Characterization of Structural Changes in Biomolecules Using Polarization Transfer from Hyperpolarized Water
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Date
2020-07-17
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Abstract
Structural changes occurring during the folding and unfolding of proteins are not directly accessible with nuclear magnetic resonance (NMR) spectroscopy due to a slower timescale of detection compared to that of the folding process. This limitation originates in part from an intrinsically low sensitivity of NMR that requires signal averaging. The hyperpolarization technique of dissolution dynamic nuclear polarization (D-DNP) can enhance NMR sensitivity by several orders of magnitude, resulting in a reduction of the measurement time. In this dissertation, the hyperpolarization of water is used to enhance the sensitivity of protein signals through polarization transfer. The mechanisms of polarization transfer between water and a fluorinated small molecule, as well as between water and a protein, are characterized. In the case of the fluorinated small molecule, polarization is transferred through the intermolecular nuclear Overhauser effect (NOE). The transferred hyperpolarization on the target allows the measurement of the NOE buildup curve in a single, rapid experiment. Cross-relaxation rates are determined, which is an indicator of molecular interactions. In the case of the protein, water polarization is most effectively transferred through chemical exchange of labile protons on the protein, and then passed to nearby protons through the intramolecular NOE. Based on fitting to proposed two- and three-site models, average exchange and cross-relaxation rates are calculated, indicating that polarization transfer from hyperpolarized water to the protein through intermolecular NOE is relatively small compared to that of proton exchange. With protein signals enhanced through hyperpolarized water, two-dimensional NMR spectra can be acquired within seconds. These spectra allow accessing the protein structure not only in the folded state but also during refolding that is initiated by the dilution of urea. In both cases, the peak pattern of the folded protein is observed. Under refolding conditions, a smaller number of residues obtain polarization through NOE compared to the folded protein. This observation is explained by the different dynamic motions and molecular contacts during the folding process. Spectra of refolding protein are further measured as a function of urea concentration. The change in urea concentration causes different folding rates. From these spectra, the structural transition from an unfolded to partially folded protein is observed.
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NMR, Hyperpolarization, Dissolution DNP, Protein folding