the mismatch repair proteins MSH2 and MSH6. KRAS was detected in both wildtype KRAS-expressing cells and mutant KRAS- expressing cells. Likewise, APC was detected in wildtype APC-expressing cells and truncated APC in cells carrying mutant APC, while p53 was only detected in mutant p53-expressing cells. BAX was not detected in MMR- deficient cell lines, LoVo or LS174T cells, which are known to possess a BAX frameshift mutation, but was detected in all wildtype BAX-expressing cells. Furthermore, MSH2 and MSH6 were not detected in LoVo cells, which express mutant forms of these genes. Our results demonstrate that shotgun proteomics can be used to identify broad differences in protein expression profiles between MMR+ and MMR- cell lines and is sensitive enough to pick up subtle changes at the individual protein level. The data suggest that global shotgun proteomic analyses can map individual protein expression patterns to corresponding genetic profiles and distinct cancer-related phenotypes.

2. Julia Koehler, "Simultaneous Prediction of Secondary Structure and Trans-Membrane Spans for Alpha-Helical Proteins As Well As Beta-Barrels Using Artificial Neural Networks." Tools for the identification of trans-membrane spans as well as secondary structure from the protein sequence are widely used in the experimental community. Computational structural biology seeks to increase the prediction accuracy of such methods since they represent a first step towards membrane protein tertiary structure prediction from the amino acid sequence. We introduce the first predictor that is able to simultaneously predict the secondary structure as well as identify trans-membrane spans from the sequence of a protein. The novelty of the approach is the application to both α-helical proteins as well as β-barrels. An artificial neural network was trained on databases of 102 membrane proteins and 3499 soluble proteins. The output is a nine-dimensional probability vector for each residue in the sequence. The probability vector contains the probabilities for all possible combinations of three secondary structure types (helix, strand, coil) with three environment types (trans-membrane, interface, solution). The prediction accuracy of simultaneously identifying secondary structure and environment correctly is 70% for nine possible states. Secondary structure prediction (three states) yields accuracies of up to 74% and trans-membrane span prediction (three states) correctly identifies 95% of the residues.

3. Robert Lavieri, "Phospholipase D (PLD) is an essential enzyme responsible for the production of the lipid second messenger phosphatidic acid (PA)." Both PLD and PA have been implicated in oncogenic signaling. PLD expression and enzymatic activity are both known to be elevated in a variety of human cancers. It has also been suggested that inhibition of PLD leads to decreased cell invasion and decreased metastasis of human cancer cells. The aforementioned evidence provided the impetus for a medicinal chemistry project focused on the development of small-molecule PLD inhibitors. The development of such inhibitors is an essential step in advancing the study of PLD as a potential oncology drug target. A group from Novartis published a report in 2007 disclosing the small-molecule halopemide as a hit from a high throughput screen for small-molecule inhibitors of PLD2. While halopemide was our initial lead compound we have explored chemical space quite divergent from halopemide. We utilized a diversity-oriented synthetic approach in order to develop a library of approximately 600 compounds. This effort has yielded the most potent, isoform-selective PLD inhibitors described to date. We are currently utilizing these compounds, coupled with a wide range of biochemical techniques, in order to more clearly characterize the role of PLD signaling in oncogenic processes such as cell cycle checkpoint override, suppression of apoptosis, cell invasion, and metastasis. Ultimately, we plan to contribute both to our basic understanding of cancer at the molecular level, and to the development of novel therapeutic strategies in oncology.

4. Rory Pruitt, "Autoprocessing of Clostridium difficile Toxin A." The nosocomial pathogen, Clostridium difficile, secretes two AB toxins which are responsible for virulence. These proteins, TcdA and TcdB, have recently been shown to employ an autoproteolytic step to release their cytotoxic component in the host cytosol. We have defined the cysteine protease domain responsible for auto-processing within toxin A and report the 1.6 Ã… X-ray crystal structure of the domain bound to its allosteric activator, inositol hexakisphosphate (InsP6). Functional studies confirm an intra-molecular mechanism of cleavage and highlight specific residues of toxin A required for InsP6-induced processing. This research will provide a platform for understanding the unusual mechanisms that TcdA and TcdB use to deliver their cytotoxic components to the target cell.

5. Reese Harry, "Hairpin DNA-Functionalized Gold Nanoparticles: Novel Intracellular mRNA Probes for Gene Expression Phenotyping of Malignant Melanoma." S. Reese Harry and David W. Wright. With approximately 70,000 new cases of melanoma in the US each year, early detection of malignancy is of utmost importance. Normal melanocytes utilize the enzyme tyrosinase (TYR) to carryout the formation of melanin. In the case of melanoma, the expression of this enzyme has been shown to be highly upregulated resulting in heavy pigmentation seen at the site of formation. Additional gene expression targets such as the S100β protein (S100b) and melanoma inhibitory activity (MIA) have also been show to be associated with malignant melanoma. Techniques such as reverse transcription polymerase chain reaction (RT-PCR) have been used to detect such melanoma gene expression targets in lymphatic and circulatory systems but with a high degree of variability. To circumvent issues associated with current detection methods, we present a novel intercellular mRNA expression probe that combines gold nanotechnology with hairpin DNA recognition methodology. These hairpin DNA-functionalized gold nanoparticles (hAuNP) utilize a 40-base modified oligonucleotide that is terminated with a fluorophore and provides a fluorescence enhancement upon binding to its target mRNA molecule. Using three known melanoma markers (TYR, S100b, and MIA) as initial screening targets, confocal microscopy was capable of accurately distinguishing SK-MEL-28 cells in complex culture with excellent specificity. Concurrent flow cytometric analysis confirmed a positive gene expression profile with elevated levels of expression for each target. Furthermore, primary melanoma cultures obtained from the Vanderbilt Ingram Cancer Center displayed analogous specificity highlighting the potential utility of these novel gene expression probes to assist in personalized medical assessments and treatment.

6. Steffen Lindert, "Folding Proteins into Medium Resolution cryoEM Density Maps." Using cryo-electron microscopy (cryoEM) numerous sub-nanometer resolution density maps of large macromolecular assemblies have been reported recently. Although generally no atomic detail is resolved in these density maps, at 7 Å resolution α-helices are observed as density rods. Here we present the development of a computational protein structure prediction algorithm that incorporates the experimental cryoEM data as restraints. The placement of helices is restricted to regions where density rods are observed in the cryoEM density map. The Monte Carlo based protein folding algorithm is further driven by knowledge based energy functions. The method has been benchmarked with ten highly α-helical proteins of known structure. The chosen proteins range in size from 250 to 350 residues. Starting with knowledge of the true secondary structure for these ten proteins, the method can identify the correct topology within the top scoring 10 models. With more realistic secondary structure prediction information, the correct topology is found within the top scoring 5 models for seven of the ten proteins. The algorithm has been applied to human adenovirus protein IIIa. This protein, for which there is no high resolution structure, is predicted to be highly α-helical. It is resolved in a 6.8 Å resolution cryoEM adenovirus structure as a bundle of 14 α-helical density rods.

7. Conbao Kang, "Solution NMR Study on Human Membrane Protein KCNE3-A Voltage-gated Potassium Channel Regulator." Congbao Kang, Carlos G. Vanoye, Jarrod A. Smith, Richard C. Welch, Jens Meiler, Charles R. Sanders. Membrane proteins such as ion channels and receptors play very important roles in cells. About one third of proteins in human genome are membrane proteins, but the structure information of these proteins is very limited because of their hydrophobic nature. KCNQ1 is a voltage-gated channel that is modulated by KCNE family proteins. Mutations in KCNQ1 or in KCNE family proteins are associated with inherited and acquired diseases such as long QT syndrome (LQTS). In the human heart, KCNQ1 associates with KCNE1 to form the cardiac IKs channel that contributes to ventricular repolarization, while KCNE3 greatly reduces the voltage dependence of KCNQ1 resulting in constitutively open channels. To understand the contrasting roles of KCNE1 and KCNE3 in regulating KCNQ1, NMR was used to characterize the structures of KCNE3 and KCNE1 in different membrane mimetic systems. The structure of KCNE1 was solved in model membrane and we are proceeding to solve the structure of KCNE3 in model membrane. Oocyte injection study shows recombinant KCNE3 from E.coli was functional following injection in model membrane system. Backbone NMR resonance assignments of KCNE3 were obtained in two model membrane systems. While the completion of KCNE3's structure is on-going, our determined KCNE1 structure, homology modeling, and ROSETTA-based docking methods strongly suggest the structural basis for the contrasting functional differences in how KCNE1 and KCNE3 modulate the KCNQ1 channel.

8. Bo Shen, "Semiaqueous Amide and Peptide Synthesis through Reversed Reactant Polarity: A Conceptually New Alternative to Traditional Condensative Methods." Bo Shen, Dawn M. Makley, and Jeffrey N. Johnston. A new synthesis of amides has been developed that involves an iodonium-mediated nitroalkane-amine coupling in wet THF. A range of a-bromo nitroalkanes are used as formal acyl donors for commercially available amine acceptors. Common functionality, including olefins, alkynes, and unprotected alcohols are tolerated, providing the amide products in good yield using equimolar amounts of reagents and mild reaction temperatures (ice water to room temperature). Our preliminary mechanistic studies suggest a pathway that contrasts most dehydrative amide couplings, in which reversed reactant polarity in the key C-N bond forming step is operative. Insofar as enantioselective peptide synthesis remains impractical, this approach has been successfully merged with chiral proton catalysis of the enantioselective aza-Henry reaction to provide a direct method to homologate N-terminal peptides with an aryl glycine residue.

9. Devin Stauff, "Insights Into the Mechanism of Signal Sensing by a Bacterial Histidine Kinase Using a Small Molecule Library Screen." During invasion of the tissues of its human host, the Gram-positive pathogen Staphylococcus aureus must acquire heme from red blood cells to support bacterial fitness. However, heme acquisition by staphylococci comes at a cost: heme is a toxic molecule capable of killing bacterial cells. S. aureus solves the heme paradox by detecting exogenous heme through the histidine kinase HssS. HssS-mediated heme sensing initiates a heme detoxification response that allows staphylococci to overcome heme toxicity. Although the role of HssS in heme detoxification has been described in detail, the mechanism by which HssS senses heme has remained elusive. To begin dissecting the means by which HssS detects heme, we have screened a small molecule library for non-heme activators of this histidine kinase. From this screen, we have identified a non-heme in vivo activator of HssS that points toward an indirect mechanism of heme sensing by HssS. These studies have begun to elucidate the uncharacterized pathway of heme toxicity in Gram-positive bacteria as well as the enigmatic mechanism of heme sensing by staphylococci.

