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

Structure determination lies at the heart of many biochemical research programs. However, the "giants": X-ray diffraction, electron microscopy, molecular dynamics simulations, and nuclear magnetic resonance, among others, leave quite a few dark spots on the structural pictures drawn of proteins, nucleic acids, membranes, and other biomacromolecules. For example, structural models under physiological conditions or of short-lived intermediates often remain out of reach of the established experimental methods. This account frames the possibility of including hyperpolarized, that is, dramatically signal-enhanced NMR in existing workflows to fill these spots with detailed depictions. We highlight how integrating methods based on dissolution dynamic nuclear polarization can provide valuable complementary information about formerly inaccessible conformational spaces for many systems. A particular focus will be on hyperpolarized buffers to facilitate the NMR structure determination of challenging systems.

Constraining and Modifying Peptides Using Pd-Mediated Cysteine Allylation.

Over the past decades, several strategies for inducing and stabilizing secondary structure formation in peptides have been developed to increase their proteolytic stability and their binding affinity to specific interaction partners. Here, we report how our recently introduced chemoselective Pd-catalyzed cysteine allylation reaction can be extended to stapling and how the resulting alkene-containing staples themselves can be further modified to introduce additional probes into such stabilized peptides. The latter is demonstrated by introducing a fluorophore as well as a PEG moiety into different stapled peptides using bioorthogonal thiol-ene and Diels-Alder reactions. Furthermore, we investigated structural implications of our allyl staples when used to replace conformationally relevant disulfide bridges. To this end, we chose a selective binder of integrin α3 ß1 (LXY3), which is only active in its cyclic disulfide form. We replaced the disulfide bridge by different stapling reagents in order to increase stability and binding affinity towards integrin α3 ß1 .

Frémy's Salt as a Low-Persistence Hyperpolarization Agent: Efficient Dynamic Nuclear Polarization Plus Rapid Radical Scavenging.

Nuclear magnetic resonance (NMR) spectroscopy is a key technique for molecular structure determination in solution. However, due to its low sensitivity, many efforts have been made to improve signal strengths and reduce the required substrate amounts. In this regard, dissolution dynamic nuclear polarization (DDNP) is a versatile approach as signal enhancements of over 10 000-fold are achievable. Samples are signal-enhanced ex situ by transferring electronic polarization from radicals to nuclear spins before dissolving and shuttling the boosted sample to an NMR spectrometer for detection. However, the applicability of DDNP suffers from one major drawback, namely, paramagnetic relaxation enhancements (PREs) that critically reduce relaxation times due to the codissolved radicals. PREs are the primary source of polarization losses canceling the signal improvements obtained by DNP. We solve this problem by using potassium nitrosodisulfonate (Frémy's salt) as polarization agent (PA), which provides high nuclear spin polarization and allows for rapid scavenging under mild reducing conditions. We demonstrate the potential of Frémy's salt, (i) showing that both 1H and 13C polarization of ∼30% can be achieved and (ii) describing a hybrid sample shuttling system (HySSS) that can be used with any DDNP/NMR combination to remove the PA before NMR detection. This gadget mixes the hyperpolarized solution with a radical scavenger and injects it into an NMR tube, providing, within a few seconds, quantitatively radical-free, highly polarized solutions. The cost efficiency and broad availability of Frémy's salt might facilitate the use of DDNP in many fields of research.

Formation and Evolution of Nanoscale Calcium Phosphate Precursors under Biomimetic Conditions.

Simulated body fluids (SBFs) that mimic human blood plasma are widely used media for in vitro studies in an extensive array of research fields, from biomineralization to surface and corrosion sciences. We show that these solutions undergo dynamic nanoscopic conformational rearrangements on the timescale of minutes to hours, even though they are commonly considered stable or metastable. In particular, we find and characterize nanoscale inhomogeneities made of calcium phosphate (CaP) aggregates that emerge from homogeneous SBFs within a few hours and evolve into prenucleation species (PNS) that act as precursors in CaP crystallization processes. These ionic clusters consist of ∼2 nm large spherical building units that can aggregate into suprastructures with sizes of over 200 nm. We show that the residence times of phosphate ions in the PNS depend critically on the total PNS surface. These findings are particularly relevant for understanding nonclassical crystallization phenomena, in which PNS are assumed to act as building blocks for the final crystal structure.

