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Structural Studies of Biomolecules by Dynamic Nuclear Polarization Solid-State NMR Spectroscopy

Conroy, Daniel William
http://orcid.org/0000-0003-4328-1634
http://rave.ohiolink.edu/etdc/view?acc_num=osu1555428362333615

Abstract Details

2019, Doctor of Philosophy, Ohio State University, Chemistry.
Solid-state NMR (SSNMR) spectroscopy is an incredibly powerful tool for studying the structure and dynamics of biomolecules and large macromolecular complexes. SSNMR has no inherent size restriction and is useful in studying non-crystalline protein such as membrane-bound protein or amyloid fibrils, DNA-protein complexes, and large protein assemblies such as the HIV-capsid. Meanwhile, techniques such as solution-state NMR and x-ray crystallography are strictly limited by molecular size and sample condition, and thus cannot study large biomolecules or insoluble protein aggregates, respectively. The first protein at the center of this study is the human prion protein (PrP) and its formation into an insoluble amyloid fibril. The formation of this fibril leads to a deposition of an insoluble plaque on the central nervous system which leads to the development of the prion protein disease, known as Gerstmann-Straussler-Scheinker (GSS) disease. The second system-of-interest is the nucleosome core particle (NCP) which is a DNA-protein complex and is the building-block of chromatin and chromosomes. An individual NCP is composed of dsDNA wrapped around an octamer of histone protein, mimicking the natural phenomena of DNA storage and compaction in eukaryotic cells. SSNMR is uniquely qualified in studying these biological systems in depth to characterize the amyloid fibril propagation and disease-causing mechanism of prion protein, and to explore nucleic acid base-pairing behavior at DNA-protein interfaces and the important interactions therein. Dynamic nuclear polarization (DNP) is a hyperpolarization technique for NMR spectroscopy which dramatically increases the overall sensitivity of these experiments. In DNP-SSNMR, hyperpolarization is achieved by applying microwave irradiation to free electrons, often in the form of stable-radicals, within a static magnetic field and inducing a polarization transfer to neighboring nuclear spins, especially spin-½ protons. These elevated levels of magnetization in protons can be passed on to other NMR-active nuclei, in particular carbon-13 (13C) and nitrogen-15 (15N), using standard SSNMR experimental protocols. This process can generate NMR signal enhancements ranging from 20 to 200 and may be generally applied to small molecules, solid-phase materials and biological samples. DNP offers significant time savings when performing complex SSNMR experiments and opens the door for studying dilute species, weak interactions, or minor populations which are typically below the threshold of standard NMR spectroscopy. The primary goal of this dissertation is to develop strategies to study the structures of prion protein amyloid fibrils and dsDNA by expanding on current DNP-SSNMR methodologies, as well as introducing a novel class of biradicals. First, new polarizing agents, peptide-based biradicals are synthesized, purified, characterized and are successfully employed to study small molecules, crystalline protein, and amyloid protein by DNP-SSNMR. These dinitroxides include TOAC-TOAC (TT), TOAC-Ser-TOAC (TST), TOAC-TOAC-Ser (TTS) and acetyl-TOAC-TOAC (ATT) and compare favorably to well-established, commercial polarizing agents TOTAPOL and AMUPol. Next, two samples of dsDNA are used as models to develop DNP-SSNMR techniques for uniquely characterizing Watson-Crick (WC) and Hoogsteen (HG) base-pairing in duplex DNA. The DNP sample preparation is optimized for studying nucleic acids and a variety of SSNMR experiments are performed to produce spectral fingerprints of WC and HG base-pairing. These methods are then applied to a nucleosome sample to study the populations of HG base-pairing present in chromatin. This analysis provides a roadmap for studying DNA base-pairing in vitro by DNP-SSNMR spectroscopy. Finally, the amyloid structure of the human prion protein (huPrP) with a single mutation, A117V, is analyzed by various structural biology techniques to characterize the fibrils and to begin understanding the GSS disease-associated, amyloid-fibrillar structure. The DNP-SSNMR strategies and experiments described herein can be generally applied to various biological systems, containing protein or DNA, as the overall techniques are not tailored specifically to these systems-of-interest. In addition, the novel class of biradical peptides offer versatility and simplicity for tailoring polarizing agents for specific biological samples. This dissertation should serve as a guide of useful experiments and methodologies for studying the structures of biomolecules by DNP-SSNMR spectroscopy.
Christopher Jaroniec (Advisor)
Rafael Bruschweiler (Committee Member)
Joshua Goldberger (Committee Member)
Marcos Sotomayor (Committee Member)
295 p.
Chemistry
Solid-State NMR Spectroscopy; Dynamic Nuclear Polarization; Dinitroxide; Biradical; Polarizing Agent; Solid-Phase Peptide Synthesis; TOAC; DNA; Hoogsteen Base-Pairing; Nucleosome Core Particle; Amyloid Fibril; Prion Protein

Recommended Citations

Citations

  • Conroy, D. W. (2019). Structural Studies of Biomolecules by Dynamic Nuclear Polarization Solid-State NMR Spectroscopy [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1555428362333615

    APA Style (7th edition)

  • Conroy, Daniel. Structural Studies of Biomolecules by Dynamic Nuclear Polarization Solid-State NMR Spectroscopy. 2019. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1555428362333615.

    MLA Style (8th edition)

  • Conroy, Daniel. "Structural Studies of Biomolecules by Dynamic Nuclear Polarization Solid-State NMR Spectroscopy." Doctoral dissertation, Ohio State University, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=osu1555428362333615

    Chicago Manual of Style (17th edition)

osu1555428362333615
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This open access ETD is published by The Ohio State University and OhioLINK.