 

 

(Poster abstracts will be added as they are submitted via online registration - see above link.)

1. Tom Bridges, Activation of muscarinic acetylcholine receptor subtype-1 (M1) has been investigated preclinically as a potential strategy for treatment of Alzheimer's disease (AD). Selective targeting of subtype-5 (M5) may also be therapeutically relevant to AD due to its role in regulation of cerebral vascular tone and certain cognitive functions as suggested by studies with M5-KO mice. Historically, high homology of the orthosteric acetylcholine-binding site across the five muscarinic receptor subtypes has made discovery and development of highly subtype-selective or preferring compounds challenging, and non-selective activation of M2 and M3 has contributed to failure of muscarinic agonists in clinical trials. To date, no ligands exhibiting high M5-preference or selectivity have been reported. Recently, functional cell-based high throughput screening was used to identify a number of novel muscarinic potentiators, including VU0119498, which enhanced acetylcholine (ACh) potency at M1, M3, and M5 via an allosteric interaction. An iterative analog library approach was used to chemically optimize VU0119498 for increased M5 activity, which identified VU0238429 as the first highly M5-preferring small molecule. In calcium mobilization assays, VU0238429 displayed an EC50 of approximately 1 µMfor potentiation of acetylcholine at M5 and an EC50 of >30 µM for potentiation at M1 and M3, with no effect at Gqi5 co-transfected M2 or M4 receptors. In similar cell-based assays, a fixed 30 µM concentration of VU0238429 induced an approximately 14-fold leftward shift of a full ACh concentration-response curve at M5. This novel allosteric potentiator represents the first highly M5-preferring small molecule ever reported and may serve as a useful pharmacological tool for probing the therapeutic potential of M5 activation within the context of AD and cerebrovascular dementia.

2. Emily Rubinson, DNA glycosylases safeguard the genome by locating and excising chemically modified DNA bases through a base flipping mechanism. AlkD is a bacterial DNA glycosylase specific for positively charged alkylpurine nucleobases 3-methyladenine and 7-methylguanine. The crystal structure of Bacillus cereus AlkD revealed that the protein represents a new superfamily of DNA repair proteins, and is composed exclusively of HEAT-like repeats perfectly shaped to accommodate an undistorted DNA duplex on the concave surface. Structural analysis of the variant HEAT repeats provided clues for how this protein scaffolding has been modified to bind DNA, but the details for DNA binding were unclear. We present here crystal structures of AlkD in complex with DNA containing 3-deaza-3-methyladenine (3d3mA) and a tetrahydrofuran (THF) reduced abasic site, representing substrate and product complexes. These structures reveal unexpectedly that AlkD makes no contact to the lesioned base, but senses the structural destabilization of the 3d3mA-T base pair through electrostatic interactions with the DNA backbone. In the product complex, both nucleotides of the THF-T pair are flipped in an extrahelical position in which the THF is pointing away from the protein and the opposite thymine is sandwiched between the DNA minor groove and the protein active site. The absence of any intercalating side chains within the DNA base stack suggests that AlkD utilizes a novel strategy to manipulate DNA in its search for alkylpurine bases. Comparison of AlkD to existing DNA glycosylase structures, along with mutational studies of 7mG excision and DNA binding activities, provides important insight into the requirements for alkylation repair within DNA.

3. Patrick Halvey, Deficiencies in DNA mismatch repair (MMR) systems occur in hereditary non-polyposis colorectal cancer (HNPCC) and in 10-15% of sporadic colorectal cancers (CRC). Loss of function mutations in MMR proteins such as MLH1 and MSH2 lead to genetic instability, which contributes to tumor formation. Widespread mutations at the genome level presumably give rise to alterations in protein expression profiles; however, little is known about the effects of MMR deficiency on the proteome. We carried out global shotgun proteomic analysis of four MMR proficient (MMR+) CRC cell lines (HT-29, COLO-205, CaCo2, SW480) and four MMR deficient (MMR-) CRC cell lines ( HCT-116, LS174T, LoVo, RKO). Tryptic peptides from whole cell lysates were separated by isoelectric focusing (IEF) and analyzed by liquid chromatography/tandem mass spectrometry (LC-MS/MS) on a linear ion trap quadrupole (LTQ) mass spectrometer. Resulting spectra were searched against protein databases using the Myrimatch search algorithms and proteins were identified using parsimonious assembly with IDpicker. When data from all cell lines were assembled together, 5047 protein groups were identified (4.8% protein FDR). Statistical analysis of group-wise differences identified a small number of proteins that differentiated MMR+ and MMR- cell lines, with notably increased spectral counts for DNA repair proteins in MMR+ cell lines (MSH2, RAD50B, MRE11 and others). We further examined protein expression levels for genes known to be mutated in these cell lines. These genes include the KRAS, p53, APC, BAX and the mismatch repair proteins MSH2 and MSH6. KRAS was detected in both wildtype KRAS-expressing cells and mutant KRAS- expressing cells. Likewise, APC was detected in wildtype APC-expressing cells and truncated APC in cells carrying mutant APC, while p53 was only detected in mutant p53-expressing cells. BAX was not detected in MMR- deficient cell lines, LoVo or LS174T cells, which are known to possess a BAX frameshift mutation, but was detected in all wildtype BAX-expressing cells. Furthermore, MSH2 and MSH6 were not detected in LoVo cells, which express mutant forms of these genes. Our results demonstrate that shotgun proteomics can be used to identify broad differences in protein expression profiles between MMR+ and MMR- cell lines and is sensitive enough to pick up subtle changes at the individual protein level. The data suggest that global shotgun proteomic analyses can map individual protein expression patterns to corresponding genetic profiles and distinct cancer-related phenotypes.

4. Surajit Banerjee, DNA alkylation by aflatoxin B1 (AFB1) yields the N7-deoxyguanosine adduct trans-8,9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B1, which hydrolyze and gives the persistent and highly mutagenic formamidopyrimidine (FAPY) derivative. The FAPY derivative equilibrates between alpha and beta deoxyribose anomers; the beta anomer is associated with the high mutagenicity of this lesion in E. coli, whereas the alpha anomer blocks DNA replication. We report the structure of the Sulfolobus solfataricus DNA polymerase Dpo4 with the template:primer 5'-TCATTGAATCCTTCCCCC-3':5'-GGGGGAAGGATTC-3', where G is the site of adduction with either the AFB1-N7-Gua or the AFB1-FAPY adduct. For both adducts, the 5'-intercalation of the AFB1 moiety relative to the alkylated dG is maintained during DNA replication. The structures with the Dpo4 polymerase show that the mutagenic beta anomer of the lesion is maintained within the active site. The structural studies are correlated with replication bypass experiments in vitro.

5. Christopher Blake Sullivan, "Phosphatidylethanolamine is modified by isoketals in cells and contributes to isoketal induced cytotoxicity," C. Blake Sullivan, Elena Matafonova, L. Jackson Roberts, Venkataraman Amarnath, and Sean S. Davies. Levuglandins are γ-ketoaldehyde eicosanoids formed by non-enzymatic rearrangement of prostaglandin H2. Additional levuglandin isomers can be formed by peroxidation of arachidonic acid via the isoprostane pathway (isoketals). Levuglandin/isoketal (IsoK) levels increase in pathological conditions including atherosclerosis and Alzheimer’s Disease and IsoKs can induce ion channel dysfunction and cell death. The mechanism for inducing cellular dysfunction has been presumed to be the rapid reaction of IsoKs with the lysyl residues of cellular proteins. However, when we added radiolabeled IsoK to HEK293 cells in order to identify the molecular targets of IsoKs, we found that a significant portion of the radiolabel fractionated with phospholipids (40%). IsoK react with a variety of primary amines, including phosphatidylethanolamine (PE) in vitro, but their abundance in vivo is unknown. We therefore sought to develop LC/MS/MS methods to measure IsoK adducts of PE (IsoK-PE) in cells. We found that phospholipase D from Streptomyces chromofuscus hydrolyzed IsoK-PE to the IsoK-ethanolamine (IsoK-Etn) which could then be quantitatively measured using stable isotope LC/MS/MS. Addition of 0-5 µM IsoK to HEK293 cells dose-dependently increased PE adduct concentrations to a similar extent as protein adduct. To test whether formation of IsoK-PE had biological significance, we treated human umbilical vein endothelial cells (HUVEC) with IsoK-PE. IsoK-PE induced dose-dependent cytotoxicity, with doses of 10 µM or greater inducing maximal cytotoxicity. These results indicate that the aminophospholipid PE is a major cellular target of IsoKs, and that formation of IsoK-PE may mediate at least some of the biological effects of IsoKs that are relevant to disease.

6. Daniel Brown, "HTS Assays for the Discovery of Bioactive Inhibitors to the Fosfomycin Resistance Enzyme, FosA, from Pseudomonas aeruginosa." The antibiotic fosfomycin is underutilized in the US, despite its broad-spectrum activity and low number of side effects, due in large part to clinically relevant bacterial resistance. Much of this resistance is enzymatic and has been found encoded on multi-drug resistant plasmids, but also in the genomes of many pathogenic bacteria, including Pseudomonas aeruginosa. An attempt is currently underway to develop inhibitors for these enzymes that could be delivered in the clinic along with the antibiotic, allowing the antibiotic to perform its cytotoxic function. This has been successful in the β-lactam/β-lactamase inhibitor combinations and should be possible for fosfomycin as well. Here we describe the development of a high throughput screen for the discovery of novel inhibitors to the genomically encoded fosfomycin resistance enzyme from P. aeruginosa. The two screens presented here work together to identify inhibitors of the purified enzyme and to test their specific bioactivity.

7. Larissa Fenn, "Integrated 'omics' on the basis of structural separations by ion mobility-mass spectrometry." Many contemporary life science studies center on different "omics"; i.e. proteomics, glycomics, and lipidomics. These analyses of various biomolecular classes are typically performed separately and then combined to derive information about a system as a whole, i.e. as in systems biology. Even though this reductionist methodology has worked for explaining numerous biological processes, it underestimates the complexity of the processes as a whole and therefore hinders current and future research. In this report, we describe the use of ion mobility-mass spectrometry (IM-MS) for the structural characterization of biomolecules for the ultimate goal of integrated "omics." IM-MS combines rapid (us-ms) structural separation by ion mobility and subsequent identification by MS.

