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Browsing Chemistry by Author "Audette, Gerald F."
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Item Open Access Crystallization Studies of the TraF Protein from the F Plasmid of Escherichia coli(2022-03-03) Samari-Kermani, Naveed; Audette, Gerald F.Crystallization of suitable crystals for diffraction analysis is a major hurdle in structure-function studies of proteins using X-ray crystallography. This is especially true when the protein may be generally hydrophobic, membrane associated or contain regions of increased flexibility. The objective of the research outlined in this thesis was to determine a more amenable set of crystallization conditions for the protein TraF from the F plasmid for X-ray diffraction and structure solution. The F plasmid of Escherichia coli utilizes a conjugative type IV secretion system (T4SS) by which the transmission of genes in bacteria occurs contributing to the current issue of antibiotic resistance. The TraF protein of this system is a periplasmic protein with a dynamic hydrophobic N-terminal tail which interacts directly with TraH and a C-terminal thioredoxin-like domain. TraF is hypothesized to chaperone and interact with several other proteins of this system. Although its exact function remains unclear, TraF is essential in in the F plasmids T4SS as a lack of function abolishes gene transfer through this system. Modification of upstream conditions, including expression and purification of a GST-tagged variant of TraF, as well as modification of buffer conditions including the detergent NP-40 allowed for an increased solubility and concentration of the protein for crystallization trials. The traditional screening process with GST-TraF resulted in two conditions that showed birefringent structures which did not yield any better results when optimized. Optimization of the original reported TraF purification and crystallization conditions did double the protein concentration, however resulted in a lack of miscibility between the protein stock and the reservoir solution during crystallization. Furthermore, a head-to-head crystallization screening was conducted incorporating 5-amino-2,4,6-triiodoisophthalic (I3C) in the crystallization drop to advance structure solution using experimental phasing methods. The head-to-head experiment led to multiple crystallization conditions that should be optimized and further assessed while validating the novel process of incorporating I3C in the screen formation process.Item Open Access Obtaining Structural Insights on Bacterial Protein Complexes Using Time-Resolved Hydrogen-Deuterium Exchange Mass Spectrometry(2015-12-16) Lento, Cristina; Audette, Gerald F.Persistent infections by Pseudomonas aeruginosa are initiated by interaction of a type IV pilus (T4P) with receptors on the mucosal cells of susceptible hosts. Here, we examine the structural changes occurring between the monomeric and dimeric states of ΔK122 using time-resolved electrospray ionization hydrogen-deuterium exchange mass spectrometry (TRESI-HDX-MS). Based on levels of deuterium uptake, the N-terminal α-helix and the loop connecting the second and third strands of the anti-parallel β-sheet contribute significantly to pilin dimerization. Conversely, the antiparallel β-sheet and αβ loop region exhibit increased flexibility, while the receptor binding domain retains a rigid conformation in the equilibrium state. Additionally, Escherichia coli are able to adapt to changing environmental conditions and develop antibiotic resistance through a process called F-plasmid conjugation, carried out through a type IV secretion system (T4SS). The F-T4SS protein TraF is of particular interest due to its involvement in pilus assembly to mediate the transfer of DNA. Dynamic analysis of a GST-TraF construct through TRESI-HDX-MS was performed to gain further insights on its structure. These studies have revealed that the C-terminal region predicted to contain the thioredoxin-like domain is quite structured compared to the more solvent accessible N-terminal region predicted to form a protein-protein interaction with companion T4SS protein TraH. Structural analysis of a GST-TraF construct is on-going to further characterize the regions responsible for protein-protein interaction and the elucidation of its three-dimensional structure.Item Open Access Part I: Crystallization of A Type IV Pilin from Pseudomonas Aeruginosa. Part II: Characterization of a Peptidyl-Prolyl-Cis,Trans-Isomerase Through X-Ray Crystallography(2018-05-28) Yaseen, Ayat; Audette, Gerald F.Within a host, pathogenic bacteria employ several mechanisms that enhance their survival and motility. These mechanisms include their ability to adhere, replicate (in order to avoid eradication), and secretion of virulent