Studying the temporal evolution of protein interactions (enzymatic reactions, biomineralization, molecular recognition) with high structural resolution is challenging. Nuclear Magnetic Resonance (NMR) spectroscopy, a key method for determining protein structures in solution, typically focuses on systems in chemical equilibrium due to weak signal intensities requiring long signal averaging, hindering time-resolved studies. To overcome this, methods that improve signal intensity while maintaining high resolution are needed. This paper introduces a method based on dissolution dynamic nuclear polarization (d-DNP) to boost NMR signals and enable time-resolved studies. Hyperpolarization techniques, particularly d-DNP, have enhanced NMR signals in residue-resolved studies of proteins and nucleic acids, offering substantial signal intensity improvements. However, while d-DNP provides signal enhancements up to 10,000-fold, enabling access to time-resolved data on milliseconds to seconds timescales, its application in biomolecular NMR has been limited. This is largely because many applications require residue-resolution, which is typically lost in faster time-resolved experiments. The current work aims to overcome this by using hyperpolarized water to enhance NMR proton signals in proteins, allowing for both time- and residue-resolved NMR spectra under near-physiological conditions.

Recent research has demonstrated the use of hyperpolarization, specifically dissolution dynamic nuclear polarization (d-DNP), to significantly enhance NMR signals in proteins and nucleic acids. Studies have shown successful applications in improving signal intensities in residue-resolved studies, leading to improved sensitivity. However, a key limitation of these methods was the trade-off between achieving high temporal resolution and maintaining residue-specific information. Previous studies focused on either high sensitivity or residue resolution but lacked both simultaneously. This paper builds upon this existing work by proposing a method that combines high sensitivity with the capability of obtaining residue-resolved, time-dependent information.

The experimental strategy combines two key concepts: hyperpolarized buffers and selective detection. Hyperpolarized buffers, created using d-DNP, enhance protein NMR signals through chemical exchange and Nuclear Overhauser Effect (NOE) interactions. The d-DNP procedure consists of three steps: 1) Preparation of hyperpolarized water protons at cryogenic temperatures and high magnetic field through off-resonance microwave irradiation. 2) Rapid dissolution of the hyperpolarized water and mixing with the protein solution in the NMR spectrometer. 3) NMR detection during which polarization transfers from the buffer to the protein, enhancing signals of solvent-exposed residues. Selective detection uses tailored NMR pulse sequences to select a small subset of hyperpolarized residues for detection. The experiment employs water-selective nuclear Overhauser spectroscopy (WS-NOESY) to quantify polarization transfer from the solvent to the protein at residue resolution. By varying the mixing time, the dependence of polarization transfer on solvent interaction of each residue is determined. This allows selection of specific residues for detection by adjusting the interscan delay in d-DNP experiments to achieve spectral sparseness. The resulting signals are so sparse that individual residues can be resolved in 1D NMR, enabling real-time monitoring of hyperpolarization levels of individual residues. The method utilizes a 1D ¹⁵N-edited ¹H spectrum, where the sparseness of the spectrum allows for the deconvolution of signals. Data analysis involves fitting the time traces of the signals to determine the apparent decay rates. NMR spectroscopy employs a WS-NOESY pulse sequence with water-selective saturation and a mixing period to control polarization transfer. Experiments were performed on Ubiquitin under near-physiological conditions.

The study successfully demonstrated residue-resolved real-time NMR of Ubiquitin (Ubq) under near-physiological conditions using hyperpolarized water. The combination of hyperpolarized buffers and selective detection resulted in spectra sparse enough to allow real-time monitoring at a 2 Hz sampling rate. The time-dependent signal intensities revealed that residues buried in hydrophobic regions exhibited faster relaxation due to less efficient hyperpolarized proton replenishment, while residues in flexible regions showed slower apparent decays. The observed decay rates varied depending on factors like water proton and ¹H relaxation rates, proton exchange rates, and pulse angles. The study found a clear correlation between the signal intensities observed in the WS-NOESY and those observed in the d-DNP experiments, indicating the efficacy of the approach. Specifically, 13 residues displayed observable signals in WS-NOESY at 0.5 s mixing time, and 10 discernible lines were seen in the 1D spectrum from a d-DNP experiment. Seven residues could be unambiguously identified in the 1D spectrum. The signal enhancements and apparent decay rates for ten fitted lines are listed in Table 1. Residues with protic side chains exhibited higher enhancements, consistent with previous findings regarding the contributions of chemical exchange and solvent-relayed NOEs to signal enhancement. The presented technique allowed for monitoring of the hyperpolarization levels of individual residues simultaneously, offering a novel approach to studying protein dynamics and interactions at high temporal and residue resolution.

The presented method successfully addresses the longstanding challenge of obtaining both high temporal and spatial resolution in protein NMR studies. By using hyperpolarized water and selective detection techniques, the authors achieved residue-resolved NMR spectra of ubiquitin with a high sampling rate of 2 Hz. This overcomes limitations of previous approaches that either compromised resolution or temporal resolution. The ability to monitor individual residues over time opens up exciting possibilities for studying protein dynamics and interactions under near-physiological conditions. This is particularly relevant for understanding protein folding, ligand binding, and enzymatic reactions. The observed differences in decay rates among different residues provide valuable insights into the local environment and dynamics of each residue. This methodology represents a significant advancement in biomolecular NMR, paving the way for more comprehensive investigations of protein behavior in various contexts.

This study presents a novel method for real-time, residue-resolved NMR spectroscopy of proteins using hyperpolarized water and selective detection. This technique enables the monitoring of individual residue hyperpolarization levels with sub-second time resolution under near-physiological conditions. The method opens avenues for investigating protein dynamics and interactions with high spatiotemporal resolution, particularly useful for studying ligand binding and enzymatic reactions. Future research could explore the applicability of this technique to a broader range of proteins, including intrinsically disordered proteins and larger protein complexes. Further optimization of experimental parameters could also enhance sensitivity and resolution.

The technique's applicability might be limited for intrinsically disordered proteins with narrow chemical shift dispersions in the proton dimension, where 2D or 3D detection is typically required for residue resolution. The signal enhancement and decay rates are influenced by multiple factors (proton exchange, NOEs, relaxation rates), potentially leading to complexities in interpreting the data. The selective detection approach may not capture all protein residues, potentially biasing the observations.