8. Randi Gant, This report focuses on the identification of sites of protein phosphorylation using several MS-based strategies, namely: HPLC-ion trap-MS, HPLC-orbitrap-MS, and ion mobility-MS (IM-MS). Each of these techniques exhibit distinct advantages and limitations in the characterization of phosphorylation sites. The specific phosphoprotein for which sites of phosphorylation are identified is human actin patch protein (APPL1). APPL1 is implicated in important cell migration regulatory pathways of particular interest in cancer biology and cell membrane curvature. Although there is increasing interest in the interactions and biological roles of this protein, the sites of phosphorylation have not been reported to date.

9. Suraj Adhikary, As DNA nucleobases are susceptible to modification by chemical, metabolic and environmental agents, cells have devised elaborate mechanisms to identify and repair these modifications. 3-methyladenine DNA glycosylase (Mag1) from Schizosaccharomyces pombe initiates the base excision repair (BER) pathway by recognizing and catalyzing excision of various damaged nucleobases from the genome. In addition to its role in the BER, Mag1 is postulated to contribute to other DNA repair mechanisms such as nucleotide excision repair (NER) and recombinational repair (RR). Mag1 shares extensive sequence similarity with 3-methyladenine glycosylases from S. cerevisiae and E. coli (MAG and AlkA, respectively). However, unlike MAG and AlkA, which recognize and excise a wide array of damaged nucleobases, Mag1 has a relatively limited number of substrates that include 3-methyladenine, 7-methylguanine, and 3-methylguanine. In order to understand the molecular basis of substrate specificity between Mag1, MAG, and AlkA, we determined the crystal structure of Mag1 bound to DNA containing an abasic site and confirmed the catalytically active residue using site directed mutagenesis. The structure verifies that Mag1 is a member of the helix-hairpin-helix superfamily of DNA glycosylases and utilizes the base-flipping mechanism for lesion recognition. Comparision of three-dimensional structures of Mag1 and AlkA help to understand the structural determinants for the substrate specificity differences between two enzymes with extraordinarily homologous active sites.

10. Michal Kliman, Imaging mass spectrometry utilizing MALDI-MS has demonstrated great potential for spatial profiling and imaging of biomolecules (e.g. proteins and lipids) in histologically relevant tissue samples. However, using conventional MALDI techniques, the spatial resolution (ca. 10-25 µm) and sample selectivity (shape of the laser irradiated area at the target) is limited. The optical arrangement described here significantly improves both spatial resolution and spatial selectivity for imaging MS. The principal component of the new optical arrangement is a digital micro-mirror array. The 1x1.5 cm mirror array of this device consists of ca. one million individually addressable 13x13 µm size mirrors that can be tilted to either reflect or deflect parts of the incident laser beam. Using this device the MALDI laser can be patterned at the target into regular or complex shapes of variable dimensions and even non-congruent spatial regions can be irradiated simultaneously. Initial experiments suggest that significant signal can be expected from patterns with submicrometer resolution at the target (ca. 0.9 x 0.9 µm per mirror at target). From 0.1 pmol of peptide RPPGFSPFR, patterns of 5 x 5 mirrors (target size 4 x 4 µm) yield 1 x 105 counts and patterns of 4 x 4 mirrors (target size 3.3 x 3.3 µm) yield 1 x 103 counts. Therefore, up to 40,000 counts per mirror can be generated from 5 x 5 mirror patterns and 6,000 counts per mirror from 4 x 4 mirror patterns. Applications of this new arrangement are illustrated in imaging MS of tissues and cells.

11. Joel Musee, The endocannabinoid 2-arachidonoyl glycerol (2-AG) is a selective substrate for the inducible isoform of cyclooxygenase (COX), COX-2, in vitro. Its turnover leads to formation of glyceryl analogs of traditional prostaglandins (PG-Gs), a subset of which elicit agonism at unique receptors, at picomolar to nanomolar concentrations. While 2-AG is approximately one-tenth as abundant as arachidonic acid (AA) in zymosan-stimulated macrophages, the ratio of PGs to PG-Gs recovered from these macrophages is about 1000:1 (Rouzer, C.A. and Marnett, L.J. (2008) J. Biol. Chem. 47:3917.). The activation of oxygenase activity in COX is dependent on the turnover of the peroxide product of AA or 2-AG oxygenation (PGG2 and PGG2-G respectively). We hypothesized that PGG2-G is a less efficient POX substrate than PGG2 and therefore a poor activator of 2-AG turnover. Here we demonstrate that while both peroxide intermediates are reduced at comparable rates, the turnover of 2-AG is exquisitely sensitive to peroxide tone in activating and sustaining oxygenase activity.

12. Niki Arinze, The characterization of peptides has seen numerous advances over the years through the application of Electrospray Ionization (ESI) and Matrix Assisted Laser Desorption Ionization (MALDI) Mass spectrometry to the field. Coupling these proteomic tools with ion mobility spectrometry (IMS) provides the ability to obtain both mass and structural information. Through the use of ion mobility mass spectrometry (IM-MS), we use collision cross section measurements to elucidate the conformation of these peptides. The capabilities of IM-MS to provide structural information for peptides are widely used in the positive ionization mode, while the negative ionization mode of IM-MS has received considerably less attention. The use of negative mode MALDI-MS has been shown to allow for the detection of many peptides not detected in the positive mode. Thus, the use of both the positive and negative modes of MALDI-IM-MS for peptide analysis may allow for greater sequence coverage. Furthermore, the utilization of both ionization modes in MALDI-IM-TOFMS, will enable the acquisition of structural information, or collision cross section information, for a wider range of peptides. Additionally for the peptides that can be detected in both ionization modes, we can begin to study the effect of charge state on gas phase conformation.

13. Xuyang Peng, "SW07001 Protects Myocytes from DNA Damage," Xuyang Peng, Emily Simon, Lin Zhong, Chengchun Cao, Laura Pentassuglia, Basak Icli, Douglas Sawyer. Objective: A compound SW07001 is purified from a plant Kudzu and has been used to treat cardiovascular disease and myocardial infarction in China. We hypothesized that SW07001 has protection effect on myocytes. Methods and results: Experiments were performed in vivo and in primary ventricular myocytes (ARVMs). Mice were treated by SW07001 in different time and dose course. ARVMs were isolated from adult rats and cultured for seven days after they started to beat. DNA damage was assessed with primary antibody to phospho-H2A.X antibody and p53 in immunofluorescence microscopy as well as western blot in ARVMs. Significant expression in phospho-H2A.X and p53 expression and activation at site 15 were induced in ARVM by FBS starvation in long term cultured myocytes. These increases and activation were inhibited by SW07001 at a dose of 100ug/ml in ARVM. Suppression of p53 activation by FBS starvation in mice heart was also detected. Moreover, we found that p53 expression and activation by FBS starvation in ARVM was suppressed in both cytoplasmic and nuclear extraction. However the suppression of p53 activation in the nuclear extraction was robust. P21, a known mediator of p53-dependent cell cycle arrest was suppressed by SW07001. Sacomere proteins synthesis in myocytes was increased to FBS starvation as assessed by staining for myomesin and phalloidin. Alpha-Actin expression by immuoblot in mice heart was increased after 6 hours of SW07001 treatment. Interestingly, increasing of expression of multiple drug resistant (MDR1) induced by FBS starvation was suppressed by SW07001 in a time and dose dependent course in the mice heart. The mechanism of SW07001 cytoprotection is still under investigation. Conclusion: These results suggest that SW07001 has cardioprotective effects in myocytes response to DNA damage induced by FBS starvation. This will potentially supply a basis for application of SW07001 in the clinic in the future.

14. Kelsey Duggan, Naproxen is a widely used over-the-counter nonsteroidal anti-inflammatory drug (NSAID) with analgesic and antipyretic properties. The pharmacological effects of NSAIDs, including naproxen, arise from the suppression of prostaglandin biosynthesis by inhibiting the cyclooxygenase enzymes, COX-1 and COX-2. COX-1 and COX-2, which share high sequence homology and similar three-dimensional structures, catalyze the formation of prostaglandin H2 from arachidonic acid (AA). Despite long-term use in the clinic, the molecular basis for COX inhibition by naproxen is not well defined. We have performed extensive mutagenesis and structure-activity relationship studies in order to establish a hypothetical binding mode of naproxen within the COX active site. Our data suggest that naproxen is bound in the canonical conformation with the carboxylate moiety making critical interactions with constriction site residues Arg-120 and Tyr-355. Interestingly, we identified a novel interaction at the top of the active site involving Trp-387, and we have synthesized derivatives of naproxen with modifications at the 6-position resulting in COX-2 preferring inhibitors. In addition to evaluating the inhibition of AA oxygenation by naproxen, we have performed studies to elucidate the ability of naproxen to inhibit the turnover of the endocannabinoid, 2-arachidonyl glycerol (2-AG). 2-AG is selectively oxygenated by COX-2 to form glyceryl prostaglandins that exhibit biological activities distinct from those of the prostaglandins. Our results indicate that compared to AA, COX-2-dependent metabolism of 2-AG is 10 to 100-fold more sensitive to inhibition by naproxen and other rapidly reversible NSAIDS.

15. Rebecca Connor, 4-Hydroxynonenal (HNE), a product of oxidative stress, has been shown to induce expression of molecular chaperones Hsp70 and BAG3 through the activation of Hsf1. Inactive Hsf1 is complexed with heat shock proteins, Hsp70 and Hsp90 in the cytosol. Upon activation, it dissociates and trimerizes into its active form, which then translocates across the nuclear membrane and activates the heat shock response. The modification by HNE of proteins within the heat shock complex is thought to induce the activation of Hsf1. In particular, Hsp90 is not only a repressor of Hsf1 activation, but also a target of HNE modification both in vivo and in cell culture at low HNE concentrations and short time scales. Using a biotinylated derivative of the Hsp90 inhibitor geldanamycin, Hsp90 isoforms were captured from a cell lysate and analyzed using liquid chromatography and tandem mass spectrometry (LC-MS/MS) on a Thermo LTQ-Orbitrap instrument to identify discrete HNE modification sites. Histidine residues on both alpha and beta Hsp90 isoforms, H450/442, were found to be modified in RKO cells treated with 250 uM HNE. These modifications were also identified on purified Hsp90 treated with HNE in vitro. Furthermore, Hsp90 residues, S231/225 and S263/254 were found to be phosphorylated in both HNE-treated and untreated RKO cells. Small molecule affinity capture enabled detection and verification of both known and induced post-translational modifications of Hsp90. The identification of HNE adduction sites on Hsp90 will inform mechanistic studies of the effects of oxidative stress on the heat shock complex.

