| dc.description.abstract | Solution nuclear magnetic resonance spectroscopy (NMR) is known as a powerful tool in many fields because of its remarkable atomic resolution. However, its applications to non-equilibrium systems are limited due to low sensitivity of detection. A new technique, dissolution dynamic nuclear polarization (D-DNP) provides a unprecedented enhancement on NMR signals and opens a variety of applications of D-DNP NMR on time-resolved measurements at subsecond to second time scales. This thesis made a first attempt to monitor a protein kinetics using D-DNP NMR. A medium-size model protein (a two-state folder, L23) was directly hyperpolarized on 13C nuclei. The focus was placed on quantitative kinetic analysis and evaluations of possibilities that may affect the observed kinetics. In addition, this thesis included developments of experimental setups and NMR acquisition methods to address encountered problems in studies of large biomolecules using D-DNP.
The refolding was triggered by changing the pH environment as a result of mixing aciddenatured
L23 solution with a buffer of high pH. Chemical shift changes were observed in a series of time-resolved 13C spectra, indicating the formation of secondary structures. The re-folding rate constant was extracted by fitting fractions of folded/unfolded forms to a two-state folding kinetic model taking into account non-instantaneous mixing of two solutions. The assumption of equal relaxation time constants for folded/unfolded L23 and spectral artifacts arose from one-sided reaction were validated and evaluated. Consequently, the rate constants are in good agreement with those obtained from fluorescence experiments.
In the above experiment, gas was used to inject the protein into an NMR tube. Due to turbulence from the rapid injection, protein samples are prone to foam which broadens NMR linewidths and leads to low signal-to-noise ratio. We have developed a new method to suppress air bubbles in which water was used to inject samples into a flow cell. Because of the incompressibility of liquid, the minimized residual sample motion was demonstrated as a parallel advantage that can alleviate signal loss in measurements using pulsed field gradients. In addition, the liquid driven injection permits studying biochemical reactions at a physiological condition since no need of using a mixture of organic solvent and water to dissolve protein sample for reducing propensity of foaming as in the above study. Conventional multidimensional NMR is not immediately compatible with the dissolution DNP to interrogate spin correlations due to the need of iterative measurements and the limitation of non-renewable hyperpolarization. We have developed a new method that integrates flow NMR into D-DNP to perform Hadamard spectroscopy and used entroy maximization algorithm to reconstruct pseudo-multidimensional spectra that contains spin correlations.
As signal enhancement is critical for experiments that directly polarize large molecules, it is beneficial to monitor hyperpolarization level in the solid state and investigate field dependence of T1 relaxation during sample delivery. For this purpose, we have constructed an NMR device based on field programmable gate arrays (FPGA) and surface mount technology as an ancillary diagnostic tool. | en |
