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Description
Membrane proteins located within or as attachments to the cell membrane play critical roles in many essential cellular functions and host-pathogen interactions. Knowledge of the structure and function of membrane proteins in pathogenic species can allow for the development of specific vaccines and therapeutic agents against the pathogen. Francisella tularensis is an intracellular pathogen that is the causative agent of the severe, life-threatening infection, tularemia, in humans and other small mammals. F. tularensis is prevalent within the environment and is a potential bioterrorism agent due to its high virulence and its ability to be spread easily as an aerosol. The CapBCA membrane protein complex has been identified as a virulence factor of F. tularensis. This project, derived from the Membrane Proteins in Infections Diseases (MPID) Project, aims to successfully express the membrane proteins CapBCA, which are crucial to the pathogenic properties of F. tularensis. To accomplish this goal, methods for in vivo recombinant expression and purification of membrane proteins are in the process of being developed. The expression of the CapA component has been successful for some time, therefore, the goal of this study is to develop an approach toward recombinant in vivo membrane protein expression of both the CapB and CapC components of the CapBCA membrane protein complex. In this study, the CapB and CapC components were expressed for the first time in vivo through the use of the novel MPID vector, pelB-MBP. The expression of the CapB and CapC components will allow for large-scale expressions to commence with the end goal of determining the crystal structures of the individual proteins or the complex. Ultimately, it is hoped that knowledge of these molecular structures can lead to the development of a vaccine or other therapeutic agents against this pathogen.
ContributorsTrimble, Kelli Lauren (Author) / Fromme, Petra (Thesis director) / Hansen, Debra (Committee member) / Barrett, The Honors College (Contributor) / Department of Chemistry and Biochemistry (Contributor) / School of Film, Dance and Theatre (Contributor)
Created2015-05
Purification, Characterization, and Structural Determination of Proteins Vital to Infectious Disease
Description
The work in this dissertation progressed the research of structural discovery for two targets critical in the fight of infectious disease. Francisella lipoprotein 3 (Flpp3) is a virulent determinant of tularemia and was the first protein of study. The proteins soluble domain was studied using a hybrid modeling theory that used small angle X-ray scattering (SAXS) in combination with computation analysis to generate a SAXS-refined structure. The SAXS-refined structure closely resembled the NMR structure (PDB: 2MU4) which contains a hydrophobic cavity inside the protein that could be used for drug discovery purposes. The full-length domain of Flpp3 purified from the outer membrane of E. coli was also studied with a combination of biophysical characterization methods. Mass spectrometry and western blot analysis confirmed Flpp3 being translocated to the outer membrane, while SDS-PAGE confirmed the purity of Flpp3 in the monomeric form after size exclusion chromatography. Using Circular Dichroism (CD) the monomeric form of Flpp3 was shown to be almost fully refolded into having a primarily β-stranded secondary structure. This information advances the progress of both tularemia research and outer membrane protein research as no natively folded outer membrane protein structures have been solved for F. tularensis.The second protein worked on in this dissertation is the nonstructural protein 15 from SARS-CoV-2, also called NendoU. Nsp15 is an endoribonuclease associated with aiding the virus responsible for the current COVID-19 pandemic in evasion of the immune system. An inactive mutant of Nsp15 was studied with both negative stain electron microscopy and cryogenic electron microscopy (Cryo-EM) in the presence of RNA or without RNA present. The initial findings of negative stain electron microscopy of Nsp15 with and without RNA showed a difference in appearance. Negative stain analysis of Nsp15 is in the presence of a 5nt RNA sequence in low salt conditions shows a conformational change when compared to Nsp15 without RNA present. As well the presence of RNA appeared to shift the electron density in Cryo-EM studies of Nsp15. This work advances the research in how Nsp15 may bind and cleave RNA and aid in the evasion of the host cell immune system.
ContributorsGoode, Matthew (Author) / Fromme, Petra (Thesis advisor) / Guo, Jia (Committee member) / Chiu, Po-Lin (Committee member) / Arizona State University (Publisher)
Created2022