16. Jennifer McKenzie, Lower morbidity rates have been found in patients with a history of “mini-strokes”, or transient ischemic attack, prior to a conventional ischemic attack. A similar effect is seen in neuronal cultures, where exposure to a sub-lethal insult up-regulates protective mechanisms, yielding a 50% survival rate upon further attack. Without the sub-lethal insult to induce metabolic stress, which initiates the production of reactive oxygen species, heat shock proteins, as well as the opening of ATP channels, the neurons are unable to protect themselves from the lethal insult. In our study, an instrument capable of simultaneous electrochemical measurement of extracellular metabolites, the multi-analyte microphysiometer (MAMP), was used to investigate the altered metabolic activity of primary neurons when exposed to lethal and sublethal insults. By studying the metabolic activity of neurons as they undergo ischemic preconditioning and attack, one can hope to better understand the underlying mechanisms and modes of action yielding neuroprotection. As this is the first time primary neurons have been studied in the MAMP, a variety of methods are employed to optimize the experimental procedures. A new pH-sensitive iridium oxide sensor is integrated into the MAMP sensor array, resulting in higher signal-to-noise compared to the previous silicon oxide-based pH sensing electrode. In these experiments, neuronal metabolic activity was monitored during exposure and recovery from a variety of stressors meant to induce or replicate ischemic conditions.

17. Patrick Robertson, "A Structural Investigation of DNA Binding by the C-Terminal Domain of MCM10." Eukaryotic DNA replication is tightly regulated during the initiation phase to ensure that the genome is copied only once and at the proper time during each cell cycle. During replication initiation, over twenty different proteins are recruited to each origin of replication to denature the DNA duplex and assemble a functional replication fork. Mcm10 is a DNA binding protein that is recruited to origins in early S-phase and is required for the activation of Mcm2-7, the replicative DNA helicase. Importantly, Mcm10 is also necessary for subsequent loading of downstream replication proteins, including Cdc45, And1, RPA, and DNA polymerase α-primase (pol α), onto chromatin. Mcm10 interacts with single- and double-stranded DNA, pol α, as well as other proteins involved in DNA synthesis. Despite its importance in both replication fork assembly and progression, the precise role of Mcm10 remains undefined. In order to better understand the importance of the molecular interactions of vertebrate Mcm10, we are carrying out a structure-function study of the protein from Xenopus laevis (XMcm10), which shares a high sequence homology with the human ortholog. XMcm10 contains three structured regions: a putative oligomerization domain at the N-terminus (NTD) and two independent DNA binding domains located in the internal (ID) and C-terminal (CTD) regions of the protein. XMcm10-CTD is stabilized by two zinc ions and binds to both single- and double-stranded DNA and to the p180 polymerase subunit of pol α. Here we present our progress towards the three-dimensional structure and DNA binding of XMcm10-CTD. NMR chemical shift perturbation data shows that ssDNA binding is exclusive to the two zinc motifs within the CTD. This analysis, together with our work on the other two domains, provides a preliminary model for the coordination of DNA and protein binding by full-length Mcm10.

18. Carol Bansbach, The DNA damage response (DDR) is a signal transduction pathway that recognizes challenges to the genome and initiates cellular programs that maintain genomic integrity. By promoting DNA repair, senescence, or apoptosis in response to genetic insults, the DDR acts as a cancer barrier inhibiting the survival and growth of genetically unstable cells. An important component of the DDR is the replication stress response (RSR). The RSR acts during every cell division cycle to deal with challenges to the genome during replication. In order to better understand the development and progression of human disease, especially cancer, it is important to identify genetic alterations that drive genome instability. Using functional genomic screens in human cell culture, we have identified genes whose deregulation activates the DDR in the absence of any added genotoxic agent. Many known regulators of DNA repair, cell cycle control, and checkpoint signaling were identified in the RNAi screen. Approximately one-third are putative tumor suppressors. Several known oncogenes were identified in the overexpression screen. A single gene SMARCAL1 was identified in both the RNAi and overexpression genes. The function of the SMARCAL1 protein is unknown, although sequence analysis suggests that SMARCAL1 is a member of the SNF2 family of chromatin remodeling ATPases. Our data indicate that SMARCAL1 is a novel replication stress response protein. RNAi silencing of SMARCAL1 causes hyper-sensitivity to replication stress agents. SMARCAL1 localizes to stalled replication forks. This localization is mediated through an interaction with replication protein A (RPA), placing SMARCAL1 activity at sites of replication. Thus, our functional genomic screens identified novel genome maintenance activities within human cells including a new replication stress response protein, SMARCAL1.

19. Ganesh Shanmugam, When γ-hydroxy-1,N2-propano-2′-deoxyguanosine (γ-OHPdG), a major deoxyguanosine adduct derived from acrolein, placed complementary to deoxycytosine in the oligonucleotide duplex, it undergoes ring-opening to the N2-(3-oxopropyl)-dG aldehyde or its hydrate. This is anticipated to facilitate Watson-Crick hydrogen bonding with incoming dCTP during DNA replication, explaining why γ-OHPdG is weakly mutagenic. Reduced N2-(3-hydroxyl-propyl)-dG (reduced γ-OHPdG) is chemically a stable analogue for the N2-(3-oxopropyl)-dG aldehyde adduct. We present structures of the reduced γ-OHPdG adduct in complex with the Sulfolobus solfataricus DNA polymerase Dpo4. Two primer:templates were examined: 5′-d(TCACXGAATCCTTCCCCC)-3′•5′-d(GGGGGAAGGATTC)-3′ (template-I) and 5′-d(TCATXGAATCCTTCCCCC)-3′•5′-d(GGGGGAAGGATTC)-3′ (template-II), where X is N2-(3-hydroxyl-propyl)-dG. Replication bypass experiments with the templates containing 5′-TXG-3′ and 5′-CXG-3′ sequences in the presence of all four dNTPs showed that the Dpo4 polymerase bypassed the reduced γ-OHPdG adduct and extended the primers to the full-length products. In the crystal structure of ternary complex (template II), the incoming dCTP paired with the reduced γ-OHPdG adduct, consistent with the single nucleotide incorporation data where dCTP was preferentially incorporated. In both ternary complexes, the incoming nucleotides dGTP or dATP did not pair with the reduced adduct, but instead with the 5′-neighbor template dC (template-I) or dT (template-II), utilizing Watson-Crick geometry. Thus both complexes were of the type II structure described for ternary complexes of the Dpo4 polymerase with native DNA. Supported by NIH grant ES-05355 (C.J.R., M.E., and M.P.S.).

20. Liping Du, "Engineering multigene expression in vitro and in vivo with small terminators for T7 RNA polymerase," Liping Du, Rong Gao and Anthony C. Forster. Engineering protein expression in vitro or in vivo is usually straightforward for single genes, but remains challenging for multiple genes because of the requirement of coordinated control. RNA and protein over expression strategies often exploit T7 RNA polymerase and its natural T? Class I terminator. However, this terminator’s inefficiency and large size (100 base pairs) are problematic for multigene construction and expression. Here, we measure the effects of tandem copies of a small (18 base pair) Class II T7 terminator from vesicular stomatitis virus on transcription in vitro and on translation in vitro and in vivo. We first test monomeric and dimeric gene constructs, then attempt extension to pentameric gene constructs. “BioBrick” versions of a pET vector and translation factor genes were constructed to facilitate cloning, and His-tags were incorporated to allow copurification of all protein products for relatively unbiased analysis and easy purification. Several results were surprising, including imbalanced expression of the pentameric constructs in vivo, illustrating the value of synthetic biology for investigating gene expression. However, these problems were solved rationally by changing the orders of the genes and by adding extra promoters to the upstream gene or by moving to a more predictable in vitro translation system. These successes were significant, given our initial unexpected results and that we are unaware of another example of coordinated over expression of five proteins. Our modular, flexible, rational method should further empower synthetic biologists wishing to over express multiple proteins simultaneously.

21. Roman Shchepin, "Identification of Novel Tricyclic Diels-Alder Tyrosine Adducts with Oxidized Lipids or Phospholipids Using LC-NMR and LC-MS/MS," Roman Shchepin, Duane M. Hatch, Donald F. Stec, Rafael Radi, Hye-Young H. Kim, and Ned A. Porter, Department of Chemistry, Vanderbilt University, Nashville, TN 37235. Protein nitration is important post-translational modification in vivo that can alter protein functions. It is associated to acute and chronic disease states and can be a predictor of disease risk and progression. 3-Nitrotyrosine of proteins is formed via metal mediated oxidation to generate tyrosyl radical. Subsequently, it is readily reactive toward other radical species such as ROS or lipidhyroperoxyl radical to be quenched. This could potentially form adducts with lipid/phospholipid either at ortho or para position. Then subsequently the adducts undergo Diels-Alder condensation to form tricyclic adducts. It has been reported that a phenolic antioxidant such as Curcumin undergoes similar chemistry to form stable tricyclic adducts with linoleate induced by azo initiator. We would like to explore the novel tyrosine adducts arising from tryosyl radical and lipids/phospholipid peroxides under oxidative condition. The preliminary study using hydrophobic tyrosine analogue and phospholipid/lipid revealed the formation of para-adducts on a tyrosine analogue. These adducts ultimately underwent Diels-Alder condensation to form relatively stable cyclized adducts. Linoleate alone forms various regio- and stereo-isomers, therefore, the resulting cyclized adducts are mixture of diasteromers that can be separated by HPLC both normal and reverse phase. The structures of isolated adducts have been identified using LC-NMR and LC-MS/MS. Ultimately, the efforts will be made to find the same adducts in proteins and develop analysis protocols using tandem mass spectrometer (LC-MS/MS).

22. Libin Xu, "Autoxidation of 7-dehydrocholesterol and the implications in Smith-Lemli-Opitz syndrome." In an effort to determine the reactivities of various lipids toward radical-catalyzed peroxidation reactions, 7-dehydrocholesterol (7-DHC) was found to be exceptionally oxidizable with a propagation rate constant in solution (2260 M-1s-1) more than 10 times that of arachidonic acid (197 M-1s-1), a polyunsaturated fatty acid that was considered to be highly susceptible to oxidation. 7-DHC is accumulated in patients with Smith-Lemli-Opitz syndrome (SLOS) due to mutations in the genes encoding 7-DHC reductase, the enzyme that catalyzes the reduction of 7-DHC to cholesterol. We propose that the unusual reactivity of 7-DHC may play a role in the pathogenesis of SLOS. Thus, autoxidation of 7-DHC initiated by 2,2'-azobis(4-methoxy-2,4-dimethylvaleronitrile) was carried out at 37°C in benzene, and a series of new oxidation products were isolated and characterized. A novel reaction mechanism was proposed to account for the formation of the oxysterols from 7-DHC. More significantly, some of the oxysterols were identified in neuro2a cell and mutant mouse SLOS models, while they are not present in control samples. Furthermore, considering the high oxidizability of 7-DHC, oxidative stress was assessed by standard methods including quantification of isoprostanes and neuroprostanes, the results of which suggested increased stress in SLOS models. Biological activities of oxysterols from 7-DHC are under investigation.

