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Cloneable' nanoparticles: identification and utilization of metal reducing enzymes as biological electron microscopy tags

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dc.contributor.authorButz, Zachary J., author
dc.contributor.authorAckerson, Christopher J., advisor
dc.contributor.authorNelson, James R., committee member
dc.contributor.authorSnow, Christopher D., committee member
dc.contributor.authorSantangelo, Thomas J., committee member
dc.date.accessioned2020-01-13T16:41:36Z
dc.date.available2020-01-13T16:41:36Z
dc.date.issued2019
dc.description.abstractThe ability to image individual proteins in biological systems has yet to be realized. The identification and utilization of 'cloneable' nanoparticles (cNP), i.e. genetically encoded tags capable of forming in situ inorganic nanoparticles from soluble inorganic precursors is the focus of this dissertation. The long-term goal of this project is to produce GFP analogues that can then be used in electron microscopy, light microscopy, and correlative light/electron microscopy. The first chapter of this dissertation explores a metal reducing enzyme capable for converting soluble inorganic materials to insoluble (nano)particulates. Glutathione reductase-like metalloid reductase, GRLMR, was first isolated from Pseudomonas moraviensis stanleyae and characterized. GRLMR was identified as not only being able to reduce the precursor selenodiglutathione to produce Se⁰ nanoparticles but was also capable of increasing a host cells resistance to 10-fold that of the cell sans GRLMR. The structure of the enzyme was then predicted using Phyre² and related to other glutathione reductases to determine possible residues important for its inherent activity. In the second chapter a dodecapeptide was identified using phage display for its ability to bind to Se⁰ nanoparticles produced by GRLMR. Fusing this peptide to the C-terminus of GRLMR resulted in unexpected enzymes characteristics. Only when concatenated to GRLMR, the Se0 binding peptide conveyed increased size control of nanoparticle product over a wide range of substrate not seen with GRLMR alone. The peptide facilitated greater affinity between the enzyme and the nanoparticle product as well. Finally, presence of the peptide on GRLMR was also able to increase the enzyme's kinetics for precursor reduction. Raman spectroscopy was used to characterize which residues on the peptide were responsible for the interaction between the peptide and the nanoparticle surface. The third chapter explores the application of GRLMR as a cNP. A cNP tag containing two concatenated copies of GRLMR and two Se⁰ binding peptides was constructed and fused to the polymerizing protein FtsZ for expression and studies in native activity. Variants of the tagged FtsZ were isolated and studied in vitro or observed in vivo. In vitro studies resulted in filaments decorated with Se⁰ nanoparticles that could be observed with and without formal staining with uranyl acetate. Images resulting from in vivo studies indicated that both the tag and FtsZ were able to function to produce filaments within cells of high contrast. The fourth chapter isolates and characterizes a Te-reducing enzyme identified from screening environmental isolates collected throughout the Colorado Mineral Belt. A specific isolate, R. erythropolis PR4 possessed resistance to a broad range of metal and metalloid species. Specifically, R. erythropolis grew exceptionally well in up to 4.5 mM of TeO3²⁻ determined by broth microdilution. The lysate from the bacteria was also incubated in different metals and metalloids to identify any proteins with metal reductase activity. Mycothione reductase, a glutathione reductase analogue was characterized with Te-reductase activity. Mycothione reductase was then isolated and characterized and could form Te⁰ nanoparticles and bundled fibers. Although mycothione reductase was able to reduce SeO3²⁻, when the enzyme was incubated with TeO3²⁻ and an excess of SeO3²⁻ the resulting particulate had a mole ratio in favor of Te.
dc.format.mediumborn digital
dc.format.mediumdoctoral dissertations
dc.identifierButz_colostate_0053A_15732.pdf
dc.identifier.urihttps://hdl.handle.net/10217/199764
dc.languageEnglish
dc.language.isoeng
dc.publisherColorado State University. Libraries
dc.relation.ispartof2000-2019
dc.rightsCopyright and other restrictions may apply. User is responsible for compliance with all applicable laws. For information about copyright law, please see https://libguides.colostate.edu/copyright.
dc.subjectelectron microscopy
dc.subjectmetal reducing enzyme
dc.subjectcloneable nanoparticle
dc.subjectmycothione reductase
dc.subjectglutathione reductase-like metalloid reductase
dc.titleCloneable' nanoparticles: identification and utilization of metal reducing enzymes as biological electron microscopy tags
dc.typeText
dcterms.rights.dplaThis Item is protected by copyright and/or related rights (https://rightsstatements.org/vocab/InC/1.0/). You are free to use this Item in any way that is permitted by the copyright and related rights legislation that applies to your use. For other uses you need to obtain permission from the rights-holder(s).
thesis.degree.disciplineChemistry
thesis.degree.grantorColorado State University
thesis.degree.levelDoctoral
thesis.degree.nameDoctor of Philosophy (Ph.D.)

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