Conformational tuning of a DNA-bound transcription factor.

Transcription factors are involved in many cellular processes that take place remote from their cognate DNA sequences. The efficiencies of these activities are thus in principle counteracted by high binding affinities of the factors to their cognate DNAs. Models such as facilitated diffusion or dissociation address this apparent contradiction. We show that the MYC associated transcription factor X (MAX) undergoes nanoscale conformational fluctuations in the DNA-bound state, which is consistent with facilitated dissociation from or diffusion along DNA strands by transiently reducing binding energies. An integrative approach involving EPR, NMR, crystallographic and molecular dynamics analyses demonstrates that the N-terminal domain of MAX constantly opens and closes around a bound DNA ligand thereby dynamically tuning the binding epitope and the mode of interaction.

Evaporation-Induced Self-Assembly of Small Peptide-Conjugated Silica Nanoparticles.

Self-assembly processes guide disordered molecules or particles into long-range organized structures due to specific supramolecular interactions among the building entities. Herein, we report a unique evaporation-induced self-assembly (EISA) strategy for four different silica nanoparticle systems obtained through peptide functionalization of the particle surface. First, covalent peptide-silica coupling was investigated in detail, starting with the grafting of a single amino acid (L-serine) and expanded to specific small peptides (up to four amino acids) and transferred to different particle types (MCM-48-type MSNs, solid nanoparticles, and newly developed virus-like nanoparticles). These materials were investigated regarding their ability to undergo EISA, which was shown to be independent of particle type and amount of peptide anchored to their surface. This EISA-based approach provides new possibilities for the design of future advanced drug delivery systems, engineered hierarchical sorbents, and nanocatalyst assemblies.

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

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).

Assessing the Onset of Calcium Phosphate Nucleation by Hyperpolarized Real-Time NMR.

We report an experimental approach for high-resolution real-time monitoring of transiently formed species occurring during the onset of precipitation of ionic solids from solution. This is made possible by real-time nuclear magnetic resonance (NMR) monitoring using dissolution dynamic nuclear polarization (D-DNP) to amplify signals of functional intermediates and is supported by turbidimetry, cryogenic electron microscopy, and solid-state NMR measurements. D-DNP can provide drastic signal improvements in NMR signal amplitudes, permitting dramatic reductions in acquisition times and thereby enabling us to probe fast interaction kinetics such as those underlying formation of prenucleation species (PNS) that precede solid-liquid phase separation. This experimental strategy allows for investigation of the formation of calcium phosphate (CaP)-based minerals by 31P NMR-a process of substantial industrial, geological, and biological interest. Thus far, many aspects of the mechanisms of CaP nucleation remain unclear due to the absence of experimental methods capable of accessing such processes on sufficiently short time scales. The approach reported here aims to address this by an improved characterization of the initial steps of CaP precipitation, permitting detection of PNS by NMR and determination of their formation rates, exchange dynamics, and sizes. Using D-DNP monitoring, we find that under our conditions (i) in the first 2 s after preparation of oversaturated calcium phosphate solutions, PNS with a hydrodynamic radius of Rh ≈ 1 nm is formed and (ii) following this rapid initial formation, the entire crystallization processes proceed on considerably longer time scales, requiring >20 s to form the final crystal phase.

Hyperpolarized NMR Metabolomics at Natural 13C Abundance.

Metabolomics plays a pivotal role in systems biology, and NMR is a central tool with high precision and exceptional resolution of chemical information. Most NMR metabolomic studies are based on 1H 1D spectroscopy, severely limited by peak overlap. 13C NMR benefits from a larger signal dispersion but is barely used in metabolomics due to ca. 6000-fold lower sensitivity. We introduce a new approach, based on hyperpolarized 13C NMR at natural abundance, that circumvents this limitation. A new untargeted NMR-based metabolomic workflow based on dissolution dynamic nuclear polarization (d-DNP) for the first time enabled hyperpolarized natural abundance 13C metabolomics. Statistical analysis of resulting hyperpolarized 13C data distinguishes two groups of plant (tomato) extracts and highlights biomarkers, in full agreement with previous results on the same biological model. We also optimize parameters of the semiautomated d-DNP system suitable for high-throughput studies.

Hyperpolarized Water Enhances Two-Dimensional Proton NMR Correlations: A New Approach for Molecular Interactions.

