Complete resonance assignment of a pharmaceutical drug at natural isotopic abundance from DNP-Enhanced solid-state NMR.

Solid-state dynamic nuclear polarization enhanced magic angle spinning (DNP-MAS) NMR measurements coupled with density functional theory (DFT) calculations enable the full resonance assignment of a complex pharmaceutical drug molecule without the need for isotopic enrichment. DNP dramatically enhances the NMR signals, thereby making possible previously intractable two-dimensional correlation NMR spectra at natural abundance. Using inputs from DFT calculations, herein we describe a significant improvement to the structure elucidation process for complex organic molecules. Further, we demonstrate that a series of two-dimensional correlation experiments, including 15N-13C TEDOR, 13C-13C INADEQUATE/SARCOSY, 19F-13C HETCOR, and 1H-13C HETCOR, can be obtained at natural isotopic abundance within reasonable experiment times, thus enabling a complete resonance assignment of sitagliptin, a pharmaceutical used for the treatment of type 2 diabetes.

19F Dynamic Nuclear Polarization at Fast Magic Angle Spinning for NMR of HIV-1 Capsid Protein Assemblies.

We report remarkably high, up to 100-fold, signal enhancements in 19F dynamic nuclear polarization (DNP) magic angle spinning (MAS) spectra at 14.1 T on HIV-1 capsid protein (CA) assemblies. These enhancements correspond to absolute sensitivity ratios of 12-29 and are of similar magnitude to those seen for 1H signals in the same samples. At MAS frequencies above 20 kHz, it was possible to record 2D 19F-13C HETCOR spectra, which contain long-range intra- and intermolecular correlations. Such correlations provide unique distance restraints, inaccessible in conventional experiments without DNP, for protein structure determination. Furthermore, systematic quantification of the DNP enhancements as a function of biradical concentration, MAS frequency, temperature, and microwave power is reported. Our work establishes the power of DNP-enhanced 19F MAS NMR spectroscopy for structural characterization of HIV-1 CA assemblies, and this approach is anticipated to be applicable to a wide range of large biomolecular systems.

Efficient 263 GHz magic angle spinning DNP at 100 K using solid-state diode sources.

The development of new, high-frequency solid-state diode sources capable of operating at 263 GHz, together with an optimized stator design for improved millimeter-wave coupling to the NMR sample, have enabled low-power DNP experiments at 263 GHz/400 MHz. With 250 mW output power, signal enhancements as high as 120 are achieved on standard samples - approximately 1/3 of the maximal enhancement available with high-power gyrotrons under similar conditions. Diode-based sources have a number of advantages over vacuum tube devices: they emit a pure mode, can be rapidly frequency-swept over a wide range of frequencies, have reproducible output power over this range, and have excellent output stability. By virtue of their small size, low thermal footprint, and lack of facility requirements, solid-state diodes are also considerably cheaper to operate and maintain than high-power vacuum tube devices. In light of these features, and anticipating further improvements in terms of available output power, solid-state diodes are likely to find widespread use in DNP and contribute to further advances in the field.

A biradical-tagged phospholipid as a polarizing agent for solid-state MAS Dynamic Nuclear Polarization NMR of membrane proteins.

A novel Dynamic Nuclear Polarization (DNP) NMR polarizing agent ToSMTSL-PTE representing a phospholipid with a biradical TOTAPOL tethered to the polar head group has been synthesized, characterized, and employed to enhance solid-state Nuclear Magnetic Resonance (SSNMR) signal of a lipid-reconstituted integral membrane protein proteorhodopsin (PR). A matrix-free PR formulation for DNP improved the absolute sensitivity of NMR signal by a factor of ca. 4 compared to a conventional preparation with TOTAPOL dispersed in a glassy glycerol/water matrix. DNP enhancements measured at 400 MHz/263 GHz and 600 MHz/395 GHz showed a strong field dependence but remained moderate at both fields, and comparable to those obtained for PR covalently modified with ToSMTSL. Additional continuous wave (CW) X-band electron paramagnetic resonance (EPR) experiments with ToSMTSL-PTE in solutions and in lipid bilayers revealed that an unfavorable conformational change of the linker connecting mononitroxides could be one of the reasons for moderate DNP enhancements. Further, differential scanning calorimetry (DSC) and CW EPR experiments indicated an inhomogeneous distribution and/or a possibility of a partial aggregation of ToSMTSL-PTE in DMPC:DMPA bilayers when the concentration of the polarizing agent was increased to 20 mol% to maximize the DNP enhancement. Thus, conformational changes and an inhomogeneous distribution of the lipid-based biradicals in lipid bilayers emerged as important factors to consider for further development of this matrix-free approach for DNP of membrane proteins.

