Date of Award

1-1-2020

Language

English

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

College/School/Department

Department of Chemistry

Content Description

1 online resource (xii, 97 pages) : color illustrations.

Dissertation/Thesis Chair

Alexander Shekhtman

Committee Members

Daniele Fabris, Jayanti Pande, Li Niu, Qiang Zhang

Keywords

Bioreactor, In-cell NMR, Nuclear magnetic resonance spectroscopy, Bioreactors, Proteins, Escherichia coli, Cell interaction, Biomolecules

Subject Categories

Chemistry

Abstract

Long has it been the goal of biochemists to observe a protein within the cellular environment allowing the true nature of a protein function to be determined within a cell. To understand the nature of a protein within a cell, the role of the cellular environment must be considered when determining the function as a protein. The cellular environment is a crowded place filled with macromolecules such as DNA, RNA and proteins that all interact with one another affecting the function of a protein. Intracellular macromolecules can reach concentrations of 300 g/L and occupy 30% of the available space in a bacteria cell leaving minimal space with in the cells for protein migration. Hydrophobic and electrostatic interactions between macromolecules have been shown to have both positive and negative effects on a proteins function within the cell. These weak, transient interactions that play a function role in the cell have been coined quinary interactions. Observing these interactions relies on a noninvasive technique that will not disturb the cellular environment. In-cell NMR is such a technique that allows for the protein to be observed within the cellular environment while maintaining the integrity of the cell. Doing so relies on incorporating stable isotopes such as 13C, 15N and 2H to aid the isolation of the NMR signal of the target protein from the complex cellular environment. With the addition of transverse relaxation-optimized spectroscopy, TROSY, and cross relaxation-induced polarization transfer, CRIPT, proteins previously too large to be observed by NMR have been able to be observed. Bioreactor technology has expanded the scope of in-cell NMR studies. We developed a bioreactor that is able to stabilize the metabolism and remove waste during NMR acquisition. Using the bioreactor we investigated the role of quinary interaction. It is not well understood how ribosome-targeted antibiotics affect a wide range of metabolic pathways. Weak functional interactions between a target protein and the cellular environment named quinary interactions, mediated by RNA and proteins inside the crowded cytosol may provide one possible mechanism for this effect. We developed a flow in-cell NMR system that allows us to monitor both the spacial and temporal changes in protein quinary interactions that occur when ribosome antibiotics are added to living cells. We show that only antibiotics binding to the small ribosomal subunit cause a change in the quinary interactions of thioredoxin. Further we improved upon the bioreactor design in hopes of allowing interactions depend on an active metabolic state to be observed by in-cell NMR. Protein-protein interactions, PPIs, underlie most cellular processes, but many PPIs depend on a particular metabolic state that can only be observed in live, actively metabolizing cells. Real time in-cell NMR spectroscopy, RT-NMR, utilizes a bioreactor to maintain cells in an active metabolic state. Improvements in bioreactor technology maintained ATP levels at >95% for up to 24 hours enabling protein overexpression and a previously undetected interaction between prokaryotic ubiquitin-like protein, Pup, and mycobacterial proteasomal ATPase, Mpa, to be detected. Singular value decomposition, SVD, of the NMR spectra collected over the course of Mpa overexpression easily identified the PPIs despite the large variation in background signals due to the highly active metabolome. Lastly we applied bioreactor technology to trace metabolic pathways. By means of a pulse-chase experiment, metabolic pathways were traced as 13C-glucose was digested by cardiomyocytes. Doing so allowed for the changes in the metabolism of glucose to be observed in the presence and absence of diaphanous homolog 1 (Diaph1). Diaph1 is a member of the Rho family of GTPases that has been hypothesized to slow the TCA cycle and decease ATP production in the heart.

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