News & Updates


Selective High-resolution DNP-enhanced NMR of Biomolecular Binding Sites

posted Feb 1, 2019, 10:06 AM by Daniel Lee

Selective High-resolution DNP-enhanced NMR of Biomolecular Binding Sites. Locating binding sites in biomolecular assemblies and solving their structures is of the utmost importance to unravel functional aspects of the system and provide experimental data that can be used for structure-based drug design. This often still remains a challenge, both in terms of selectivity and sensitivity for X-ray crystallography, cryo-electron microscopy and NMR. In this work, we introduce a novel method called Selective Dynamic Nuclear Polarization (Sel-DNP) that allows selectively highlighting and identifying residues present in the binding site. This powerful site-directed approach relies on the use of localized paramagnetic relaxation enhancement induced by a ligand-functionalized paramagnetic construct combined with difference spectroscopy to recover high-resolution and high-sensitivity information from binding sites. The identification of residues involved in the binding occurs using spectral fingerprints obtained from a set of high-resolution multidimensional spectra with varying selectivity. The methodology is demonstrated on the galactophilic lectin LecA, for which we report well-resolved DNP-enhanced spectra with linewidth between 0.5-1 ppm, which enable the de novo assignment of the binding interface residues, without using previous knowledge of the binding site location. Since this approach produces clean and resolved difference spectra containing a limited number of residues, resonance assignment can be performed without any limitation with respect to the size of the biomolecular system and only requires the production of one protein sample (e.g. 13C,15N labeled protein).

Sparsely Pillared Graphene Materials for High Performance Supercapacitors: Improving Ion Transport and Storage Capacity

posted Jan 17, 2019, 9:49 AM by Daniel Lee   [ updated Feb 7, 2019, 3:57 AM ]

Sparsely Pillared Graphene Materials for High Performance Supercapacitors: Improving Ion Transport and Storage Capacity. Graphene-based materials are extensively studied as promising candidates for supercapacitors (SCs) owing to the high surface area, electrical conductivity, and mechanical flexibility of graphene. Reduced graphene oxide (RGO), a close graphene-like material studied for SCs, offers limited specific capacitances (100 F.g-1) as the reduced graphene sheets partially restack through π-π interactions. This paper presents pillared graphene materials designed to minimize such graphitic restacking by cross-linking the graphene sheets with a bi-functional pillar molecule. Solid-state NMR, X-ray diffraction, and electrochemical analyses reveal that the synthesized materials possess covalently cross-linked graphene galleries that offer additional sites for ion sorption in SCs. Indeed, high specific capacitances in SCs are observed for the graphene materials synthesized with an optimized number of pillars. Specifically, the straight-forward synthesis of a graphene hydrogel containing pillared structures and an inter-connected porous network delivered a material with gravimetric capacitances two times greater than that of RGO (200 F.g-1 vs. 107 F.g-1) and volumetric capacitances that are nearly four times larger (210 F.cm-3 vs. 54 F.cm-3). Additionally, despite the presence of pillars inside the graphene galleries, the optimized materials show efficient ion transport characteristics. This work therefore brings perspectives for the next generation of high-performance SCs.

De novo prediction of cross-effect efficiency for magic angle spinning dynamic nuclear polarization

posted Jan 11, 2019, 5:27 AM by Daniel Lee   [ updated Feb 7, 2019, 3:57 AM ]

De novo prediction of cross-effect efficiency for magic angle spinning dynamic nuclear polarization. Magic angle spinning dynamic nuclear polarization (MAS-DNP) has become a key approach to boost the intrinsic low sensitivity of NMR in solids. This method relies on the use of both stable radicals as polarizing agents (PAs) and suitable high frequency microwave irradiation to hyperpolarize nuclei of interest. Relating PA chemical structure to DNP efficiency has been, and is still, a long-standing problem. The complexity of the polarization transfer mechanism has so far limited the impact of analytical derivation. However, recent numerical approaches have profoundly improved the basic understanding of the phenomenon and have now evolved to a point where they can be used to help design new PAs. In this work, the potential of advanced MAS-DNP simulations combined with DFT calculations and high-field EPR to qualitatively and quantitatively predict hyperpolarization efficiency of particular PAs is analyzed. This approach is demonstrated on AMUPol and TEKPol, two widely-used bis-nitroxide PAs. The results notably highlight how the PA structure and EPR characteristics affect the detailed shape of the DNP field profile. We also show that refined simulations of this profile using the orientation dependency of the electron spin-lattice relaxation times can be used to estimate the microwave B1 field experienced by the sample. Finally, we show how modelling the nuclear spin-lattice relaxation times of close and bulk nuclei as well as accounting for PA concentration allows the approximation of DNP enhancement factors and hyperpolarization build-up times.