23. Wei Liu, "Novel Chemical Mechanism of 4-Hydroxynonenal (4-HNE) Formation: Cross-Chain Peroxyl Radical Addition and Decomposition in Phospholipids." Over the past two decades the 4-hydroxyalkenal (4-HNE) has been one of the most studied reactive aldehydes derived from lipid peroxidation due to its diverse biological activities ranging from modulation of multiple signal transduction pathways to modification of proteins and DNA. However, the mechanisms that lead to the formation of 4-HNE in vivo is much less well-understood. Recently a novel chemical mechanism for the HNE formation has been proposed that involves intermolecular dimerization and oligomerization of fatty acid derivatives as key intermediates. In this presentation we provide mass spectrometry (MS) evidence that these key intermediates play an important role in the formation of 4-HNE from autooxidation of a biologically relevant phospholipid, di-linoleoyl-phosphatidylcholine (DLPC). The level of 4-HNE from autooxidation of DLPC is much higher than that from the same molar concentration of 1-palmitoyl-2-linoleoyl-phosphatydylcholine (PLPC). The key intermediates that are consistent with the cross-chain peroxyl radial addition and decomposition have also been identified using liquid chromatography (LC)-MS. Our studies provide the first evidence for the formation 4-HNE from phospholipids via the novel mechanism that may involve cross-chain peroxyl radical addition and decomposition.

24. Edward Prage, Prostaglandin E2 (PGE2) is a lipid mediator molecule that plays diverse signaling roles in a variety of tissues throughout the body. In its induced form, PGE2 is a principal mediator of pain, fever, and inflammation. Microsomal PGE2 synthase 1 (MPGES1) is the terminal enzyme in the induced PGE2 synthesis pathway. As such, MPGES1 shows promise as a therapeutic target in the treatment of inflammatory diseases, such as rheumatoid arthritis. Our goal is to utilize amide hydrogen/deuterium exchange mass spectrometry to locate inhibitor-binding sites, as well as to determine conformational changes that occur upon inhibitor binding. Our ultimate goes is to contribute to the creation of MPGES1-specific inhibitors and a new class of drugs to combat inflammatory diseases.

25. Yu Du, "Bacteria-based Assay for pfKASIII Inhibitor." The human malaria parasite, Plasmodium falciparum, uses type II fatty acid synthases (FAS II) to produce fatty acids. Even though erythrocytic stage malaria parasites scavenge fatty acids from the host, FAS II is still essential for their liver-to-blood cycle transition and is necessary for the production of certain fatty acids and related compounds such as important co-factor lipoate. β-Ketoacyl-ACP synthase III (PfKASIII) is one of the key enzymes in the initiating steps of FAS II pathway possessing two functions: catalyzes the decarboxylative condensation of malonyl-pfACP and various acyl-CoAs (KAS activity) and catalyzes the acyl-CoA:ACP transacylase reaction (ACAT activity) Earlier studies indicate that it is a promising target for anti-malarial drugs. Here we report the construction and characterization of a hybridized Lactococcus lactis strain, CL112/pfKASIII, which uses pfKASIII instead of its own KASIII to synthesize fatty acid. CL112/pfKASIII has comparable growth rate and fatty acid profile, suggesting that pfKASIII can use L.lactis ACP as substrate and perform normal function in L.lactis cells. This strain not only could be used as a bacterial model for pfKASIII inhibitor screening, but also provides an example of bacteria-based screening model for other anti-malarial compounds.

26. Leena Maddukuri, "Translesion DNA Synthesis Across the M₁dG Lesion Catalyzed by Y-Family Human DNA Polymerase Kappa (κ)." M₁dG (pyrimidopurinone deoxyguanosine adduct, 3-(2'-deoxy-beta-D-erythro-pentofuranosyl)pyrimido-[1,2-a]purin-10(3H)-one), a major and stable DNA-adduct of MDA (malondialdehyde) and basepropenal is known to block replication in vitro. The ability of polymerase κ to bypass M1dG lesions has been tested. Steady-state kinetic parameters and LC/MS/MS analysis showed that the insertion of dCTP opposite the M₁dG lesion is most favorable event (only 50-fold less efficient than dCTP opposite dG). Pol κ also inserts dGTP and dTTP opposite the M₁dG lesion, 7- to 8-fold less efficiently than dCTP incorporation. These data suggests that pol κ may bypass M₁dG lesions more accurately and efficiently than pol η. One possible explanation for this accurate bypass is might be the involvement of N-clasp domain of pol κ, which helps the polymerase to encircle the DNA at the primer-template active site and could conceivably be involved in the ring opening of M₁dG through stabilization of the nascent dCTP:M₁dG base pair.

27. Anh Hoang, The mechanism of formation for hemozoin, a detoxification byproduct of several blood-feeding organisms (including malaria parasites), has been a subject of debate; however, recent studies suggest that neutral lipids may serve as a catalyst. In this study, a model system of neutral lipid nanospheres was employed to investigate the formation of β-hematin, synthetic hemozoin, at the lipid-water interface. A solution of monoglyceride (monostearoylglycerol (MSG) or monopalmitoylglycerol (MPG) ) dissolved in acetone and methanol was introduced to an aqueous surface. Imaging (fluorescent, confocal and transmission electron microscopy (TEM)) and dynamic light scattering analysis of samples obtained from beneath the surface confirmed the presence of solid lipid particles existing in two major populations: lower micrometer and lower hundred nanometer. The introduction of Fe(III)PPIX to this lipid particle system under physiological conditions (37C, pH 4.8) produced β-hematin with an average half life of 0.5 min-1. TEM of extruded monoglyceride (MSG or MPG) with Fe(III)PPIX through a 200nm filter produced β-hematin crystals position along and parallel to the interface. TEM data coupled with methyl laurate and docosane studies which demonstrated that the OH and C=O is necessary for nucleation, suggested that β-hematin crystallizes via epitaxial nucleation at the lipid-water interface through interaction of Fe(IIII)PPIX with the polar head group. Once nucleated, the crystal grows parallel to the interface until growth is hindered by the geometry of the lipid particle. The hydrophobic nature of the mature crystal favors an interior transport so that crystals are aligned parallel to the lipid-water interface and each other.

28. Lilu Guo, The 18 amino acid peptide Ac-DWFKAFYDKVAEKFKEAF-NH2 (4F), is an HDL mimetic that was recently shown to reduce atherosclerotic lesions when injected in a mice model of atherosclerosis. We want to develop a facile method to chronically deliver 4F peptide as a treatment for atherosclerosis. Our strategy is to transform the probiotic bacteria,E. coli Nissle 1917 (EcN), to secrete 4F peptide within the GI tract of the affected individual. Long-term colonization by 4F expressing EcN could provide a chronic source of therapeutic 4F. For efficacy, 4F peptide must cross the GI tract and enter circulation. We therefore designed a synthetic gene constitutively expressing a fusion peptide containing an amylase secretion leader sequence, a penatratin transcytosis sequence, and 4F, followed by a 6xHis tag (AP4F). A similar gene lacking the penatratin sequence was also synthesized (A04F). Supernatant from EcN transformed with AP4F or A04F were purified on nickel beads and peptide levels quantified by anti-HIS tag ELISA. Treatment with nickel purified AP4F or A04F dose-dependently inhibited LPS stimulated adhesion of THP-1 cells to HUVEC, similar to what has been reported for 4F. In CaCo-2 monolayer transwell experiments to model transcytosis across gut wall, no detectable amounts of AP4F or A04F were found in the basalateral compartment. MALDI/MS analysis of nickel purified AP4F found extensive fragmentation and the loss of the penetratin sequence. Therefore, while EcN can express a 4F-like peptide with appropriate bioactivity, additional optimization of bacterially expressed 4F peptide is required to reduce proteolysis and improve transcytosis.

29. Joshua Swartz, Variability among most malarial rapid diagnostic tests (RDTs), extreme sensitivity to thermal storage conditions, and poor performance at low parasitemia have exemplified the need for new approaches to rapid malarial diagnostics. To overcome these limitations, a simple alternative diagnostic design based on the rapid, radial transport and deposition of small particles at the edge of an evaporating drop has been developed. Several different sizes of fluorescent polystyrene microspheres (0.5-6.3 ?m) were functionalized with Ni-NTA moieties that are traditionally used in metal affinity chromatography for the purification of His-tagged proteins. Ni-NTA has a reported high binding affinity to histidine-rich protein 2 (HRP-II), a protein highly expressed in Plasmodium falciparium. As a model, a peptide containing three repeat motifs (AHHAHHAAD) found in HRP-II (HRP) was synthesized using standard FMOC peptide chemistry and tagged with a fluorescent TAMRA on the N-terminus. Upon incubation with the Ni-NTA micropsheres, HRP localizes to the microspheres and is easily detected using fluorescence microscopy. However, when the particles were deposited onto both glass and Ni-NTA coated glass slides, HRP does not induce a visible change of deposition when compared to control particles, likely due to the small size of the peptide. Current work involves synthesizing 15 nm Ni-NTA containing gold nanoparticles (AuNP) to explore their aggregation behavior in the presence of HRP or poly-L-histidine (PLH). Since AuNP’s exhibit specific Plasmon resonance absorption dependant on their size and concentration, it is expected that a distinct color change will be detected when aggregation occurs as a result of target binding.

30. Rong Gao, "Modularities of domains of transfer RNA in translation," Rong Gao and Anthony C. Forster, Department of Pharmacology and Vanderbilt Institute of Chemical Biology, Vanderbilt University Medical Center. During protein synthesis, tRNAs transfer specific amino acids to a growing polypeptide chain on the ribosome. There are four standardized modules in aminoacyl-tRNAs: an amino acid, an invariant 3'-terminal CCA, a three-base anticodon and a tRNA body. Here, the variability of each module of tRNA is investigated using a published pure translation assay1 and tRNA^Ala. Comparison of natural tRNA^Ala with an in vitro synthesized version shows that the absence of RNA modifications has little effect on incorporation of multiple amino acids. Replacing the 3'-terminal CCA with CdCA (useful for incorporating unnatural amino acids) moderately reduces translation efficiency. However, substituting the anticodon dramatically inhibits translation. Surprisingly, there is little effect of exchanging alanine to two unnatural amino acids, allyl glycine and methyl serine. These results define the modularity of individual domains of tRNA in translation and suggest a strategy for improving incorporation of unnatural amino acids.

