2009 Honors Recipients
Majors in other disciplines often do honors research with Chemistry Faculty and those major departments are listed in parenthesis after their names.
Rachel Ackerman, '09 (Biochemistry)
Title: Genetic Sequencing and Distribution of C-type Allatostatin Neuropeptides in the Maine Lobster, Homarus americanus, Using Mass Spectral and Immunohistochemical Methods
Advisors: Elizabeth Stemmler and Patsy Dickinson
Abstract: The C-type allatostatin neuropeptide was originally characterized in insects to have PISCF C-terminal conservation, a pyroglutamine blocked N-terminus, and a disulfide bridge between its cysteine residues. It was found to inhibit both the synthesis of juvenile hormone and contractions of the gut. Recently, an EST encoding a novel C-type-like allatostatin has been identified in the Maine lobster, Homarus americanus. In this study the gene for the C-type-like allatostatin in lobsters was sequenced and the processing of the peptide from the preproprotein was predicted. This peptide shares SCF C-terminal conservation and a disulfide bridge between cysteines with the insect C-type allatostatin, but it has an unblocked N-terminus and an amidated C-terminus. A comparison of the distributions within the lobster of the insect and C-type-like allatostatins was conducted via MALDI-FT mass spectrometry and immunohistochemistry with antibodies directed against the two peptides. The C-type-like allatostatin was localized to the brain, commissural ganglia, eyestalk, and pericardial organ, while the insect C-type allatostatin was localized to those tissues as well as to the posterial midgut caecum. Althought the insect and C-type-like allatostatins have similar structures, their differing distributions suggest that they may be found to have distinct physiological functions.
Elizabeth Barton, '09
Title: A Physiology and Mass Spectrometric Investigation of Orcokinin Neuropeptides: Gut Activity and Peptide Modifications
Advisor: Elizabeth Stemmler and Patsy Dickinson
Abstract: The orcokinins comprise a family of neuropeptides found in crustaceans and insects. While orcokinins have been found in many nervous system and endocrine tissues in the American lobster, Homarus americanus, a physiological role has yet to be definitively determined. In this study we compared the physiological response of the mid/hindgut of two decapod crustaceans (H. americanus and Procambarus clarkii) to the
orcokinin, NFDEIDRSGFGFN (Orc[Asn13]). We found that application of a 10-6 M exogenous Orc[Asn13] standard did not significantly change the contraction rate of the mid/hindgut of H. americanus compared to the control. However, the contraction rate did increase significantly compared to the control for the red swamp crayfish P. clarkii when Orc[Asn13] was applied. It is surprising, given the close phylogenetic relation of
lobsters and crayfish, that this orcokinin does not seem to affect gut motility in lobsters.
In addition to physiological studies, we performed MALDI-FTMS studies to investigate post-translational processing of orcokinins to truncated, modified forms. We consistently detected a methyl-esterified orcokinin isoform, NFDEIDRSGFG-OMe (Orc[1-11]-OMe) upon extraction of H. americanus eyestalk tissues. We confirmed the identity of this isoform using SORI-CID and the analysis of a peptide standard and showed that the isoform is not NFDEIDRSGFA, an isomer that had been identified in previous studies. We found that formation of the C-terminal methyl esterified Orc[1-11] occurs during extraction when the peptide is present in lobster eyestalk tissue, but not when the methanol extraction solvent is presented to the Orc[1-11] synthetic standard. This suggests that a component of the tissue plays a key role in the reaction. This hypothesis was supported by the extraction of specific regions of eyestalk tissues, which showed that methyl esterification occurs only when certain types of tissues are extracted. While the reaction still occurred when tissues were frozen with liquid nitrogen prior to extraction, a protease inhibitor cocktail inhibited methyl esterification. Overall, our results suggest that the methyl esterification may result from reaction with an activated intermediate by means of an enzyme. We proposed that Orc[1-11]-OMe is detected because the extraction procedure converts the activated intermediate into Orc[1-11]-OMe. The selective methyl esterification of Orc[1-11] at the C-terminus may occur as full length orcokinins are converted, through an enzyme-catalyzed pathway, into a biologically active orcokinin isoform.
