Structural Biology in Cellular Environments Using Sensitivity Enhanced NMR

This dissertation describes the general applicability of DNP solid state NMR to mammalian cells. DNP NMR can detect proteins at their physiological concentrations within cellular environment in tractable times. However, these experiments were limited to cellular lysates, which did not recapitulate the intracellular environment completely. Studying proteins within their native cellular environment required in-cell NMR. This work focuses on developing methodology for in-cell DNP NMR and applying the technique to investigate the conformational ensemble of Tau. Chapter 1 provides an overview of in-cell NMR in biological systems and intrinsically disordered proteins. A brief primer of NMR, DNP and EPR is also provided in this chapter. Chapter 2 describes the development of methodology to maintain biological integrity of mammalian cells during DNP NMR. Description of a novel method to insert samples into a spectrometer is added as a supplementary chapter 2. Chapter 3 is based on a comparative study between different cryoprotectants to optimize biological integrity for in-cell DNP NMR. Chapter 4 focusses on the characterization of the reduction process of a polarizing agent, AMUPol inside intact and lysed HEK293 cells using electron paramagnetic resonance (EPR) spectroscopy. In the supplementary chapter 4, a novel DNP radical AsymPol-POK and its reduction kinetics within intact HEK293 cells is discussed. Chapter 5 deals with the application of in-cell DNP NMR to study the conformational ensemble of tau protein within cellular environment. The in-cell DNP NMR methodology developed in this work can be potentially applied to various biological systems. Additionally, the strategies and experiments described here to investigate the conformational ensemble of an intrinsically disordered protein, tau, within cellular environment, can be tailored to any other IDP or protein of interest within normal or pathological cellular milieu. Also, the EPR experiments described here will be a useful guide to test reduction kinetics of novel polarizing agents for in-cell DNP NMR. Overall, this dissertation should serve as a useful literature for technical advancement and biological application of in-cell DNP NMR.

Author dc:creator

• Ghosh, Rupam

Contributors dc:contributor

• Rizo-Rey, José
• Frederick, Kendra
• Diamond, Marc
• Liszczak, Glen

• Magnetic Resonance Spectroscopy
• Single-Cell Analysis
• Intrinsically Disordered Proteins

Language dc:language

en

Identifier

1482732344

OAI identifier oai:identifier

oai:utswmed-ir.tdl.org:2152.5/10454