proteins. The study of proteins involved in bacterial pathogenesis provides us with a more thorough understanding of their mechanism and function, which can lead to the development of more effective therapeutics. In this study proteins secreted by gram-negative bacteria are explored, specifically those from Pseudomonas aeruginosa and Helicobacter pylori. P. aeruginosa is a common opportunistic pathogen associated with 10% of hospital infections, mainly owed to their ability to bind to biotic and abiotic surfaces. Type IV pili secreted by P. aeruginosa are associated with adhesion, motility and DNA transfer. Part I details the cloning to crystallization of KB7, a pilin secreted by P. aeruginosa. The second bacteria studied in Part II is H. pylori, associated with gastric ulcers and gastric inflammation compromising 50% of the global population, where severity of infection is highly dependent on the strain of H. pylori, and the individual infected. The secreted protein HP0175 from H. pylori binds to Toll-Like Receptor 4 and activates a cascade of mechanisms leading to apoptosis, as well as triggering the innate immune response. HP0175 is also classified as a peptidyl-prolyl cis,trans-isomerase involved in the isomerization of proline peptide bonds preceding the N-terminal. Here, apo-HP0175 was crystallized to 2.09 in space group P3221 with one monomer in the asymmetric unit; the dimer is generated through symmetry mates. A comparison to indole-2-carboxylic acid bound HP0175 shows N- and C- terminal helix extensions upon interaction of the catalytic residues in the binding pocket. Helix extension supports other parvulin findings that N- and C- terminal helices stabilize proteins undergoing catalysis by protein-protein interaction.Item Open Access Protein Structure Dynamics In Gram Negative Bacterial Secretion Systems(2025-04-10) Nicholas Joseph Bragagnolo; Audette, Gerald F.Bacteria use a variety of secretion systems that enhance their virulence. The conjugative type IV secretion system attributes transmission of mobile DNA elements in bacteria, whereas type IV pili are akin to the type II secretion system and are responsible for host adhesion and twitching motility. Understanding the structure and function of components in these systems has the potential to elucidate the mechanisms of bacterial commensalism and antagonism, and would aid in the development of inhibitors to bacterial pathogenesis by disrupting the tools virulent species use for stable, long-term infections. The entry exclusion protein of the F-like type IV secretion system, TraG, consists of a membrane-bound N-terminal domain and a periplasmic C-terminal domain, denoted TraG*. TraG* is essential in preventing redundant DNA transfer through interaction with a cognate TraS in the inner membrane of the recipient cell to prevent conjugation when the recipient cell carries the same plasmid. Using a multitude of biophysical methods including thermofluor, circular dichroism, collision induced unfolding mass spectrometry, and small angle X-ray scattering, the structural dynamics of TraG* was investigated. This allowed for the characterisation of the 50 N-terminal residues, the 77 C-terminal residues, and the TraS-interacting region of the protein as highly dynamic, providing evidence for the mechanism of the long-distance interactions made by TraG. This dissertation also describes the 1.3 Å crystal structure of the N-terminally truncated Type IV pilin of Pseudomonas aeruginosa from strain P1 (ΔP1), the first structure of its phylogenetically linked group (group Ia) to be reported. The structure was solved from X-ray diffraction data collected 20 years ago with a molecular replacement search model generated using AlphaFold. Comparisons of the ΔP1 structure to other Type IV pilins using ProSMART indicated that there are cases of higher structural homology between different phylogenetic groups of P. aeruginosa than there are between pilins from the same phylogenetic group, indicating that the classification of pilins based on structural homology may be necessary in developing anti-virulence drugs and vaccines.Item Open Access Structural and Functional Insights into the F Plasmid Type IV Secretion System proteins TrbI and TrbB(2024-03-16) Apostol, Arnold Jan; Audette, Gerald F.Bacteria have evolved elaborate mechanisms to thrive in stressful environments. One mechanism that bacteria utilize are secretion systems that can traverse protective lipid cell membranes and serve as mediators for a diverse set of goals, including the secretion of toxins implicated with target host pathogenesis. F-like plasmids in gram-negative bacteria encode for the multi-protein Type IV Secretion System (T4SSF) that is functional for bacterial proliferation and adaptation through the process of conjugation. The periplasmic