Book chapter: MAS‐DNP Enhancements: Hyperpolarization, Depolarization, and Absolute Sensitivity

posted Jan 11, 2019, 5:24 AM by Daniel Lee   [ updated Feb 7, 2019, 3:59 AM ]

MAS‐DNP Enhancements: Hyperpolarization, Depolarization, and Absolute Sensitivity. Dynamic nuclear polarization at high magnetic fields has made significant progress over the last decades, and this hyperpolarizing technique is currently revolutionizing the impact of solid‐state NMR for the study of complex systems in chemistry, material science, and biology. In this article, we emphasize the importance and difficulty in quantifying sensitivity from DNP under magic‐angle spinning. To this end, we provide insight into the cross effect, the current main MAS‐DNP mechanism. This includes a description of the microwave‐induced hyperpolarization phenomenon but also of the reduction of the NMR signal prior to microwave irradiation for samples doped with polarizing agents (bleaching and depolarization effects). We highlight the importance of the nuclear hyperpolarization buildup time in the evaluation of MAS‐DNP efficiency. Finally, we discuss other experimental parameters affecting sensitivity in DNP‐enhanced spectra and propose a guideline for its proper characterization depending on the type of investigation.

Structural Fingerprinting of Protein Aggregates by Dynamic Nuclear Polarization-Enhanced Solid-State NMR at Natural Isotopic Abundance

posted Nov 9, 2018, 3:43 AM by Daniel Lee   [ updated Jan 11, 2019, 5:19 AM ]

Structural Fingerprinting of Protein Aggregates by Dynamic Nuclear Polarization-Enhanced Solid-State NMR at Natural Isotopic Abundance. A pathological hallmark of Huntington’s disease (HD) is the formation of neuronal protein deposits containing mutant huntingtin fragments with expanded polyglutamine (polyQ) domains. Prior studies have shown the strengths of solid-state NMR (ssNMR) to probe the atomic structure of such aggregates, but have required in vitro isotopic labeling. Herein, we present an approach for the structural fingerprinting of fibrils through ssNMR at natural isotopic abundance (NA). These methods will enable the spectroscopic fingerprinting of unlabeled (e.g., ex vivo) protein aggregates and the extraction of valuable new long-range 13C–13C distance constraints.

Computationally Assisted Design of Polarizing Agents for Dynamic Nuclear Polarization Enhanced NMR: The AsymPol Family

posted Nov 9, 2018, 3:32 AM by Daniel Lee

Computationally Assisted Design of Polarizing Agents for Dynamic Nuclear Polarization Enhanced NMR: The AsymPol Family. We introduce a new family of highly efficient polarizing agents for dynamic nuclear polarization (DNP)-enhanced nuclear magnetic resonance (NMR) applications, composed of asymmetric bis-nitroxides, in which a piperidine-based radical and a pyrrolinoxyl or a proxyl radical are linked together. The design of the AsymPol family was guided by the use of advanced simulations that allow computation of the impact of the radical structure on DNP efficiency. These simulations suggested the use of a relatively short linker with the intention to generate a sizable intramolecular electron dipolar coupling/J-exchange interaction, while avoiding parallel nitroxide orientations. The characteristics of AsymPol were further tuned, for instance with the addition of a conjugated carbon–carbon double bond in the 5-membered ring to improve the rigidity and provide a favorable relative orientation, the replacement of methyls by spirocyclohexanolyl groups to slow the electron spin relaxation, and the introduction of phosphate groups to yield highly water-soluble dopants. An in-depth experimental and theoretical study for two members of the family, AsymPol and AsymPolPOK, is presented here. We report substantial sensitivity gains at both 9.4 and 18.8 T. The robust efficiency of this new family is further demonstrated through high-resolution surface characterization of an important industrial catalyst using fast sample spinning at 18.8 T. This work highlights a new direction for polarizing agent design and the critical importance of computations in this process.


