Protein folding occurs on a timescale that is not directly observable with most traditional nuclear magnetic resonance (NMR) spectroscopy methods. The insights into this complex process that can potentially be gained from the high site resolution of NMR has led to developments such as stopped-flow NMR, incorporation of specific isotope labels, and pulse sequences tailored for rapid data acquisition.' Hyperpolarization, the generation of a non-equilibrium spin state, shows significant promise to enhance the sensitivity and, by removing the need for signal averaging, dramatically decrease signal acquisition time. For the study of protein folding, chemically induced dynamic nuclear polarization (CIDNP) has been used to hyperpolarize tryptophan residues that undergo a cyclic reaction with a photo-sensitizer. A different technique, dynamic nuclear polarization (DNP), hyperpolarizes nuclei throughout a molecule via an admixed stable free radical. Combined with solid state NMR, DNP provides unique information on protein structure. Using dissolution DNP, NMR in the liquid state would be sensitive to structural changes across the entire macromolecule during the folding process. Liquid-state NMR signals of a DNP hyperpolarized, denatured protein, the ribosomal protein L23, have recently been observed.
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