Master of Science / Biochemistry and Molecular Biophysics / John M. Tomich / NC-1007 (CK₄-M2GlyR) (PARVGLGITTVLTMTTQSSGSRAKKKK) is a synthetic peptide modeled after the second transmembrane segment of the spinal cord glycine receptor’s α-subunit, and has demonstrates the capacity to oligomerize to form transmembrane channels with Cl[superscript]- permselectivity. While studies into the effects of truncation on both CK[subcript]4 (C-terminal tetra-lysl adducted) and NK[subscript]4 (N-terminal tetra-lysl adducted) led to more control over solution aggregation in the NK[subscript]4 variant, the work presented explore whether C-terminal sequential substitutions with a tryptophan residue could similarly stabilize the aqueous structure in monomeric form or further define the pore registry in such a way as to promote an increase ion permeability. Tryptophan was substituted for amino acids in the 18[superscript]th, 19[superscript]th, 20[superscript]th, and 21[superscript]st positions of the peptide sequence (SSGS, respectively), and changes in aggregation profiles, secondary structure, and channel ion permeability were observed. Synthesized peptides show circular dichroism spectral profiles indicating that the studied tryptophan substitutions did not result in a reduction of the characteristic helicity of the peptide; however, the tryptophan substitution also did little to decrease solution aggregation as demonstrated by comparative studies by reverse-phase high- performance liquid chromatography. All peptides demonstrated channel activity, directly measured by recordings of transepithelial short-circuit current. with profiles that suggest trends in electrostatic interactions and membrane registry relative to substitution position. One peptide in particular, NC-1007 S21W displayed atypical activity, which could not be effectively described by the standard Hill-based model but may be indicative of an ill-defined registry due to the substituted peptide’s proximity to another strongly pore-defining residue. Further studies in the effects of sequence modification to channel-forming peptides will elucidate how sequences may be altered to optimize synthetic peptide solubility, resistance to in-solution aggregation, and ability to form selective and permeable ion channels. The understanding gained from this study will improve our ability to develop peptides that could serve as a therapeutic treatments for a number of endogenous channelopathies.

Master of Science / Department of Biochemistry and Molecular Biophysics / Phillip Klebba / Iron acquisition is a component of Gram-negative bacteria pathogenesis, therefore as a form of 'nutritional immunity' host organisms sequester iron. To obtain iron bacteria secrete siderophores that scavenge iron. The E. coli outer membrane protein FepA actively transports the siderophore ferric enterobactin into the periplasm. We observe this uptake reaction by fluorescently labeling FepA in live bacteria, monitoring quenching that occurs upon binding of FeEnt, and then fluorescence recovery during transport. Energy poisons azide, arsenate, and 2,4-dinitrophenol were evaluated to determine sensitivity to known transport inhibitors. We developed and optimized methods to screen for iron transport inhibitors using a cell-based high-throughput screening platform. These inhibitors may have broad spectrum bacteriostatic antibiotic properties.

Master of Science / Department of Chemistry / Ping Li / Biodegradable polyhydroxybutyrates (PHBs) produced by a wide range of bacteria have been considered as an ideal alternative to petroleum-based plastics. Two types of mechanistic probes have been synthesized in order to understand the mechanism of PHB synthases (PhaCs). The first type is oxo analogs in which the sulfur in the coenzyme A (CoA) thioester has been replaced with an oxygen atom. A series of 3-R-hydroxybutyryl oxo CoA analogs, (HB)[subscript]nOCoA (n = 1, 2 and 3), were synthesized chemoenzymatically in good yields. Two models involving covalent catalysis with Cys have been proposed for the chain elongation catalyzed by PhaCs. The first involves an active site composed of two monomers in which the growing hydroxybutyrate (HB) chain alternates between Cys on each monomer. The second involves noncovalent intermediates (HB)[subscript]nCoA (n ≥ 2). Here the substrate analog HBOCoA was successfully employed to trap the noncovalent intermediates in the reactions catalyzed by class III PhaC from Allochromatium Vinosum, which supports our preferred second mechanistic model. Furthermore, it is also the first time that a wild-type (wt) synthase was used to investigate the chain elongation models. The other type of mechanistic probes is 3-R-hydroxyalkyl CoA that was used to investigate the substrate specificity of PhaCs from different classes. Substrate availability has been a challenge to study PHB synthases in vitro. Starting with commercially available dimethyl S-malate, the intermediate S-ethyl 2-(oxiran-2-yl) acetate 23 was synthesized via a ring-opening reaction involving lactone 21 and trimethylsilyl iodide followed by an oxidation reaction involving silver oxide. The regiospecific ring-opening reaction of epoxide 23 with different organometallic reagents afforded a straightforward access to ethyl 3-R-hydroxybutanoates attached with a variety of side chains. The final CoA compounds were obtained through the thiotransesterification reaction between corresponding benzenethioesters and the thiol group in CoA. This synthetic approach provides a new avenue to modifications of alkyl groups in 3-R-hydroxyalkyl CoA in an efficient manner.