Matthew Eshelman, '09
Title: Conformational Analysis of a Catalytic, Enantioselective, Acyl-Transfer Tetrapeptide Using 1-D and 2-D Nuclear Magnetic Resonance and Computational Modeling
Advisor: Daniel O'Leary
Abstract: Conformational analysis was performed on a peptide designed to catalyze a longrange desymmetrization of a bisphenol. This peptide successfully catalyzes the longrange reaction, giving a single enantiomer product in high yield. 1D and 2D NMR spectra were used to estimate conformational constraints, which were incorporated into modeling
calculations to determine the three dimensional structure of the peptide. Unconstrained searches of the ethylated peptide and unmodified peptide indicated that a conformational change occurs when the peptide is ethylated. Constrained searches of the unmodified peptide gave structures that were different from structures obtained from the unconstrained search. The constrained search results show that the peptide adopts one
conformational family stabilized by four hydrogen bonds.
Han Guo, '09 (Biochemistry)
Title: Synthesis of Unnatural Azidosugars Designed to Hijack Helicobacter pylori's Pseudaminc Acid Biosynthestic Pathway
Advisor: Danielle Dube
Abstract: The gram negative bacterium Helicobacter pylori colonizes the gastrointestinal tract of humans and causes ulcers and gastric cancer. Unfortunately, current treatment methods to clear H. pylori infection are not effective. We plan to chemically target H. pylori by virtue of a unique sugar, pseudaminic acid (PseNAc), which is found on H. pylori but is absent from humans. PseNAc is essential for formation of functional flagella of H. pylori, and H. pylori that lack the ability to make PseNAc are avirulent. We plan to hijack the PseNAc biosynthetic pathway with an unnatural, azide-containing sugar. I started to synthesize the azido analog of the starting material of PseNAc biosynthesis - UDP-GlcNAz. The synthesis of UDP-GlcNAz is comprised of six steps and the first three steps have been completed successfully. I encountered difficulties when conducting the fourth step - synthesis of diallyl phosphorylated Ac3GlcNAz. Diallyl phosphonate was formed as a side reaction product due to the presence of water and/or impure reagent, m-chloroperoxybenzoic acid (mCPBA). Therefore, conducting this synthesis in a dry condition and using purified mCPBA are crucial to achieve a high yield of the desired product. Once diallyl phosphorylated Ac3GlcNAz is available, the synthesis of UDP-GlcNAz can proceed and finally this unnatural azidosugar will be available for further experiments to test its flexibility with the PseNAc biosynthetic enzymes and H. pylori.
Adam Hall, 08
Title: Retention of Phosphorus by Sediments in Merrymeeting Bay
Advisor: Dharni Vasudevan
Abstract: Sediments from Merrymeeting Bay, a freshwater tidal ecosystem at the confluence of the Kennebec and Androscoggin Rivers, and from Gulf Island Pond, an impoundment on the Androscoggin River, were investigated to determine their role in modulating availability of increased anthropogenic phosphorus loading. Sequential extractions divided sediment phosphorus into fractions representing different degrees of biological availability based on the mechanisms by which that phosphorus is retained in the sediment. Comparison of the two sites showed greater concentrations of phosphorus in Gulf Island Pond, closer to the paper mills, illustrating the impact of paper mill effluents on phosphorus availability in the Androscoggin. That impact was especially apparent in extraction with a reducing solution, which removed significantly greater amounts of iron and phosphorus from the sediment from Gulf Island Pond suggesting that differences in sediment properties and composition are responsible for the greater magnitude of phosphorus retention by iron oxides in this sediment. Because iron oxide bound phosphorous is sensitive to release under anoxic conditions this finding suggests the possibility that removal of oxygen diffusers currently operated in the Androscoggin, and subsequent decrease in oxygen availability, could result in a release of iron bound phosphorus. The same sequential extraction was applied to sediments from a depth profile in Merrymeeting Bay representing sediments deposited prior to industrial activity, during the mid 20th century, and in recent years. Consideration of fractions which would provide a durable record of past phosphorus availability supported the conclusion that phosphorus availability in the ecosystem appears to have increased with loading of phosphorus from anthropogenic sources. Greater amounts of phosphorus retained in organic matter in the more recently deposited sediments suggest that the ecosystem is not currently P. Furthermore, there was no indication that P availability has decreased since the mid-20th century.
Lindsay Heuser, 09
Title: GC/MS Characterization of Products Formed in the Heterogeneous Reaction of Oleic Acid with Atmospheric Oxidants
Advisor: Laura Voss
Abstract: Bulk oleic acid and oleic acid monolayers were oxidized by ozone to approximate atmospheric processing of fat-coated aerosols surfaces at the air-water interface. The products of the oxidation reaction were separated using gas chromatography and analyzed using mass spectrometry. To facilitate GC/MS analysis of these oxidized products, solid phase extraction and derivatization techniques were developed. GC/MS analysis of oxidized bulk oleic acid and monolayer samples indicated formation of primary ozonolysis products. While previous studies show evidence of secondary polymer formation during the oxidation process, this was not observed in our experiments. Mass spectra of oxidized bulk products instead exhibited fragmentation consistent with autoxidation products, which suggested an interaction between oleic acid and radical species. Experimental conditions during bulk oleic acid oxidation may have inhibited polymer formation and promoted autoxidation reactions.