31. Takashi Suzuki, "Novel Eicosanoids Derived from Convergence of the 5-LOX and COX-2 Pathways," Takashi Suzuki, Markus Griesser, Noemi Tejera Hernandez and Claus Schneider. Leukotrienes and prostaglandins are lipid mediators with distinct physiological roles in tissue homeostasis but also in inflammation and cancer. The initial oxygenation of arachidonic acid by either 5-LOX or COX-2 is recognized as the committed step towards leukotriene or prostaglandin biosynthesis. We recently provided in vitro evidence for a possible cross-over of the 5-LOX and COX-2 biosynthetic pathways: the 5-LOX product, 5S-hydroxyeicosatetraenoic acid (5S-HETE) is a selective and efficient substrate for COX-2. The major oxygenation product is a bicyclic di-endoperoxide. In activated RAW 264.7 macrophage cells incubated with 5S-HETE we have now detected two novel eicosanoids that are derived from the bicyclic di-endoperoxide intermediate. Using LC-MS, UV, and NMR spectroscopy we identified these products as eicosanoids containing a hemiketal ring moiety. We detected a different ratio between the two hemiketal eicosanoids in RAW264.7 cells as compared to non-enzymatic transformation of the di-endoperoxide implicating enzymatic contribution in the synthesis of the hemiketal eicosanoids from the di-endoperoxide. Further characterization of the cross-over of the 5-LOX and COX-2 pathways and the biological activity of the hemiketal eicosanoid products could shed new light on the etiology and regulation of common inflammatory diseases like asthma and cancer.

32. Richard Watts, We have recently published the rates of ribosome-catalyzed peptide formation between certain pairs of amino acids1. Using formyl-Met-tRNA as the electrophile, the order of nucleophile reactivity was Phe > Ala > Pro > N-methyl-Phe >> N-butyl-Phe, apparently independent of the adaptor tRNA. Because this order correlated roughly with the steric bulk of the amine nucleophile, we hypothesized that peptide bond formation is rate-limiting in these reactions. To test this hypothesis, here we examine similar reactions under aqueous, uncatalyzed conditions. Amino acid amides are used to model the nucleophiles, and highly activated formyl-Met-N-Hydroxy-Succinimide ester (fMet-NHS) at 4ºC is used to best model the electrophiles. The order of reactivity is Gly > Phe > Ala > Pro > N-Me-Gly >> N-Me-Phe, correlating perfectly with that in the ribosome-catalyzed reactions. When the results are controlled for the different measured nucleophile pKa’s, the order of reactivity becomes Gly > Ala > Phe > Pro > N-methyl-Gly >> N-methyl-Phe, correlating with increasing steric bulk. These data support our hypotheses that the rates of peptide-bond formation on the ribosome are heavily influenced by the sterics and pKa of the nucleophile amine, and that peptide bond formation is the rate-limiting step in translation.

33. Noemi Tejera Hernandez, "Identification and stereochemical analysis of dihydroxy arachidonic acids formed in the reaction of COX-2 and aspirin treated COX-2 with 5S-hydroxy arachidonic acid (5S-HETE)," Noemi Tejera Hernandez, Surafel Mulugeta, Takashi Suzuki, Claus Schneider. COX-2 is a key enzyme that catalyses the first committed step in the synthesis of prostaglandins from fatty acid precursors. Under normal physiologic conditions, prostaglandins have essential homeostatic functions, but they are also implicated in a number of pathological conditions, such as inflammation, cardiovascular disease and cancer. Several drugs, including aspirin, can block their formation, playing an important role in prevention of pain, fever, and inflammation. It has been shown that acetylation of a critical serine residue in the active site of COX-2 by aspirin abrogates prostaglandin synthesis and triggers formation of 15R-HETE as the sole product of oxygenation of arachidonic acid. Recently, 5S-HETE has been described as an alternative substrate for COX-2, forming a novel di-endoperoxide as the main product. Here, we identified the by-products of this reaction as 5S,15S-diHETE, 5S,15R-diHETE, and 5S,11R-diHETE using LC-MS, UV, NMR, and CD spectroscopy, and HPLC. We also investigated product formation of acetylated COX-2 with 5S-HETE as a substrate. Aspirin attenuated formation of the di-endoperoxide and resulted in the formation of an abundant product that we identified as 5S,15R-diHETE. We conclude that aspirin treatment of COX-2 has similar effects on the oxygenation of 5S-HETE and arachidonic acid; resulting in 15R-oxygenation of either substrate. In addition, the by-products of the conversion of 5S-HETE by the untreated enzyme are the 5-hydroxy analogues of the HETE by-products formed from arachidonic acid.

34. Kellen Harkness, Thiolate-protected gold nanoparticles (AuNPs) are a versatile nanomaterial which serves as a scaffold for a rapidly expanding range of applications such as catalysis, biosensing, and biomimicry. Many biomolecules with a free thiol, such as glutathione and cysteine-bearing peptides, can be employed as a ligand. The ability to quickly and precisely characterize bound thiolate species is key to any biological application of AuNPs. An optimal characterization technique will provide three important pieces of information: the number, the placement, and the structure of thiolated molecules on the AuNP surface. However, this information is far beyond the capabilities of current analytical techniques. We propose that matrix-assisted laser desorption/ionization-ion mobility-mass spectrometry (MALDI-IM-MS) is a characterization technique which may be capable of providing the most useful information in a quick and facile platform. Current results reveal the capability of relative quantitation when AuNPs are decorated with tiopronin and glutathione. The quantitation matches well with the results obtained from nuclear magnetic resonance (NMR) spectroscopy. In addition, relative quantitation of tiopronin and (11-mercaptoundecyl)hexa(ethylene glycol) ligands, which is not possible by NMR spectroscopy, is made possible by MALDI-IM-MS. Future development of methodology for characterizing ligand placement and ligand structure will also be discussed.

35. Cody Goodwin, The complexity associated with natural products poses a challenge for structural determination, but carries promise for potential therapeutic applications. Natural products often contain non-standard amino acids, cyclic ring structures, amino acid isomers, and other unique chemical components. The organisms from which these compounds are elicited have evolved in environments where it is necessary to be both frugal with resources available and intelligent with defense systems necessary for the proliferation of the species, which lends to the uncommon nature and high degree of bioactivity of the extracted compounds. The biomolecules studied are naturally occurring cyclic peptides obtained from hypogeal organisms, which has remarkably constant temperature and humidity. It is of interest to investigate the natural products of these non-ribosomal organisms, which have evolved under naturally controlled settings, since it has been argued that the unique structure of these biomolecules lends to the high degree of bioactivity. In this study, collisional cross-sections for various cyclic peptide natural products, purified from organisms inhabiting hypogeal environments have been obtained using ion mobility-time of flight mass spectrometry. The collision cross-section data was then correlated for specific cyclic peptides with conformations generated from sampling enhanced molecular dynamic simulations. Low energy conformers that corresponded to collision cross-sections obtained were then investigated for structural similarities that could explain gas-phase behavior that is causal of the unique drift-times that fall below the common peptide trendline. The resulting data is potentially beneficial to understanding the bioactivity of these natural products, or for identifying cyclic peptides from preparations from different organisms.

36. Julie Field, The human serotonin (5-HT) transporter (hSERT) is an important target for both antidepressants, such as the serotonin-selective reuptake inhibitors (SSRIs), and drugs of abuse, such as cocaine and ecstasy (MDMA). The determination of a crystal structure of an SLC6 gene family member, the leucine transporter LeuTAa, and subsequent homology modeling have provided new insight into hSERT structure and ligand binding, implicating TMs 1, 3, 6 and 8 as potentially critical helices for substrate binding and translocation. Although we have provided evidence that TMs 1 and 3 of hSERT coordinate specific antidepressants (e.g. citalopram) at sites within or near the 5-HT binding pocket, the contributions of TMs 6 and 8 to ligand binding and translocation have not been characterized in depth. We subjected TM6 to a substituted cysteine-accessibility method (SCAM) analysis, identifying residues that significantly compromise SERT function in response to cysteine substitution, that can be protected by ligands, and that change their accessibility in response to ligand. In addition, both TM6 and TM8 were subjected to RosettaLigand-guided mutagenesis and subsequent ligand screening to assess potential impact on substrate and SSRI interactions. In this study, we provide evidence of interactions between residues in TM1 and TM6 and between TM1 and TM8, that are at, or below, the level predicted for the 5-HT binding pocket, and that appear to influence substrate translocation and antagonist potency. Our studies uncover new interactions that we propose help stabilize hSERT in distinct conformations necessary for efficient 5-HT transport and high-affinity antagonist interactions.

37. Hao Huang, "Successive molecular hand-off events coordinated by the SV40 helicase direct initiation of SV40 DNA replication." SV40 DNA replication, a simple but powerful model system, can be reconstituted with purified recombinant proteins in vitro, providing an opportunity to identify at the atomic level how a small number of eukaryotic proteins interact to initiate replication at a defined origin. The viral helicase T antigen (Tag) is the heart of the viral replisome, assembling a 1.2 MDa pre-replication complex on the viral origin, melting duplex DNA, and recruiting RPA and DNA polymerase alpha-primase (pol-prim) for initiation. Previous work from our labs and others suggests that origin melting leads to a transient ternary complex of Tag, RPA, and ssDNA. This complex dissociates as longer ssDNA emerges from the helicase, generating RPA-ssDNA parental template. Tag associated with pol-prim is proposed to then remodel the bound RPA into a weaker binding mode, allowing pol-prim to gain access to the exposed ssDNA and begin primer synthesis (EMBO J 25, 5516; NSMB 12, 332). This model predicts specific contacts of pol-prim with the helicase domain of Tag that are required for initiation of replication. To further test this model, we determined the structure of the Tag-interacting domain of pol-prim regulatory subunit p68, characterized the binding surfaces of p68 and Tag, and will present evidence that interaction between these surfaces is needed for initiation in a reconstituted system. Possible implications for cellular initiation will be discussed.