Efficient Cross-Effect Dynamic Nuclear Polarization without Depolarization in High-resolution MAS NMR

posted Oct 6, 2017, 8:51 AM by Daniel Lee   [ updated Oct 23, 2017, 12:27 AM ]

Efficient Cross-Effect Dynamic Nuclear Polarization without Depolarization in High-resolution MAS NMR. Dynamic nuclear polarization (DNP) has the potential to enhance the sensitivity of magic-angle spinning (MAS) NMR by many orders of magnitude and therefore to revolutionize atomic resolution structural analysis. Currently, the most widely used approach to DNP for studies of chemical, material, and biological systems involves the cross-effect (CE) mechanism, which relies on biradicals as polarizing agents. However, at high magnetic fields (≥ 5 T), the best biradicals used for CE MAS-DNP are still far from optimal, primarily because of the nuclear depolarization effects they induce. In the presence of bisnitroxide biradicals, magic-angle rotation results in a reverse CE that can deplete the initial proton Boltzmann polarization by more than a factor of 2. In this paper we show that these depolarization losses can be avoided by using a polarizing agent composed of a narrow-line trityl radical tethered to a broad-line TEMPO. Consequently, we show that a biocompatible trityl-nitroxide biradical, TEMTriPol-1, provides the highest MAS NMR sensitivity at ≥ 10 T, and its relative efficiency increases with the magnetic field strength. We use numerical simulations to explain the absence of depolarization for TEMTriPol-1 and its high efficiency, paving the way for the next generation of polarizing agents for DNP. We demonstrate the superior sensitivity enhancement using TEMTriPol-1 by recording the first solid-state 2D 13C-13C correlation spectrum at natural isotopic abundance at a magnetic field of 18.8 T.

Efficient 2D double-quantum solid-state NMR spectroscopy with large spectral widths

posted Aug 4, 2017, 1:30 AM by Daniel Lee   [ updated Oct 23, 2017, 12:40 AM ]

Efficient 2D double-quantum solid-state NMR spectroscopy with large spectral widths. 2D double-quantum single-quantum correlation spectra with arbitrary spectral widths can be recorded with SR26 and related supercycled recoupling sequences when applying Supercycle-Timing-Compensation (STiC) phase shifts. This concept widely extends the applicability of supercycled sequences, most importantly for obtaining long-range distance constraints for structure determination with solid-state NMR.

Book chapter: High-Field Solid-State NMR with Dynamic Nuclear Polarization

posted May 8, 2017, 2:15 AM by Daniel Lee

High-Field Solid-State NMR with Dynamic Nuclear Polarization. Microwave-induced dynamic nuclear polarization (DNP) can produce hyperpolarization of nuclear spins, leading to substantial signal enhancement in NMR. This chapter discusses the contemporary application of DNP for solid-state NMR spectroscopy at high magnetic fields. The main mechanisms and polarizing agents that enable this hyperpolarization are presented, along with more practical aspects such as the effect of decreasing sample temperature and analyzing the absolute sensitivity gain from these experiments. Examples of the exploitation of DNP for studies of biomolecules, biominerals, pharmaceuticals, self-assembled organic nanostructures, and mesoporous materials are given as is an outlook as to the future of this powerful technique.

Solvent signal suppression for high-resolution MAS-DNP

posted Mar 30, 2017, 3:05 AM by Daniel Lee

Solvent signal suppression for high-resolution MAS-DNP. Dynamic nuclear polarization (DNP) has become a powerful tool to substantially increase the sensitivity of high-field magic angle spinning (MAS) solid-state NMR experiments. The addition of dissolved hyperpolarizing agents usually results in the presence of solvent signals that can overlap and obscure those of interest from the analyte. Here, two methods are proposed to suppress DNP solvent signals: a Forced Echo Dephasing experiment (FEDex) and TRAnsfer of Populations in DOuble Resonance Echo Dephasing (TRAPDORED) NMR. These methods reintroduce a heteronuclear dipolar interaction that is specific to the solvent, thereby forcing a dephasing of recoupled solvent spins and leaving acquired NMR spectra free of associated resonance overlap with the analyte. The potency of these methods is demonstrated on sample types common to MAS-DNP experiments, namely a frozen solution (of L-proline) and a powdered solid (progesterone), both containing deuterated glycerol as a DNP solvent. The proposed methods are efficient, simple to implement, compatible with other NMR experiments, and extendable past spectral editing for just DNP solvents. The sensitivity gains from MAS-DNP in conjunction with FEDex or TRAPDORED then permits rapid and uninterrupted sample analysis.

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