Atomic Resolution Structure of Monomorphic Aß42 Amyloid Fibrils.

Amyloid-ß (Aß) is a 39-42 residue protein produced by the cleavage of the amyloid precursor protein (APP), which subsequently aggregates to form cross-ß amyloid fibrils that are a hallmark of Alzheimer's disease (AD). The most prominent forms of Aß are Aß1-40 and Aß1-42, which differ by two amino acids (I and A) at the C-terminus. However, Aß42 is more neurotoxic and essential to the etiology of AD. Here, we present an atomic resolution structure of a monomorphic form of AßM01-42 amyloid fibrils derived from over 500 (13)C-(13)C, (13)C-(15)N distance and backbone angle structural constraints obtained from high field magic angle spinning NMR spectra. The structure (PDB ID: 5KK3 ) shows that the fibril core consists of a dimer of Aß42 molecules, each containing four ß-strands in a S-shaped amyloid fold, and arranged in a manner that generates two hydrophobic cores that are capped at the end of the chain by a salt bridge. The outer surface of the monomers presents hydrophilic side chains to the solvent. The interface between the monomers of the dimer shows clear contacts between M35 of one molecule and L17 and Q15 of the second. Intermolecular (13)C-(15)N constraints demonstrate that the amyloid fibrils are parallel in register. The RMSD of the backbone structure (Q15-A42) is 0.71 ± 0.12 Å and of all heavy atoms is 1.07 ± 0.08 Å. The structure provides a point of departure for the design of drugs that bind to the fibril surface and therefore interfere with secondary nucleation and for other therapeutic approaches to mitigate Aß42 aggregation.

Tailoring of Polarizing Agents in the bTurea Series for Cross-Effect Dynamic Nuclear Polarization in Aqueous Media.

A series of 18 nitroxide biradicals derived from bTurea has been prepared, and their enhancement factors ɛ ((1)H) in cross-effect dynamic nuclear polarization (CE DNP) NMR experiments at 9.4 and 14.1 T and 100 K in a DNP-optimized glycerol/water matrix ("DNP juice") have been studied. We observe that ɛ ((1)H) is strongly correlated with the substituents on the polarizing agents, and its trend is discussed in terms of different molecular parameters: solubility, average e-e distance, relative orientation of the nitroxide moieties, and electron spin relaxation times. We show that too short an e-e distance or too long a T1e can dramatically limit ɛ ((1)H). Our study also shows that the molecular structure of AMUPol is not optimal and its ɛ ((1)H) could be further improved through stronger interaction with the glassy matrix and a better orientation of the TEMPO moieties. A new AMUPol derivative introduced here provides a better ɛ ((1)H) than AMUPol itself (by a factor of ca. 1.2).

Sensitivity-enhanced solid-state NMR detection of expansin's target in plant cell walls.

Structure determination of protein binding to noncrystalline macromolecular assemblies such as plant cell walls (CWs) poses a significant structural biology challenge. CWs are loosened during growth by expansin proteins, which weaken the noncovalent network formed by cellulose, hemicellulose, and pectins, but the CW target of expansins has remained elusive because of the minute amount of the protein required for activity and the complex nature of the CW. Using solid-state NMR spectroscopy, combined with sensitivity-enhancing dynamic nuclear polarization (DNP) and differential isotopic labeling of expansin and polysaccharides, we have now determined the functional binding target of expansin in the Arabidopsis thaliana CW. By transferring the electron polarization of a biradical dopant to the nuclei, DNP allowed selective detection of (13)C spin diffusion from trace concentrations of (13)C, (15)N-labeled expansin in the CW to nearby polysaccharides. From the spin diffusion data of wild-type and mutant expansins, we conclude that to loosen the CW, expansin binds highly specific cellulose domains enriched in xyloglucan, whereas more abundant binding to pectins is unrelated to activity. Molecular dynamics simulations indicate short (13)C-(13)C distances of 4-6 Å between a hydrophobic surface of the cellulose microfibril and an aromatic motif on the expansin surface, consistent with the observed NMR signals. DNP-enhanced 2D (13)C correlation spectra further reveal that the expansin-bound cellulose has altered conformation and is enriched in xyloglucan, thus providing unique insight into the mechanism of CW loosening. DNP-enhanced NMR provides a powerful, generalizable approach for investigating protein binding to complex macromolecular targets.