Protein and peptide interactions are characterized in the liquid state by multidimensional NMR spectroscopy experiments, which can take hours to record. We show that starting from hyperpolarized HDO, two-dimensional (2D) proton correlation maps of a peptide, either free in solution or interacting with liposomes, can be acquired in less than 60 s. In standard 2D NMR spectroscopy without hyperpolarization, the acquisition time required for similar spectral correlations is on the order of hours. This hyperpolarized experiment enables the identification of amino acids featuring solvent-interacting hydrogens and provides fast spectroscopic analysis of peptide conformers. Sensitivity-enhanced 2D proton correlation spectroscopy is a useful and straightforward tool for biochemistry and structural biology, as it does not recur to nitrogen-15 or carbon-13 isotope enrichment.

A DNP-hyperpolarized solid-state water NMR MASER: observation and qualitative analysis.

We present observations of an NMR MASER (microwave amplification by stimulated emission of radiation) of hyperpolarized 1H nuclei by dynamic nuclear polarization (DNP) at 1.2 K and in a magnetic field of 6.7 T. The sustained maser pulses originate from the interplay between radiation damping (RD) due to the large 1H magnetization, and the remagnetization to a negative value by the DNP process. NMR signals lasting for several tens of seconds are thus observed on an ensemble of dipolar-coupled nuclear spins. Magnetization dynamics are analyzed in terms of the combined Bloch-Maxwell and Provotorov (BMP) equations for RD and DNP. Insight into the long time evolution of the magnetization is provided by a theoretical analysis of this nonlinear dynamical system, and by fitting the NMR signal to a simplified version of the BMP equations.

Transport of hyperpolarized samples in dissolution-DNP experiments.

Dissolution dynamic nuclear polarization (D-DNP) experiments rely on the transfer of a sample between two high-field magnets. During this transfer, samples might experience passage through regions where the stray fields of the magnets are very weak, can approach zero, and even change their sign. This can lead to unexpected spectral features in spin systems that undergo transitions from weak- to strong-coupling regimes and vice versa, much like in field cycling nuclear magnetic resonance experiments. We herein demonstrate that the spectral features observed in D-DNP experiments can be rationalized, provided the time-dependence of the spin Hamiltonian upon field cycling is sufficiently adiabatic. Under such conditions, a passage through a weak static field can lead to the emergence of a long-lived state (LLS) based on an imbalance between the populations of singlet and triplet states in pairs of nuclei that are strongly coupled during the passage through low field. The LLS entails the appearance of anti-phase multiplet components upon transfer to a high-field magnet for observation of NMR signals.

High-Resolution NMR of Folded Proteins in Hyperpolarized Physiological Solvents.

Hyperpolarized 2D exchange spectroscopy (HYPEX) to obtain high-resolution nuclear magnetic resonance (NMR) spectra of folded proteins under near-physiological conditions is reported. The technique is based on hyperpolarized water, which is prepared by dissolution dynamic nuclear polarization and mixed in situ in an NMR spectrometer with a protein in a physiological saline buffer at body temperature. Rapid exchange of labile protons with the hyperpolarized solvent, combined with cross-relaxation effects (NOEs), leads to boosted signal intensities for many amide 1 H-15 N correlations in the protein ubiquitin. As the introduction of hyperpolarization to the target protein is mediated via the solvent, the method is applicable to a broad spectrum of target molecules.

Rates of Chemical Reactions Embedded in a Metabolic Network by Dissolution Dynamic Nuclear Polarisation NMR.

The isomerisation of 6-phosphogluconolactones and their hydrolyses into 6-phosphogluconic acid form a non enzymatic side cycle of the pentose-phosphate pathway (PPP) in cells. Dissolution dynamic nuclear polarisation can be used for determining the kinetic rates of the involved transformations in real time. It is found that the hydrolysis of both lactones is significantly slower than the isomerisation process, thereby shedding new light onto this subtle chemical process.

Relaxation of long-lived modes in NMR of deuterated methyl groups.

