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dc.contributor.advisorAckerson, Christopher J.
dc.contributor.authorBorgognoni, Kanda
dc.contributor.committeememberNeilson, James
dc.contributor.committeememberKennan, Alan J.
dc.contributor.committeememberTsunoda, Susan
dc.date.accessioned2021-06-07T10:21:31Z
dc.date.available2021-06-07T10:21:31Z
dc.date.issued2021
dc.descriptionIncludes bibliographical references.
dc.description.abstractA meaningful understanding of biochemistry requires that we understand the function of proteins, which is heavily dependent on their structure and location within an organism. As the Resolution Revolution of cryo-electron microscopy gains unprecedented ground largely due to the recent development of commercially available direct electron detectors, energy filters, and high-end computation, thousands of protein structures have been solved at atomic or near-atomic resolution, with the highest resolution structure to date being solved at 1.2 Å. A major challenge that has limited the broad use of cryo-electron tomography (cryo-ET) is locating a protein of interest in an organism, as no commercially available high-contrast markers which can be generated in vivo exist. Herein, we present a breakthrough study which aims to solve this problem by synthesizing high contrast metal nanoparticles labeling desired proteins in situ. We isolated a Glutathione Reductase-like Metalloid Reductase (GRLMR), which can reduce selenite and selenate into selenium nanoparticles (SeNPs), from Pseudomonas moraviensis stanleyae found in the roots of a Se hyperaccumulator Stanleya pinnata, or Desert Princes’ Plume. A recombinant variant, denoted as a clonable Selenium NanoParticle (cSeNP), was fused to filamentous temperature sensitive protein Z (FtsZ), and the chimera was expressed in vivo using a T7 expression system in model organism E. coli for a proof-of-concept study. Because the SeNPs biogenically produced are amorphous, they exist in a quasistable state and are composed of polymeric Sen in the form of chains and rings that are constantly breaking and reforming. To stabilize the particles during cellular preservation ex aqua, a disproportionation-like reaction can be done either in vivo or as a post-fixation step to form crystalline metal selenide (MSe) NPs that can withstand the processing liquids used. Thereafter, electron tomography was used to acquire a tilt series that was reconstructed into a tomogram and segmented using IMOD, generating a model representing MSeNPs labeling FtsZ filaments. As such, we have demonstrated the potential of using cSeNP as a high resolution marker for cryo-ET. While our study relied on traditional preservation and embedment techniques, we anticipate that for cells preserved via vitrification, cloned SeNPs can be used without subsequent transformation to MSeNPs, as the amorphous particles are stable in aqueous media. Prospectively, we expect that clonable nanoparticle technology will revolutionize cryo-ET, allowing us to localize proteins in vivo at high resolution while maintaining organism viability through metal immobilization. Furthermore, this technique can be expanded to other imaging modalities, such as light microscopy and X-ray tomography, through the discovery and engineering of other clonable nanoparticles.
dc.format.mediumborn digital
dc.format.mediumdoctoral dissertations
dc.identifierBorgognoni_colostate_0053A_16574.pdf
dc.identifier.urihttps://hdl.handle.net/10217/232630
dc.languageEnglish
dc.publisherColorado State University. Libraries
dc.relation.ispartof2020- CSU Theses and Dissertations
dc.rightsCopyright of the original work is retained by the author.
dc.titleA CLONABLE SELENIUM NANOPARTICLE IN ACTION: HIGH RESOLUTION LOCALIZATION OF FTSZ USING ELECTRON TOMOGRAPHY
dc.typeText
dcterms.rights.dplaThe copyright and related rights status of this Item has not been evaluated (https://rightsstatements.org/vocab/CNE/1.0/). Please refer to the organization that has made the Item available for more information.
thesis.degree.disciplineChemistry
thesis.degree.grantorColorado State University
thesis.degree.levelDoctoral
thesis.degree.nameDoctor of Philosophy (Ph.D.)


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