Biogenic nanoparticles and their application in biological electron microscopy
Date
2018
Authors
Nemeth, Richard S., author
Ackerson, Christopher, advisor
Yao, Tingting, committee member
Bjostad, Louis, committee member
Peersen, Olve, committee member
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Abstract
Interest in nanomaterials has seen a dramatic increase over the past twenty years. In recent years many have turned toward proteins to aid in developing novel materials due to the mild reaction conditions, functionalization, and novel synthetic control of the resulting inorganic structures. Proteins have the ability to direct aggregation of inorganic nanostructures, while some enzymes are able to perform oxio/reductase activity to synthesize the materials as well. These two general properties are not always mutually exclusive and the dual function of certain proteins in nanoparticle synthesis is at the core of this work. Of all the applications for biogenic nanoparticles, generating tools for biological electron microscopy is one of the most appealing. The contrast issue, specifically with in vivo biological sample in the electron microscope has drastically limited the information obtainable by this method. An ideal biogenic nanoparticle would operate analogously to GFP in optical microscopy and contain the dual function characteristics stated above. More specifically it would have to fulfill three criteria: i) reduction of a metal precursor, ii) product size control, iii) product retention. To discover such a clonable contrast tag we must deepen our understanding of biogenic nanoparticle formation in tandem with discovering and developing novel dual function enzymes. This work encapsulates both aspects necessary for the development of a successful clonable nanoparticle for biological electron microscopy. Current biogenic synthetic methods produce nanomaterials with less desirable properties than their inorganic counterparts. Conducting fundamental research and establishing a set of rules and guidelines for biogenic methods will ultimately get us closer to mimicking the control nature has already developed. This dissertation contains 3 chapters. Chapter 2 focuses on the use of protein crystals as scaffolds for nanomaterial synthesis. Herein porous protein crystals were used to control the gold nanocluster seeded growth of gold nanorods in an attempt to help establish guidelines for biogenic nucleation controlled nanomaterial synthesis. High aspect gold nanorod products were generated from within the crystal pores. Subsequent dissolving of the crystals allowed for release of these rods from their template. The following two chapters focus on metalloid reductase nanoparticle synthesis in which we have discovered and characterized a novel selenophile bacteria. Through purification and mass spectrometry we found a glutathione reductase like enzyme to be responsible for Se nanoparticle formation. A commercially available glutathione reductase from yeast was used for Se nanoparticle formation in vitro. This mechanism was characterized and the system was assessed for potential use as a clonable tag. The native enzyme was sequenced and isolated, followed by its own characterization. Our kinetic findings suggest this enzyme is the first documented metalloid reductase due to its specificity for selenium substrates. The enzymes transportability to foreign organisms demonstrates its potential use as a clonable contrast tag for electron microscopy.
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Subject
enzymes
nanoparticles
selenium
gold nanorods
electron microscopy
protein crystal templates