Long-lived imbalances of spin state populations can circumvent fast quadrupolar relaxation by reducing effective longitudinal relaxation rates by about an order of magnitude. This opens new avenues for the study of dynamic processes in deuterated molecules. Here we present an analysis of the relaxation properties of deuterated methyl groups CD3. The number of coupled equations that describe cross-relaxation between the 27 symmetry-adapted spin states of a D3 system can be reduced to only 2 non-trivial "lumped modes" by (i) taking the sums of the populations of all states that equilibrate rapidly within each irreducible representation of the symmetry group, and (ii) by combining populations that have similar relaxation rates although they belong to different irreducible representations. The quadrupolar relaxation rates of the spin state imbalances in CD3 groups are determined not by the correlation time of overall tumbling of the molecule, but by the frequency of jumps of methyl groups about their three-fold symmetry axis. We access these states via dissolution dynamic nuclear polarization (D-DNP), a method that allows one to populate the desired long-lived states at cryogenic temperatures and their subsequent detection at ambient temperatures after rapid dissolution. Experimental examples of DMSO-d6 and ethanol-d6 demonstrate that long-lived deuterium spin states are indeed accessible and that their lifetimes can be determined. Our analysis of the system via "lumped" modes allows us to visualize different possible spin-state populations of symmetry A, B, or E. Thus, we identify a long-lived spin state involving all three deuterons in a CD3 group as an A/E imbalance that can be populated through DNP at low temperatures.

Sample Ripening through Nanophase Separation Influences the Performance of Dynamic Nuclear Polarization.

Mixtures of water and glycerol provide popular matrices for low-temperature spectroscopy of vitrified samples. However, they involve counterintuitive physicochemical properties, such as spontaneous nanoscopic phase separations (NPS) in solutions that appear macroscopically homogeneous. We demonstrate that such phenomena can substantially influence the efficiency of dynamic nuclear polarization (DNP) by factors up to 20 % by causing fluctuations in local concentrations of polarization agents (radicals). Thus, a spontaneous NPS of water/glycerol mixtures that takes place on time scales on the order of 30-60 min results in a confinement of polarization agents in nanoscopic water-rich vesicles, which in return affects the DNP. Such effects were found for three common polarization agents, TEMPOL, AMUPol and Trityl.

Hydrophobic Collapse of the Intrinsically Disordered Transcription Factor Myc Associated Factor X.

The conformational space of the proto-oncogenic transcription factor Myc associated factor X (MAX) comprises a dynamic equilibrium between a stably folded coiled-coil homodimer and an intrinsically disordered ensemble of states. We show by means of nuclear magnetic resonance spectroscopy that the intrinsically disordered ensemble samples structures that are even as compact as the folded dimer. These extremely dense, hydrophobically collapsed globules might be of importance for interconversion between different conformations of intrinsically disordered proteins.

Single-Scan 13 C Diffusion-Ordered NMR Spectroscopy of DNP-Hyperpolarised Substrates.

Diffusion-ordered NMR spectroscopy (DOSY) is a powerful approach for the analysis of molecular mixtures, yet its application range is limited by the relatively low sensitivity of NMR. We show here that spectrally resolved 13 C DOSY data can be collected, in a single scan, for substrates hyperpolarised by dissolution dynamic nuclear polarisation (D-DNP), which provides signal enhancements of several orders of magnitude. For this we use a convection-compensation pulse scheme, which we also analyse by numerical simulation. The proposed method further allows the acquisition of several consecutive DOSY spectra in a single D-DNP experiment.

NMR probing and visualization of correlated structural fluctuations in intrinsically disordered proteins.

A novel statistical analysis of paramagnetic relaxation enhancement (PRE) and paramagnetic relaxation interference (PRI) based nuclear magnetic resonance (NMR) data is proposed based on the computation of correlation matrices. The technique is demonstrated with an example of the intrinsically disordered proteins (IDPs) osteopontin (OPN) and brain acid soluble protein 1 (BASP1). The correlation analysis visualizes in detail the subtleties of conformational averaging in IDPs and highlights the presence of correlated structural fluctuations of individual sub-domains in IDPs.

Communication: Dissolution DNP reveals a long-lived deuterium spin state imbalance in methyl groups.

We report the generation and observation of long-lived spin states in deuterated methyl groups by dissolution DNP. These states are based on population imbalances between manifolds of spin states corresponding to irreducible representations of the C3v point group and feature strongly dampened quadrupolar relaxation. Their lifetime depends on the activation energies of methyl group rotation. With dissolution DNP, we can reduce the deuterium relaxation rate by a factor up to 20, thereby extending the experimentally available time window. The intrinsic limitation of NMR spectroscopy of quadrupolar spins by short relaxation times can thus be alleviated.