Brute-Force Hyperpolarization for NMR and MRI.

Hyperpolarization (HP) of nuclear spins is critical for ultrasensitive nuclear magnetic resonance (NMR) and magnetic resonance imaging (MRI). We demonstrate an approach for >1500-fold enhancement of key small-molecule metabolites: 1-(13)C-pyruvic acid, 1-(13)C-sodium lactate, and 1-(13)C-acetic acid. The (13)C solution NMR signal of pyruvic acid was enhanced 1600-fold at B = 1 T and 40 °C by pre-polarizing at 14 T and ∼2.3 K. This "brute-force" approach uses only field and temperature to generate HP. The noted 1 T observation field is appropriate for benchtop NMR and near the typical 1.5 T of MRI, whereas high-field observation scales enhancement as 1/B. Our brute-force process ejects the frozen, solid sample from the low-T, high-B polarizer, passing it through low field (B < 100 G) to facilitate "thermal mixing". That equilibrates (1)H and (13)C in hundreds of milliseconds, providing (13)C HP from (1)H Boltzmann polarization attained at high B/T. The ejected sample arrives at a room-temperature, permanent magnet array, where rapid dissolution with 40 °C water yields HP solute. Transfer to a 1 T NMR system yields (13)C signals with enhancements at 80% of ideal for noted polarizing conditions. High-resolution NMR of the same product at 9.4 T had consistent enhancement plus resolution of (13)C shifts and J-couplings for pyruvic acid and its hydrate. Comparable HP was achieved with frozen aqueous lactate, plus notable enhancement of acetic acid, demonstrating broader applicability for small-molecule NMR and metabolic MRI. Brute-force avoids co-solvated free-radicals and microwaves that are essential to competing methods. Here, unadulterated samples obviate concerns about downstream purity and also exhibit slow solid-state spin relaxation, favorable for transporting HP samples.

Dynamic nuclear polarization of membrane proteins: covalently bound spin-labels at protein-protein interfaces.

We demonstrate that dynamic nuclear polarization of membrane proteins in lipid bilayers may be achieved using a novel polarizing agent: pairs of spin labels covalently bound to a protein of interest interacting at an intermolecular interaction surface. For gramicidin A, nitroxide tags attached to the N-terminal intermolecular interface region become proximal only when bimolecular channels forms in the membrane. We obtained signal enhancements of sixfold for the dimeric protein. The enhancement effect was comparable to that of a doubly tagged sample of gramicidin C, with intramolecular spin pairs. This approach could be a powerful and selective means for signal enhancement in membrane proteins, and for recognizing intermolecular interfaces.

Efficient Dynamic Nuclear Polarization at 800 MHz/527 GHz with Trityl-Nitroxide Biradicals.

Cross-effect (CE) dynamic nuclear polarization (DNP) is a rapidly developing technique that enhances the signal intensities in magic-angle spinning (MAS) NMR spectra. We report CE DNP experiments at 211, 600, and 800 MHz using a new series of biradical polarizing agents referred to as TEMTriPols, in which a nitroxide (TEMPO) and a trityl radical are chemically tethered. The TEMTriPol molecule with the optimal performance yields a record (1) H NMR signal enhancement of 65 at 800 MHz at a concentration of 10 mM in a glycerol/water solvent matrix. The CE DNP enhancement for the TEMTriPol biradicals does not decrease as the magnetic field is increased in the manner usually observed for bis-nitroxides. Instead, the relatively strong exchange interaction between the trityl and nitroxide moieties determines the magnetic field at which the optimum enhancement is observed.

NMR-based structural biology enhanced by dynamic nuclear polarization at high magnetic field.

Dynamic nuclear polarization (DNP) has become a powerful method to enhance spectroscopic sensitivity in the context of magnetic resonance imaging and nuclear magnetic resonance spectroscopy. We show that, compared to DNP at lower field (400 MHz/263 GHz), high field DNP (800 MHz/527 GHz) can significantly enhance spectral resolution and allows exploitation of the paramagnetic relaxation properties of DNP polarizing agents as direct structural probes under magic angle spinning conditions. Applied to a membrane-embedded K(+) channel, this approach allowed us to refine the membrane-embedded channel structure and revealed conformational substates that are present during two different stages of the channel gating cycle. High-field DNP thus offers atomic insight into the role of molecular plasticity during the course of biomolecular function in a complex cellular environment.