Master of Science / Department of Biochemistry / Michael R. Kanost / Resilin is a rubber-like cuticular protein found in many insect species. Resilin is important for jumping and flying of those insects due to the properties of high elasticity and efficient energy storage. Some recombinant proteins or peptides derived from resilin sequences have been synthesized to produce biomaterials that mimic the remarkable properties of resilin. This research focused on resilin in the red flour beetle, Tribolium castaneum. A cDNA for T. castaneum resilin was inserted into plasmid vectors for expression of resilin in Escherichia coli or Bacillus subtilus. Resilin produced in E. coli was used as antigen to produce a rabbit antiserum. Resilin synthesized by B. subtilis as a secreted protein was purified and used for biochemical studies. Resilin is highly expressed in the late pupal stage, and in hind wings, but not found in elytra of pharate adults, indicated by RT-PCR and immunoblot analysis. Recombinant resilin could be cross-linked in the presence of horseradish peroxidase and hydrogen peroxide, detected by appearance of a high molecular weight band on SDS-PAGE, which had blue fluorescence under ultraviolet light, presumably due to dityrosine linkages. RNA interference was used to knock down resilin expression in T. castaneum. Immunoblot and RT-PCR analyses indicated that resilin expression was successfully decreased by RNAi. However, the knockdown adults exhibited no apparent differences in morphology, behavior or life span from control beetles. Blue fluorescence under ultraviolet illumination has frequently been used as an indication of the presence of resilin containing dityrosine cross-links in insect tissues such as wings, wing tendons and leg joints. A similar blue fluorescence was observed in hind wings of T. castaneum. However, this fluorescence was not decreased in hind wings of beetles in which resilin expression was knocked down by RNA interference. There was a blue fluorescence in the hind wings of knockdown beetles, which was similar in distribution to that in wings of control insects. This result suggests that the observed blue fluorescence in T. castaneum hind wings is derived not only from cross-linked resilin but also from components other than resilin, perhaps other cuticular proteins that contain dityrosine cross-links.

Master of Science / Department of Biochemistry and Molecular Biophysics / Michal Zolkiewski / ClpB is a molecular chaperone that is essential for infectivity and pathogen survival in a host. It belongs to the AAA+ protein family, which cooperates with the DnaK chaperone system to reactivate aggregated proteins. In this study, we purified and then studied the biochemical properties of the apicoplast targeted ClpB isoform from the malaria parasite Plasmodium falciparum: PfClpB1. Plasmodium falciparum is the parasite responsible for the most severe form of malaria. In contrast to the parasitophorous vacuole targeted PfClpB2 from Plasmodium falciparum which contains all characteristic AAA+ sequence motifs, PfClpB1 also includes a 52-residue long non-conserved insert in the middle domain. The ATPase activity study shows that PfClpB1 hydrolyzes ATP in presence of Poly-lysine and α-casein. Similar to most AAA+ ATPases, addition of ATP induces hexamer formation in PfClpB1. Lastly, PfClpB1 reactivates aggregated firefly luciferase. However, PfClpB1 is unable to efficiently reactivated luciferase in the presence of the E. coli DnaK chaperone system or human Hsp70 and Hsp40 (Hdj1). This can be explained by the extra middle domain sequence of PfClpB1. Our data may suggest that PfClpB1 activity is essential for Plasmodium falciparum survival by preserving the activity of apicoplast proteins.

