Toward protein NMR at physiological concentrations by hyperpolarized water—Finding and mapping uncharted conformational spaces

Abstract

Nuclear magnetic resonance (NMR) spectroscopy is a key method for determining the structural dynamics of proteins in their native solution state. However, the low sensitivity of NMR typically necessitates nonphysiologically high sample concentrations, which often limit the relevance of the recorded data. We show how to use hyperpolarized water by dissolution dynamic nuclear polarization (DDNP) to acquire protein spectra at concentrations of 1 μM within seconds and with a high signal-to-noise ratio. The importance of approaching physiological concentrations is demonstrated for the vital MYC-associated factor X, which we show to switch conformations when diluted. While in vitro conditions lead to a population of the well-documented dimer, concentrations lowered by more than two orders of magnitude entail dimer dissociation and formation of a globularly folded monomer. We identified this structure by integrating DDNP with computational techniques to overcome the often-encountered constraint of DDNP of limited structural information provided by the typically detected one-dimensional spectra.

Missing Pieces in Structure Puzzles: How Hyperpolarized NMR Spectroscopy Can Complement Structural Biology and Biochemistry

“…Such altered conditions can significantly impact the structure and function of a biomolecule. DDNP can provide the structures of many targets under conditions that are much closer to physiological compared to conventional NMR in thermal equilibrium [89–91] The interactions, conformational transitions, and molecular recognition events of many substrates proceed on the millisecond to low seconds timescale, and it is notoriously difficult to follow their kinetics.…”

“…Such altered conditions can significantly impact the structure and function of a biomolecule. DDNP can provide the structures of many targets under conditions that are much closer to physiological compared to conventional NMR in thermal equilibrium [89–91] …”

“…Adapted with permission from ref. [91]. Copyright: 2022, the American Association for the Advancement of Science.…”

“…DDNP can provide the structures of many targets under conditions that are much closer to physiological compared to conventional NMR in thermal equilibrium. [89][90][91] 3) The interactions, conformational transitions, and molecular recognition events of many substrates proceed on the millisecond to low seconds timescale, and it is notoriously difficult to follow their kinetics. With DDNP it is possible to achieve rapid real-time monitoring of such events while maintaining one main advantage of biomolecular NMR, namely residue resolution.…”

“…1) A transcription factor (the Myc-associated factor X, MAX) was studied at concentrations that are typically too low for conventional thermal equilibrium NMR, namely, 1 μM. [91] With dissolution DNP and HyperW, a 1D 1 H spectrum could yet be acquired under these conditions with a good signal-to-noise ratio. Peaks were detected that remained below the detection threshold in conventional NMR even after extensive signal averaging over one day.…”