Jason Iskarpatyoti, '09
Title: Computational and Experimental Studies of Tris-(3,3'bis(5,6-dimethyl-1,2,4-triazine))ruthenium(II) and Related Compounds
Advisor: Jeffrey Nagle
Abstract: Tris-(2,2'-bipyridine)ruthenium(II) (Ru(bpy)32+) and its many derivatives have been studied for their potential as light absorption sensitizers. In this research, computational results using the Amsterdam Density Functional (ADF) program for Ru(bpy)32+ and tris-(3,3'-bis(5,6-dimethyl-1,2,4-triazine))ruthenium(II) (Ru(taz)32+) are compared to experimental results. For these two RuII complexes, the geometry optimization results from ADF are in excellent agreement with experimental results, while the time dependent density functional theory calculated absorption spectra are in reasonable agreement with the relative energies and intensities seen in the experimental absorption spectra. This agreement allows us to make confident predictions for several potential new RuII-taz based complexes. Specifically, the computations suggest that two of these complexes should have interesting potential as light absorption sensitizers. Furthermore, three complexes of the type Ru(bpy)2(LL)2+ (where LL is a taz-based ligand) also have interesting absorption features that reflect their mixed-ligand character.
In addition to the computational analysis, syntheses of two new RuII-taz based complexes were attempted. The characterization of the products shows that milder conditions will be required to synthesize these complexes to prevent the decomposition of the taz-based ligand.
Maria Koenigs, '09 (Biochemistry)
Title: Metabolic Profiling of Helicobacter pylori Glycosylation
Advisor: Danielle Dube
Abstract:Helicobacter pylori is a pathogenic bacterium that is responsible for gastrointestinal diseases such as gastritis, duodenal ulcers, and some gastric cancers. Recent studies have revealed that H. pylori contains a unique glycan that is linked directly to its virulence. Here we set out to further explore the glycobiology of H. pylori by profiling this pathogen's glycome. We employed a technique termed metabolic oligosaccharide engineering that has been utilized extensively to profile glycosylation of mammalian cells. H. pylori cells were grown in liquid culture supplemented with the unnatural azide-containing sugar peracetylated N-azidoacetylglucosamine (Ac4GlcNAz). H. pylori cells and lysates were then probed via Staudinger ligation to assess azide incorporation. Western blotting reveals that azides are metabolically incorporated into several intracellular H. pylori proteins. Further experiments reveal that azide-dependent signal is diminished by treatment with peptide-N-glycosidase-F and by treatment with multiple O-glycosidases, suggesting that the azide is metabolically incorporated into both N- and O-linked glycans and that protein glycosylation in H. pylori is far more abundant than previously believed. Immunoprecipitation results reveal that Ac4GlcNAz is not incorporated into FlaA and FlaB, the only known glycoproteins in H. pylori. Future work will further establish the metabolic fate of Ac4GlcNAz in H. pylori and the identity of labeled proteins.
Mark Newman, '09
Title: Aromatic and Aliphatic Amine Sorption to Aluminosilicates
Advisors: Dharni Vasudevan
Abstract: The cationic amine functionality is an important structural moiety within many potentially harmful compounds that are released into the environment. The sorption of cationic amines to soil influences the route and extent of exposure to humans and ecosystems. Therefore, the goal of this study was to examine the influence of chemical structure and concentration on cationic aromatic amine sorption to negatively charged aluminosilicates, a ubiquitous mineral in soil systems. To this end, isotherms for aniline, N-methylaniline, and N,N-dimethylaniline sorption to montmorillonite and kaolinite were obtained and quantum chemical calculations were used to simulate electrostatic attraction and potential intermolecular interactions in this system. The influence of molecular structure was clearly evident as N,N-dimethylaniline sorbed to a greater extent than N-methylaniline which generally sorbed to a greater extent than aniline to both kaolinite and montmorillonite. Quantum chemical calculations of the attraction between cationic amines and anionic chloride were used to simulate the interaction between a cationic amine and a negatively charged surface site. The decrease in the energy of amine-chloride complex with the subsequent addition of methyl groups to the amine nitrogen allowed us to postulate that higher extents of sorption observed for N-methylaniline and N,N-dimethylaniline was due to the charge distribution within the entire molecular structure facilitating a greater electrostatic attraction. Consideration of the distinct kaolinite and montmorillonite sorption isotherms coupled with indirect evidence from desorption experiments suggested that aniline sorbed to the external surface of montmorillonite, while N-methylaniline and N,N-dimethylaniline sorbed to both the external surface and the interlayer of montmorillonite. In addition, Nvi methylaniline and N,N-dimethylaniline exhibited sigmoidal isotherms indicative of potential intermolecular interactions. Isotherm shapes at intermediate dissolved concentrations (≈2.00 x 10-5 – 3.44 x 10-4 M) and quantum chemical calculations of potential intermolecular ∏ interactions pointing to the feasibility of cation – ∏, polar – ∏, and ∏–∏ interactions helped explain the significantly higher extent of sorption of Nmethylaniline and N,N-dimethylaniline as compared with aniline. Finally, at high dissolved concentrations (>3.44 x 10-4 M), aniline, N-methylaniline, and N,Ndimethylaniline all sorbed to an extent close to or greater than the CEC of montmorillonite. Sorption above the CEC was postulated to occur by a mechanism other than electrostatic attraction because the CEC represents the availability of negatively charged surface sites. Therefore, following saturation of negatively charged sites, we postulated that the montmorillonite surface was modified to a non-polar surface, which allowed for additional sorption via hydrophobic exclusion of the cationic amine from aqueous solution onto the non-polar aluminosilicate surface.