Doctor of Philosophy / Biochemistry Interdepartmental Program / Antje Philine Wangemann / Mutations of SLC26A4 are the most common cause of the hearing loss associated with enlargement of the vestibular aqueduct. SLC26A4 encodes pendrin, an anion exchanger expressed in the cochlea, the vestibular labyrinth, and the endolymphatic sac of the inner ear. Slc26a4Δ/Δ mice, devoid of pendrin expression, develop an enlarged membranous labyrinth which leads to the failure to develop hearing, thereby recapitulating the human disease. Identifying the ionic composition of the endolymph and evaluating the importance of pendrin expression at various sites are initial steps towards developing strategies for preventing enlargement of the endolymph volume and subsequently restoring the inner ear functions. The major aims of the present study are 1) To determine the ionic composition of inner ear fluids during the developmental phase in which the enlargement of the endolymph volume occurs; 2) To test the hypothesis that pendrin expression in the endolymphatic sac is more important than its expression in the cochlea and the vestibular labyrinth. Here, we determined the Na+ and K⁺ concentrations in the cochlea and the endolymphatic sac with double-barreled ion-selective electrodes and generated a mouse model that restores pendrin expression in the endolymphatic sac while lacking expression in the cochlea and the vestibular labyrinth. High Na⁺ and low K⁺ concentrations were found in the cochlear endolymph during the embryonic stage. A rise of the K⁺ concentration along with a decline of the Na⁺ concentration occurred shortly before birth. The site-specific restoration of pendrin to the endolymphatic sac prevented enlargement and rescued hearing and balance. In conclusion, these data demonstrate that endolymph, in the phase of luminal enlargement during the embryonic development, is a Na⁺-rich fluid that is modified into a K⁺-rich fluid just before birth; restoration of pendrin in the endolymphatic sac is sufficient for developing normal inner ear function. Furthermore, these data suggest enlargement of endolymph volume caused by the loss of Slc26a4 is a consequence of disrupted Na⁺ absorption. Moreover, pharmacological strategies that correct fluid transport, as well as spatially and temporally limited restorations of pendrin, might restore normal inner ear functions in humans carrying mutations of SLC26A4.

Master of Science / Department of Biology / Kathrin Schrick / Steryl glucosides (SGs) are a common conjugate of sterols found in the plasma membranes of most plants and fungi, but their cellular functions remain largely unknown. Glycosylation of the C3 hydroxyl group of the sterol nucleus is catalyzed by UDP-glucose:steryl glucosyltransferase 80 (UGT80) enzymes. Two genes encoding UGT80A2 and UGT80B1 are responsible for most SG production in Arabidopsis thaliana, while UGT80C1 presents a putative third enzyme. In Arabidopsis, seed imbibition signals the epidermal seed coat cells to secrete an encapsulating mucilage that consists primarily of hydrated polysaccharides. Cellulose has been identified in the inner layer of the mucilage, providing a convenient model to study cellulose synthesis since seed mucilage is dispensable for viability and pectin and cellulose staining dyes are readily available. A reverse genetics and biochemical approach was used to characterize the role of UGT80 enzymes and their impact on cellulose synthesis in seed mucilage. ugt80B1 mucilage was found to have elongated cellulosic rays, but no defects in pectin synthesis. A double mutant of ugt80B1 and mum3-1, a mutant allele of CELLULOSE SYNTHASE 5 (CESA5), displays a novel phenotype with irregular cellulose patterning and extreme shedding of the pectinaceous layer surrounding the seed coat. The observed mucilage defects may be indicative of disrupted cellulose synthesis and a mechanistic relationship between SGs and the cellulose synthase machinery. UGT80A2 and B1 demonstrate glycosylation activity with a multitude of plant sterols. The two enzymes do display some substrate specificity, however, with UGT80A2 producing the large majority of sitosterol and stigmasterol glucoside compared to B1. UGT80C1 shows little or no sterol glucosyltransferase activity in vitro or in vivo and likely has evolved a different function from the two other genes. GFP:UGT80C1 expressed either from the constitutive 35S or from its native promoter was localized to lipid droplets and possibly chloroplasts as well, creating a new perspective on the role of the protein in plant lipid metabolism. This study extends the currently limited view of SGs as ubiquitous components of the plasma membrane to active regulators of cellulose synthesis in seeds. Evidence presented here changes the perceived role of the plant conserved protein, UGT80C1, from a putative steryl glucosyltransferase enzyme to having a function in intracellular lipid droplets.

Master of Science / Department of Biochemistry / Gerald Reeck / Aphids mount a remarkable salivary secretion to overcome plant host defenses. Our group has previously reported a gene unique to aphids enriched in the salivary glands of the pea aphid A. pisum, C002, which is required for successful feeding on its host plant Vicia fava. Here I present an analysis of genetic variation within the available EST data for C002 in pea aphids. From 596 total ESTs, 332 are full-length, and segregate into 8 validated haplotypes based on the criteria I set in place to access the quality of EST data. Additionally, Armet, is a putative multi-functional gene implicated as a neurotrophic factor during development, and as a part of the unfolded protein response during stress. I employ RNA interference in the model organism T. castaneum to determine the effect of transcript knockdown during development from early in-star larval stages, through pupation, and its effect on adult emergence. I report that knockdown of Armet transcript significantly hinders the ability for beetles to emerge from the pupae.