38. Gulfem Guler, "Recruitment of DNA Helicase B to Chromatin in Response to DNA Damage during S Phase: Pathways for DNA repair and replication are highly integrated to ensure genomic stability." DNA helicase B (DHB), a robust 5'-3' DNA helicase of superfamily I conserved among vertebrates, is necessary for initiation of DNA replication in both mouse and human cells. In accordance with a second role in preserving genomic stability, human DHB (HDHB) silencing delays recovery from camptothecin treatment and elevates aphidicolin-induced chromosomal breaks. Exposure of tumor cells with genotoxins that arrest replication forks leads to accumulation of HDHB on chromatin, most prominently in S phase. This accumulation is not diminished by ATM or ATR silencing or by wortmannin treatment, suggesting that HDHB recruitment is ATM/ATR-independent. RPA silencing reduces genotoxin-induced HDHB recruitment to chromatin. HDHB interacts physically with RPA through the N-terminal domain of RPA70 subunit (RPA70N), which serves as a recruitment scaffold for damage response proteins p53, ATRIP, Mre11, and Rad9. RPA70N interacts with an acidic motif in the helicase domain, that has sequence similarity to the RPA70N binding peptides from ATRIP, p53 and Rad9. This motif is conserved among vertebrate DHB proteins but absent in the bacterial superfamily 1 helicase RecD. Further analysis is in progress to test a working model that physical interaction of RPA70N with this motif recruits HDHB to sites of DNA damage during S phase.

39. Sarah Scott, Phospholipase D (PLD) is an essential enzyme responsible for the production of the lipid second messenger phosphatidic acid. Phosphatidic acid participates in both G protein-coupled receptor and receptor tyrosine kinase signal transduction networks. The lack of potent and isoform-selective inhibitors has limited progress in defining the cellular roles of PLD. We used a diversity-oriented synthetic approach and developed a library of PLD inhibitors with considerable pharmacological characterization. Here we report the rigorous evaluation of that library, which contains highly potent inhibitors, including the first isoform-selective PLD inhibitors. Specific members of this series inhibit isoforms with >100-fold selectivity both in vitro and in cells. A subset of inhibitors was shown to block invasiveness in metastatic breast cancer models. These findings demonstrate the power of diversity-oriented synthesis combined with biochemical assays and mass spectrometric lipid profiling of cellular responses to develop the first isoform-selective PLD inhibitors--a new class of antimetastatic agents.

40. Timothy Panosian, Phosphopentomutases (PPMs) catalyze the interconversion of D-ribose-5-phosphate and α-D-ribose-1-phosphate. PPM activity is Mn2+ dependent and is greatly enhanced in vitro by pretreatment with a bisphosphate activator. To explore the mechanism of PPM, the X-ray crystal structure of a PPM from Bacillus cereus was determined in the apo state to 1.85 Å and bound with either the substrate R5P or activator α-D-glucose-1,6-bisphosphate, to 1.9 Å and 2.3 Å respectively. PPMs are members of the alkaline phosphatase-like core domain superfamily of proteins, and their structure shares the common architecture of a highly homologous core domain attached to a unique cap domain by two flexible linker strands. Interestingly, electron density for both the substrate, D-ribose-5-phosphate, and product, α-D-ribose-1-phosphate were observed in an electropositive cleft at the interface of the two domains. Substrate was observed in only one of the three monomers in the asymmetric unit. Large changes in the positions of side chains of Arg-208 and Arg-212 between the monomers alter the electrostatic surface of the active site and likely lead to the variation in substrate binding. In the active site of all determined structures, Thr-85 was phosphorylated. Treatment with α-D-glucose-1,6-bisphosphate increased the population of phosphorylated Thr-85 and the relative enzymatic activity increased directly with increased phosphorylation. These results suggest that the function of the bisphosphate activator is to prime Thr-85 with phosphate and that the reaction mechanism of PPM proceeds through a bisphosphate intermediate that must be reoriented during catalytic turnover.

41. Colleen McGrath, "Characterization of Lipid Electrophile-Mediated Inhibition of the THP-1 Macrophage Inflammatory Response," Colleen E. McGrath, Keri A. Tallman, Simona G. Codreanu, and Lawrence J. Marnett. A major component of the inflammatory response is the recruitment and activation of macrophages, which secrete factors that mediate elimination of foreign material and resolution of inflammation. Inflammation leads to the production of reactive oxygen species that oxidize lipids and generate a number of reactive electrophiles, which inhibit the activation of macrophages. One such reactive electrophile, 4-hydroxynonenal (HNE), has been studied by our laboratory for its ability to covalently modify protein targets and modulate cellular stress pathways in human colorectal carcinoma cells. We are currently examining the modulation of a process related to tumor development (inflammation) in macrophages in response to treatment with various lipid electrophile components of oxidized LDL. Herein we investigate the ability of HNE, 4-oxononenal (ONE), and several oxidized derivatives of phosphatidylcholine to modulate the inflammatory response in differentiated human acute monocytic leukemia (THP-1) cells. ELISA and western blot assays demonstrated that acute treatment with subcytotoxic concentrations (5-10mM) of HNE and ONE inhibited the production of the pro-inflammatory cytokines IL-1b, IL-6, and TNFa in lipopolysaccharide (LPS) activated macrophages. Inhibition ranged from 20-80% of control for the various cytokines tested. Alkynyl derivatives of HNE were used in conjunction with azido-biotin and click chemistry to affinity-tag and enrich HNE modified proteins. These studies revealed that the inflammation associated transcription factor, signal transducer and activator of transcription 1 (Stat1), was modified by HNE in a dose-dependent manner. Furthermore, such modification contributed to a decrease in phosphorylation at Stat1 residues Y701 and S727, which may contribute to the ability of lipid electrophiles to modulate the inflammatory response of activated macrophages.

42. Vamessa Phelan, "Mass Spectrometry for Adenylation Enzyme Specificity." Anthramycin is a potent antitumor and antibiotic benzodiazepine alkaloid produced by the thermophilic actinomycete Streptomyces refuineus. Chemical complementation studies of gene knockouts provide evidence methylation occurs after biosynthesis of the intermediate 3-hydroxyanthranilic acid (3-HA), a known primary metabolite. The anthramycin cluster contains two nonribosomal peptide synthetase (NRPS) genes, one of which, orf21, shows little sequence similarity to known adenylation domains(A-domains) including actinomycin synthetase, which has previously been demonstrated to activate 4-methyl-3-hydroxyanthranilic acid (MHA). We have heterologously expressed orf21 and established the A-domain specificity using a rapid, highly sensitive mass spectrometry based method measuring the isotopic back exchange of unlabeled pyrophosphate into γ-¹⁸O₄-ATP via MALDI-ToF-MS, ESI-LC/MS and ESI-LC/MS/MS. This low volume (6μL) method detects as little as 0.01% (600fmol) exchange comparable to previously reported radioactive assays.

43. Dawn Makley, "An Umpolüng Approach to Amide Synthesis: Coupling of α-Bromo Nitroalkanes to Amines and its Application in Peptide Synthesis," Dawn M. Makley, Bo Shen, Jeffrey N. Johnston. A novel amide bond forming reaction involving an umpolüng-type coupling of α-bromo nitroalkanes and activated amines is presented. 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. The inherent reversal of reactivity in this reaction should provide unique opportunities in peptide synthesis. 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. The discovery and scope of this reaction, as well as initial applications will be described.

44. Aroop Chandra, "Total Synthesis of the Lycopodium Alkaloid (+)-Serratezomine A," Aroop Chandra, Julie A. Pigza, Jeong-Seok Han, Daniel Mutnick, and Jeffrey N. Johnston. (+)-Serratezomine A, a Lycopodium alkaloid, was isolated from the club moss L. serratum var. serratum in 2000 by Kobayashi et al. Serratezomine A displays moderate cytotoxicity against murine lymphoma L1210 cells (IC50 = 9.7 µg/mL) and human epidermoid carcinoma KB cells (IC50 >10 µg/mL) and exhibits strong acetyl-choline esterase (AChE) inhibition. Serratezomine A possesses a seco-serratinine-type skeleton containing six contiguous stereocenters and an all-carbon spirocyclic center embedded within its four connected ring systems. The first total synthesis of (+)-serratezomine A is presented. Overall, 15 steps (longest linear sequence) are required to prepare the natural product from a commercially available starting material, and assembly of the contiguous array of sixstereocenters is accomplished with high stereocontrol. Apart from the brevity of the current synthesis, there are a number of prominent features: (1) application of the free radical-mediated vinyl amination to construct the pyrrolidine ring; (2) a highly stereoselective intramolecular Michael addition to construct the cyclohexane ring; (3) the use of an oxidative allylation promoted by cerium(IV) (CAN) to establish the all carbon quaternary chiral center with the proper configuration; (4) a tandem saponification/intramolecular SN2 cyclization to form the bridging lactone; and (5) minimal use of the protecting groups.

45. Hubert Muchalski, "Alkylate and oxygenate before you protonate: novel reactivity of α-diazo imide system," Hubert Muchalski, Timothy L. Troyer and Jeffrey N. Johnston. The 1,2-amino alcohol is a widespread motif in therapeutically important small molecules, including aminopeptidase inhibitors like bestatin, recent isolates like the microsclerodermins and pedeins, as well as historically and clinically important antitumor agents such as Taxol. Many synthetic methods exist for the synthesis of 1,2-amino alcohols. Among them olefin functionalizations (e.g. Sharpless aminohydroxylation) are probably most reliable. Construction of this functional group via carbon-carbon bond formation is usually approached by addition of glycolic acid derivatives to azomethine which is commonly reffered to as the glycolate Mannich reaction. This strategy must diastereoselectively establish both stereocenters in a single step which represents a major challenge when developing these methods. Here we demonstrate a conceptually new approach to this motif that involves Brønsted acid activation of an imine and novel α-diazo imide. After initial carbon-carbon bond formation between azomethine and diazoalkane, a nucleophilic oxygen terminates the addition reaction by cyclization to the diazo carbon. The net result is a highly diastereoselective and efficient equivalent to a glycolate Mannich reaction. In order to extend substrate scope we turned our attention to metal based Lewis acids which uncovered novel reactivity of α-diazo imide towards imines, significantly different from commonly used ethyl diazoacetate. Details of these studies will be discussed.

