High-Resolution 2D NMR of Disordered Proteins Enhanced by Hyperpolarized Water

Abstract

This study demonstrates the usefulness derived from relying on hyperpolarized water obtained by dissolution DNP, for site-resolved biophysical NMR studies of intrinsically disordered proteins. Thanks to the facile amide-solvent exchange experienced by protons in these proteins, 2D NMR experiments that like HMQC rely on the polarization of the amide protons, can be enhanced using hyperpolarized water by several orders of magnitude over their conventional counterparts. Optimizations of the DNP procedure and of the subsequent injection into the protein sample are necessary to achieve these gains while preserving state-of-the-art resolution; procedures enabling this transfer of the hyperpolarized water and the achievement of foamless hyperpolarized protein solutions are demonstrated. These protocols are employed to collect 2D N-H HMQC NMR spectra of α-synuclein, showing residue-specific enhancements ≥100× over their thermal counterparts. These enhancements, however, vary considerably throughout the residues. The biophysics underlying this residue-specific behavior upon injection of hyperpolarized water is theoretically examined, the information that it carries is compared with results arising from alternative methods, and its overall potential is discussed.

Laser- and cryogenic probe-assisted NMR enables hypersensitive analysis of biomolecules at submicromolar concentration

“…1 H-13 C CT-SE-HSQC and 1 H-13 C CT-SOFAST-HMQC Reference Experiments. The 1 H-13 C CT-SE-HSQC and 1 H-13 C CT-SOFAST-HMQC reference experiments were run at 24°C on an Avance III HD 900 MHz (21.2 T) spectrometer equipped with a 1 H { 13 C, 15 N} cryogenic probe. The 2D 1 H-13 C CT-SE-HSQC data were collected/processed in echo-antiecho mode (53), and 2D 1 H-13 C CT-SOFAST-HMQC data were acquired/ processed in States-TPPI mode.…”

“…Methods implemented over the years to overcome the low sensitivity of NMR in liquids include the use of high applied magnetic fields, data acquisition in the time domain followed by Fourier transform, fast data collection schemes, and cryogenic probes (1)(2)(3). More recently, nuclear-spin hyperpolarization including Overhauser dynamic nuclear polarization (4,5), optical pumping (6)(7)(8)(9), parahydrogen-induced polarization (10,11), signal amplification by reversible exchange (12), and dissolution dynamic nuclear polarization (D-DNP) (13)(14)(15)(16)(17) have displayed significant potential (3).…”

“…The 1D13 C PREPRINT photo-CIDNP spectra of Trp in an E. coli cellfree system. Dark (i.e., laser-off) and light (i.e., laser-on) NMR data on15 N-13 C-Trp (25 μM) are shown in the 1 H (A) aromatic and (B) aliphatic region of a diluted cell-free solution prepared as described in SI Appendix, section 2.5, with data collected and processed as described in SI Appendix, section 2.6.Okuno et alPNAS | June 11, 2019 | vol. 116 | no.…”

“…C,15 N-Trp (Cambridge Isotopes) as a small-molecule model system and fluorescein (Sigma-Aldrich) as a photosensitizer. Aspergillus niger GO and bovine liver CAT were purchased as freeze-dried powders from Sigma-Aldrich.…”

Sensitivity-enhanced three-dimensional and carbon-detected two-dimensional NMR of proteins using hyperpolarized water

“…Hyperpolarized HDO produced using dissolution-dynamic nuclear polarization (D-DNP) can serve as a polarization reservoir, enhancing the 1 H signals of sites undergoing chemical-or polarization exchange with the hyperpolarized solvent. This approach, which has been referred to as HYPEX (Kadeřávek et al 2018) or HyperW (Szekely et al 2018), is applicable to a broad range of aqueous systems without requiring any special properties or chemical modification of the molecules under study, and makes it possible to achieve over 100-fold signal enhancements in spectra of folded or intrinsically disordered proteins (Kadeřávek et al 2018;Szekely et al 2018;Kurzbach et. al.…”