Doctor of Philosophy / Grain Science and Industry / Scott R. Bean / Hulya Dogan / The role of sorghum protein cross-linking into high M[subscript w] polymeric groups in grain hardness was investigated using a number of protein analytical techniques to study the protein composition (reduced and non-reduced) of isolated vitreous and floury endosperm. The relative molecular weight distributions of polymeric proteins within two of three differentially extracted fractions were determined by size exclusion chromatography (SEC). The proteins in vitreous endosperm showed more protein cross-linking and a larger M[subscript w] distribution than found in the floury endosperm. An improved method for fractionating sorghum proteins designed to obtain intact disulfide linked protein polymers was developed. Three protein fractions obtained by application of the method represented proportionally different protein polymer contents as evidenced by comparative SEC and provides an improved tool for polymeric protein content comparison and measurement. The improved method was applied to a highly diverse non-tannin wild-type sorghum sample set spanning a range of in-vitro protein digestibility (IVPD) values to determine polymers involved with and influencing IVPD. Grain traits other than cross-linked proteins were also investigated for significant relationships to IVPD. Three protein fractions (F1, F2, F3) containing intact protein polymers were obtained for analysis by SEC and RP-HPLC. Proteins represented by four of five individual SEC peaks from F3 were significantly negatively correlated to IVPD, with three of the correlated peaks being polymeric. A 2-dimensional (2-D) technique involving peak collection after size exclusion chromatography followed by reverse phase high performance liquid chromatography (SEC x RP-HPLC) of the collected peaks was applied to protein polymers previously determined to be correlated with IVPD. RP-HPLC chromatogram patterns unique to each collected SEC peak from three selectively extracted protein fractions allowed qualitative and quantitative comparisons of protein polymer components. A pair of early eluting peaks appearing in the [gamma]-kafirin region of 2nd-dimension RP-HPLC chromatograms from a protein fraction with the largest M[subscript w] distribution were significantly correlated to IVPD. The correlated peak of interest was collected and characterized using SDS-PAGE and was preliminarily identified as 27kDa [gamma]-kafirin. By combining techniques using differing selectivity’s (solvent based, molecular size based, hydrophobicity based), it was possible to disassemble and compare components of protein polymers significantly correlated to IVPD.

Master of Science / Department of Biological and Agricultural Engineering / Mei He / Thermobifida fusca, an aerobic moderately thermophilic, filamentous soil bacterium is capable of producing butyric acid. Butyric acid is a 4-carbon short chain fatty acid that is widely used in the chemical, food, and pharmaceutical industries. Currently, butyric acid is primarily produced through petroleum-based chemical synthesis, but could be a candidate to be produced by fermentation. By producing through a fermentation platform, production of butyric acid can be shifted from a non-renewable to a renewable source. In an effort to make T. fusca produce a high yield of butyric acid, multiple fermentation parameters were explored and optimized. The effect of different carbon sources (mannose, xylose, lactose, cellobiose, glucose, sucrose, and acetates) on butyric acid production was studied, where cellobiose produced the highest yield of 0.67 g/g C (g-butyric acid/g-carbon input). The best stir speed and aeration rate for butyric acid production were found to be 400 rpm and 2 vvm in a 5-L fermentor. The maximum titer of 2.1 g/L butyric acid was achieved on 9.66 g/L cellulose. Fermentation was performed on ground corn stover as a substrate to evaluate the production of butyric acid on lignocellulosic biomass, and the optimized conditions resulted in a titer of 2.37 g/L butyric acid. The butyric acid synthesis pathway was identified involving five genes that catalyzed reactions from acetyl-CoA to butanyol-CoA in T. fusca. A study into the transcriptomics of T. fusca was begun by growing T. fusca under a variety of fermentation conditions, isolating the messenger RNA, and performing a sequence of the mRNA using whole transcriptome shotgun sequencing. The results of sequencing of various samples were plotted to determine correlation across numerous fermentation parameters. This correlation based analysis determined that the carbon to nitrogen ratio has the largest overall impact on gene transcription of T. fusca among all of the fermentation parameters studied. Overall, the work from this study proves that production of butyric acid is possible from a renewable cellulosic feedstock.