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

Gregory L Olsen,Or Szekely,Lucio Frydman,Roberta Pierattelli,Fabien Ferrage,Robert Konrat,Dennis Kurzbach,Geoffrey Bodenhausen,Isabella C Felli,Borja Mateos,Pavel Kadeřávek

doi:10.1007/s10858-020-00301-5

Abstract

Signal enhancements of up to two orders of magnitude in protein NMR can be achieved by employing HDO as a vector to introduce hyperpolarization into folded or intrinsically disordered proteins. In this approach, hyperpolarized HDO produced by dissolution-dynamic nuclear polarization (D-DNP) is mixed with a protein solution waiting in a high-field NMR spectrometer, whereupon amide proton exchange and nuclear Overhauser effects (NOE) transfer hyperpolarization to the protein and enable acquisition of a signal-enhanced high-resolution spectrum. To date, the use of this strategy has been limited to 1D and 1H-15N 2D correlation experiments. Here we introduce 2D 13C-detected D-DNP, to reduce exchange-induced broadening and other relaxation penalties that can adversely affect proton-detected D-DNP experiments. We also introduce hyperpolarized 3D spectroscopy, opening the possibility of D-DNP studies of larger proteins and IDPs, where assignment and residue-specific investigation may be impeded by spectral crowding. The signal enhancements obtained depend in particular on the rates of chemical and magnetic exchange of the observed residues, thus resulting in non-uniform ‘hyperpolarization-selective’ signal enhancements. The resulting spectral sparsity, however, makes it possible to resolve and monitor individual amino acids in IDPs of over 200 residues at acquisition times of just over a minute. We apply the proposed experiments to two model systems: the compactly folded protein ubiquitin, and the intrinsically disordered protein (IDP) osteopontin (OPN).

Highlights

Nuclear magnetic resonance (NMR) spectroscopy is one of the principal tools for the investigation of the structural dynamics and structure–function relationships of proteins in solution (Theillet et al 2016; Reichheld et al 2017; Gil et al.1 3 Vol.:(0123456789)Journal of Biomolecular NMR (2020) 74:161–1712013; Yuwen and Skrynnikov 2014)
To quantify the signal intensities obtained with hyperpolarized HDO, we instead report signal-to-noise ratios (SNR) for the hyperpolarized spectra. (As an approximate lower bound for ε, one can assume that ε ≥ SNR for instances where the corresponding reference signal remains below the limit of detection)
We demonstrated how solvent hyperpolarization can be combined with rapid 2D 1H-15N HMQC spectroscopy (Schanda et al 2005) of folded proteins and small intrinsically disordered protein (IDP) (Kadeřávek et al 2018; Szekely et al 2018; Kurzbach et al 2017; Olsen et al 2016; Kim et al 2017)

Summary

Introduction

Nuclear magnetic resonance (NMR) spectroscopy is one of the principal tools for the investigation of the structural dynamics and structure–function relationships of proteins in solution (Theillet et al 2016; Reichheld et al 2017; Gil et al. Vol.:(0123456789)Journal of Biomolecular NMR (2020) 74:161–1712013; Yuwen and Skrynnikov 2014). Hyperpolarized HDO produced using dissolution-dynamic nuclear polarization (D-DNP) can serve as a polarization reservoir, enhancing the 1H 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 2017; Olsen et al 2016; Kim et al 2017; Ragavan et al 2017; Wang and Hilty 2019; Doll et al 2012; Viennet et al 2016)

Methods

Results

Conclusion