protein TrbB is believed to have a stabilizing chaperone role in the T4SSF assembly, with TrbB exhibiting disulfide isomerase (DI) activity. In the current report, we demonstrate that residues W57-K181, which include the active thioredoxin motif, are sufficient for DI activity. Moreover, a structural model of GST-TrbBWT based on ColabFold-AlphaFold2 and Small Angle X-Ray Scattering data indicate that TrbBWT’s N-terminus is disordered, and this disordered nature likely contributes to the protein’s dynamicity and recalcitrance to crystallization. A truncation construct, TrbB57-181, was designed and found to exhibit higher physicochemical stability using 1H-15N Heteronuclear Single Quantum Correlation spectroscopy and Circular Dichroism spectroscopy. Binding studies of TrbB and other T4SSF proteins TrbI and TraW were performed, and results do not support the inference of a stable complex forming in vitro. Comparative studies of TrbB, TraF, and TrbI also provide insights into the structure of these T4SSF component proteins. Lastly, crystallization trials of GST-TrbBWT and GST-TrbI provide leads for future crystallization campaigns.Item Open Access Structural and Functional Studies of TraG From the F Plasmid and the Major Pilin From Coxiella Burnetii(2017-07-26) Erdogan, Fettah; Audette, Gerald F.The Escherichia coli F-plasmid houses all genes necessary for the assembly of its conjugative machinery to facilitate successful DNA transfer. TraG is one of the largest proteins (102 kDa) encoded in the transfer (tra) region of the F-plasmid that facilitates conjugative transfer. The N-terminal region of TraG is involved in pilus synthesis and assembly, while the C-terminal periplasmic domain is required for plasmid-specific entry exclusion and mating pair stabilization. In order to provide structural insights into TraGs function, we have expressed, purified and initiated crystallization studies of the C-terminal region of TraG (designated TraG*, 52.7 kDa) and have obtained initial crystals of TraG*. Our experiments on functional and dynamic characterization of TraG* using liquid chromatography coupled mass spectrometry (LC-MS), size exclusion chromatography coupled multi-angle light scattering (SEC-MALS) and protein gel electrophoresis has given significant insight into TraG* crystallization strategies as well as potential TraG mechanism of action during conjugation.Item Open Access Structural Studies of the Receiver Domain of LytR from Staphylococcus Aureus and Interaction Studies of TraW and TrbC from the F Plasmid of Escherichia Coli(2015-01-26) Shala, Agnesa; Audette, Gerald F.The emergence of the multi-drug resistant bacteria and the evolutionary spread of the antibiotic resistance genes have become a great concern to human health. Bacteria use various systems in combination or alone in response to environmental stressors and stimuli to adapt to changing conditions. Such systems include the two-component regulatory system (TCS) and the type IV secretion system (T4SS). Microbes use TCSs to regulate important functions such as sporulation, chemotaxis, as well as autolysis that leads to biofilm formation. In addition, microbes utilize T4SSs to deliver DNA, protein substrates, toxin, and virulence factors, from a donor to a recipient cell. These systems are complex, versatile and have a significant impact to human health as they are a major driving force for infection and the spread of antibiotic resistance. The LytSR TCS from Staphylococcus aureus has been found to regulate murein hydrolase activity and autolysis by controlling the expression of the lrgAB and cidABC genes. LytS is predicted to autophosphorylate upon a membrane potential change, consequently transferring the phosphoryl group to the conserved Asp53 residue on the N-terminal domain of the response regulator LytR, leading to the transcription of lrgAB genes. In this study we present the X-ray crystal structure of the N-terminal domain of LytR in apo form and in complex with beryllium fluoride. The identification of dimerization interface residues can be utilized to predict modes of dimerization and therefore activation of the protein. A second project focuses on a representative of the conjugative T4SSs, is the F plasmid from Escherichia coli, which is responsible for the transfer of virulence and antibiotic resistance genes. Two proteins encoded by the F plasmid, TrbC and TraW, are unique to the F-like plasmids and are essential for the transfer of DNA from a donor cell to a recipient cell but their mechanism and function is not known. Interaction studies of TrbC and TraW, as well as crystallization experiments are reported. These studies suggest that the roles of TrbC and TraW are related, and that their interaction is important for the functional transfer of DNA in the conjugative process.