46. David Nanneman, Nucleoside analogs comprise a large proportion of compounds applied to the treatment of HIV, hepatitis and other viral infections. Their broad use and applicability, however, is in contrast to the expensive price tag associated with these treatments and significant research effort is applied to identify and streamline the synthesis of these compounds. We have characterized and improved enzymes in a semi-synthetic pathway for 2’,3’-dideoxyinosine (Didanosine, Videx®), a generic anti-HIV drug. Purine nucleoside phosphorylase, the ultimate protein in the biosynthetic portion of the pathway, has been improved through computationally-guided redesign and directed evolution to achieve >40-fold turnover in E.coli cell free extracts. Phosphopentomutase from B. cereus, the penultimate protein, has been characterized biochemically and the structure solved by x-ray crystallography, identifying residues in the binding pocket for targeted mutagenesis. Additionally, intermediates in the pathway have been chemically synthesized allowing for stepwise improvement of each enzyme in the pathway. Methods developed in this work could be applied to the synthesis of other nucleoside analogs facilitating large-scale, affordable treatment of patients with HIV and hepatitis.

47. Jashim Uddin, Coupling of purine analogs with deoxyriboside has been employed for the synthesis of isotopically substituted deoxynucleosides, antitumor agents, and related biologically active molecules and 6-chloropurine-2′-deoxyriboside is a key intermediate for the synthesis of oligonucleotides bearing adducts of purine nucleosides. The chemical synthesis of this compound is problematic because of the instability of intermediates to the conditions of multi-step transformations that results limited successes. Here, we report an efficient synthesis of 6-chloropurine-2′-deoxyriboside from the reaction 6-chloropurine with 2’-deoxycytidine in presence of E. Coli nucleoside-2′-deoxyribsyltransferase (E.C. 2.4.2.6) enzyme in water in 60% yield. The structure, regiochemistry and absolute configuration were unambiguously confirmed by 2D-NMR spectroscopy and X-ray crystal analysis. The availability of a facile enzymetic route to this compound should enable its adoption for a broad range of application in molecular toxicology, cancer biology and related fields.

48. Megan Wadington, "Functional and Structural Studies of Two Glutathione Transferase Paralogs from Escherichia Coli K-12." Escherichia coli K-12 encodes nine members of the glutathione transferase (GST) superfamily. The biochemical and biological functions of most of these proteins remain to be elucidated. We have characterized two E. coli GST paralogues (YghU and YfcG) both structurally and functionally. Both enzymes possess a unique disulfide bond reductase activity, in that no cysteine residues on the protein participate in redox chemistry. Crystallographic analysis reveals structural evidence for this enzymatic activity. Two molecules of glutathione are bound in the active site of the YghU protein while a single molecule of glutathione disulfide is bound in the active site of the YfcG protein. The two structures overlay with an RMSD of only 1.163 angstroms. The glutathione substrates are bound in a remarkably similar confirmation, suggesting that the structures represent a disulfide bond reductase trapped in the oxidized and reduced forms.

49. Joshua Kerr, Simple and accurate methods of relative quantitation have gained increased interest mirroring the rise in interest of proteomics. While many methods exist, each has its own challenges and strong points. Multiplexed methods (i.e. analysis of many samples in a single mass spectrum), as afforded by ion mobility-mass spectrometry (IM-MS), exhibit increasing utility as sample complexity grows and limit of detection is lowered. The two-dimensional separation of IM-MS data allows for the use of lanthanide based shift reagents, which can shift signals, corresponding to biomolecules with specific functionalities, to an area in the spectrum not predicted to contain those signals in the absence of labeling. This report focuses on the ionization and collision cross section consequences of using lanthanide-based shift reagents.

50. Stephanie Hirst, Site-Directed Spin-Labeling Electron Paramagnetic Resonance (SDSL-EPR), in combination with the Rosetta protein folding algorithm (Rohl et al, 2004), could serve as an alternative method in structure elucidation of proteins that continue to evade traditional techniques, such as x-ray crystallography and NMR. A spin-label “motion-on-a-cone” model was used during de novo folding of T4-lysozyme and αA-crystallin, which resulted in full-atom models at 1.0Å and 2.6Å to the experimental structures, respectively (Alexander et al, 2008). This spin-label model and already-existing EPR distance data have been used to generate a knowledge-based potential, which we plan to implement into Rosetta as a constraints function. In addition, we have introduced a rotamer library of the methanethiosulfonate spin-label (MTSSL). Spin-label rotamers have been derived from conformations observed in crystal structures of spin-labeled T4-lysozyme. The method was benchmarked using T4-lysozyme where the spin-label was positioned at various levels of exposure. The results indicate that the method is able to recover important aspects of spin-label orientation with up to 0.4Å RMSD. In particular, experimental distances and distance distributions observed for T4-lysozyme were reproduced with relatively high accuracy.

51. Nathan Alexander, More than 50% of pharmaceuticals target membrane proteins, and it is estimated that membrane proteins make up 30-40% of all proteins. Therefore, a novel method was developed for membrane protein structure determination taking advantage of alternative experimental techniques which complement NMR and X-ray crystallography. These alternative experimental techniques, such as electron paramagnetic resonance (EPR) and cryo-electron microscopy, provide sparse or low resolution structural data, but cannot alone uniquely define a protein’s structure. In order to obtain atomic detail models, the method incorporates one or more types of sparse or low resolution experimental data into a protein structure prediction algorithm. The method will be benchmarked on a set of membrane proteins with known structure using real or simulated sparse or low resolution data in order to demonstrate the feasibility of obtaining membrane protein models of biologically relevant quality.

52. Elizabeth Dong, Voltage-gated potassium channels hERG and KCNQ1 are key in the repolarization of the cardiac action potential. Several drugs have been known to bind these channels and cause cardiac arrhythmias such as long QT syndrome (LQTS). Computational structure prediction methods provide an opportunity to determine the interaction between small molecules and these potassium channels. The objectives of the project are: 1) Parameter optimization of BCL::Align, a multiple sequence alignment tool that uses a customizable scoring function for sequence alignment and fold recognition, 2) Refine BCL::Align for membrane proteins, 3) Work with Rosetta to refine its use for membrane proteins and create structural models for hERG and KCNQ1, 4) Dock drug molecules to comparative models in order to understand the structural determinants for drug-protein interactions at atomic detail. A Monte Carlo algorithm was used to determine optimized weight sets for BCL::Align. For 100 iterations, we adjusted the parameters by a random value between -0.2 and 0.2, maximizing the Cline score for sequence alignment and the area under the ROC curve for fold recognition parameter optimization. In an evaluation of sequence alignment performance, BCL::Align ranked best in alignment accuracy when compared with other alignment methods. ROC curve analysis indicates BCL::Align 's ability to correctly recognize protein folds with over 80% accuracy. The flexibility of BCL::Align allows it to be optimized for use with potassium channels involved in LQTS. Continuing research seeks to determine the interactions between hERG and KCNQ1 with small molecules that hold therapeutic implications.

53. Mariusz Butkiewicz, "Enhancing Quantitative Structure Activity Relationships for Allosteric Metabotropic Glutamate Receptors." Metabotropic glutamate receptors play a key role in modulating synaptic activity in the Central Nervous System. As such they represent viable targets for treating neuronal disorders like schizophrenia, Parkinson's disease and others. mGluRs of subtype 5 bind to glutamate, an excitatory neurotransmitter, that modulates the response of mGluRs. Starting from a set of assay data for mGluR5 potentiators, machine learning approaches like artificial neural network and support vector machines were trained to predict the mGluR5 potentiation (EC50) for small molecules. The input data consists of scalar molecular descriptors, autocorrelation functions, and radial distribution functions. Prior to the training process, an enhanced refinement protocol to optimize the trained QSAR models was applied. The Presentation shows the methods developed and implemented to train those machine learning approaches and discuss the results of this test.

54. Jordan Willis, Trimers of gp-120 and gp-41 heterodimers make up a mature envelope spike on the surface of an human Immunodeficiency virus (HIV) particle. This envelope spike is the target of many of the characterized broadly neutralizing antibodies found in HIV-infected patients. Neutralization occurs when the virus loses its infectious properties. It is hypothesized that neutralization can be correlated to binding affinity through a currently unidentified relationship. Looking at the free-energy terms given in the Rosetta Dock Design Scripter and the binding affinity given through various laboratory techniques such as ITC (isothermal titration calorimetry) this relationship between neutralization, binding affinity, and Rosetta scoring terms can be established. If a tight relationship could be established, we could pursue the rational redesign of antibodies that have lost their neutralizing capacity for escape variants of HIV.

55. Steven Combs, The human serotonin transporter (hSERT) is a transporter responsible for the reuptake of serotonin (5ht) from the synapse. In order to combat depression, several antidepressants have been developed that block the function of SERT. Unfortunately, the crystal structure of hSERT has yet to be solved. We have built a comparative model of hSERT based off of the crystal structure from Aquifex aeolicus of the leucine transporter. In this study, S-citalopram has been docked into the homology model of hSERT using RosettaLigand. The results have been analyzed in light of the experimental data. We suggest mutations to interrogate the identified binding modes.

56. Kristian Kaufmann, Docking small molecules to comparative models is common place. However, quantitative studies examine the accuracy of docking small molecules to comparative models. The Crictical Assessment of Structure Prediction (CASP) experiment occurring biennial tests the performance of the best structure prediction techniques including those of comparative modeling. Last year CASP8 included predictions of 9 structures containing cofactors. We quantify the performance of RosettaLigand on comparative models, by docking the cofactors into the top blind predictions from the CASP 8 competetion.

57. Indriati Hood, "Acinetobacter baumannii regulates antibiotic resistance in response to extracellular NaCl.” Acinetobacter baumannii has recently emerged as an important cause of nosocomial infections. While extensive antimicrobial resistance has been described clinically, the molecular determinants mediating resistance and the mechanisms involved in regulation of these determinants are poorly defined. We sought to identify environmental signals encountered in the hospital setting or within the human host, which alter the resistance phenotype of A. baumannii. In this regard, we have identified NaCl as an important environmental signal that modulates antibiotic resistance in A. baumannii. A. baumannii cultured in NaCl concentrations ranging from 50mM-300mM show increased resistance to aminoglycosides, quinolones and colistin. The global transcriptional response to NaCl was determined by microarray analyses suggesting a role for efflux in mediating resistance to antibiotics. Specifically, 14 transporters with putative roles in antibiotic efflux were significantly up-regulated in response to NaCl. To identify genes responsible for this phenomenon, a transposon insertion library was generated and screened for mutants that failed to show increased resistance to antibiotics in response to NaCl. To date this screen has identified several candidates including genes with predicted roles in transcriptional regulation, resistance to oxidative stress and maintenance of membrane structure/stability. Taken together, these data demonstrate an adaptive response to NaCl resulting in resistance to antibiotics of distinct classes. Future work will be focused on using mutants identified in the transposon screen to define the molecular mechanisms governing NaCl-induced antibiotic resistance in A. baumannii.