Nicholas Selden, '09 (Biology)
Title: Towards Enqymatic Synthesis of a Novel Helicobacter pylori Antibiotic
Advisor: Danielle Dube
Abstract: Helicobacter pylori are gram-negative pathogenic bacteria that cause gastric cancers, ulcers, and gastritis and are found in the gastrointestinal tract of roughly 50% of humans worldwide. Although H. pylori can be treated with an array of antibiotics, new treatment methods are necessary because of the bacteria's ability to develop resistance. We are developing an antibiotic strategy based on the presence of the unique sugar pseudaminic acid (PseNAc) on the surface of H. pylori but not on human cells. PseNAc is essential for formation of functional flagella on H. pylori, and H. pylori that lack the ability to make PseNAc are avirulent. In a two-step strategy, this work attempts to (1) hijack H. pylori's PseNAc biosynthetic pathway with unnatural azidosugars, then (2) covalently deliver therapeutic compounds to azide-covered H. pylori via Staudinger ligation to perturb the pathogens. For this strategy to be viable, H. pylori's PseNAc biosynthetic pathway must be permissive of azide-containing substrates. We wish to examine the substrate flexibility of PseNAc biosynthetic enzymes by comparing in vitro kinetic data of natural versus azide-containing substrates. The five enzymes (PseB, PseC, PseH, PseG, and PseI) required in this pathway have been expressed by E. coli and the first two (PseB and PseC) have been purified. The enzymatic activity of PseB on its natural substrate has been confirmed. The remaining enzymes need to be purified and tested on their natural sugars before all enzymes can be tested on their azidosugar derivatives. These data are preliminary results that will help assess the substrate flexibility of PseNAc biosynthetic enzymes to azides.
Matthew Shew, '09 (Biochemistry)
Title: Validating the Golgi Two-Hybrid Assay's Utility in Studeying Glycosylated Proteins
Advisors: Danielle Dube
Abstract: Despite its utility in detecting interactions among nuclear and cytosolic proteins, the traditional yeast two-hybrid assay (Y2H) systematically misses the interactions between secreted and cell surface proteins that traffic through the secretory pathway en route to the outside of the cell. To overcome this limitation, the Kohler lab developed the Golgi two-hybrid assay (G2H)1 which tests secretome protein-protein interactions in the Golgi, where proteins have their full complement of post-translational modifications. In the G2H, protein-protein interactions are linked to a selectable parameter within the Golgi - Och1 mannosyltransferase activity. Previous results dy Dube and Kohler demonstrate that the G2H can successfully detect the known protein-protein interaction between MyoD and Id2 in the Golgi. Here we set out to validate the G2H's ability to detect the known secretome dependent interaction between polypeptide N-acetylgalactosaminyltransferase-2 (ppGalNAcT-2) and its substrate Muc5AC22 using two readouts; a growth assay at high temperatures and/or with congo red, and a fluorimeter based assay. Both readouts in the G2H assay indicate that the known secretome interaction between ppGalNAcT-2 and its substrate Muc5 can be detected, however distinguishable background growth in the negative control of ppGalNAcT-2 is of concern. This study demonstrates that the Golgi-two hybrid assay is amenable to the study of secretome dependent protein interactions; however the assay will need further calibration in order to do so.