Selective host-guest interaction between metal ions and metal-organic frameworks using dynamic nuclear polarization enhanced solid-state NMR spectroscopy.

The host-guest interaction between metal ions (Pt(2+) and Cu(2+) ) and a zirconium metal-organic framework (UiO-66-NH2 ) was explored using dynamic nuclear polarization-enhanced (15) N{(1) H} CPMAS NMR spectroscopy supported by X-ray absorption spectroscopy and density functional calculations. The combined experimental results conclude that each Pt(2+) coordinates with two NH2 groups from the MOF and two Cl(-) from the metal precursor, whereas Cu(2+) do not form chemical bonds with the NH2 groups of the MOF framework. Density functional calculations reveal that Pt(2+) prefers a square-planar structure with the four ligands and resides in the octahedral cage of the MOF in either cis or trans configurations.

Dynamic nuclear polarization study of inhibitor binding to the M2(18-60) proton transporter from influenza A.

We demonstrate the use of dynamic nuclear polarization (DNP) to elucidate ligand binding to a membrane protein using dipolar recoupling magic angle spinning (MAS) NMR. In particular, we detect drug binding in the proton transporter M2(18-60) from influenza A using recoupling experiments at room temperature and with cryogenic DNP. The results indicate that the pore binding site of rimantadine is correlated with previously reported widespread chemical shift changes, suggesting functional binding in the pore. Futhermore, the (15)N-labeled ammonium of rimantadine was observed near A30 (13)Cß and G34 (13)Cα, suggesting a possible hydrogen bond to A30 carbonyl. Cryogenic DNP was required to observe the weaker external binding site(s) in a ZF-TEDOR spectrum. This approach is generally applicable, particularly for weakly bound ligands, in which case the application of MAS NMR dipolar recoupling requires the low temperatures to quench dynamic exchange processes. For the fully protonated samples investigated, we observed DNP signal enhancements of ~10 at 400 MHz using only 4-6 mM of the polarizing agent TOTAPOL. At 600 MHz and with DNP, we measured a distance between the drug and the protein to a precision of 0.2 Å.

Dynamic nuclear polarization enhanced natural abundance 17O spectroscopy.

We show that natural abundance oxygen-17 NMR of solids could be obtained in minutes at a moderate magnetic field strength by using dynamic nuclear polarization (DNP). Electron spin polarization could be transferred either directly to (17)O spins or indirectly via (1)H spins in inorganic oxides and hydroxides using an oxygen-free solution containing a biradical polarization agent (bTbK). The results open up a powerful method for rapidly acquiring high signal-to-noise ratio solid-state NMR spectra of (17)O nuclear spins and to probe sites on or near the surface, without the need for isotope labeling.

Higher order amyloid fibril structure by MAS NMR and DNP spectroscopy.

Protein magic angle spinning (MAS) NMR spectroscopy has generated structural models of several amyloid fibril systems, thus providing valuable information regarding the forces and interactions that confer the extraordinary stability of the amyloid architecture. Despite these advances, however, obtaining atomic resolution information describing the higher levels of structural organization within the fibrils remains a significant challenge. Here, we detail MAS NMR experiments and sample labeling schemes designed specifically to probe such higher order amyloid structure, and we have applied them to the fibrils formed by an eleven-residue segment of the amyloidogenic protein transthyretin (TTR(105-115)). These experiments have allowed us to define unambiguously not only the arrangement of the peptide ß-strands into ß-sheets but also the ß-sheet interfaces within each protofilament, and in addition to identify the nature of the protofilament-to-protofilament contacts that lead to the formation of the complete fibril. Our efforts have resulted in 111 quantitative distance and torsion angle restraints (10 per residue) that describe the various levels of structure organization. The experiments benefited extensively from the use of dynamic nuclear polarization (DNP), which in some cases allowed us to shorten the data acquisition time from days to hours and to improve significantly the signal-to-noise ratios of the spectra. The ß-sheet interface and protofilament interactions identified here revealed local variations in the structure that result in multiple peaks for the exposed N- and C-termini of the peptide and in inhomogeneous line-broadening for the residues buried within the interior of the fibrils.

A slowly relaxing rigid biradical for efficient dynamic nuclear polarization surface-enhanced NMR spectroscopy: expeditious characterization of functional group manipulation in hybrid materials.

A new nitroxide-based biradical having a long electron spin-lattice relaxation time (T(1e)) has been developed as an exogenous polarization source for DNP solid-state NMR experiments. The performance of this new biradical is demonstrated on hybrid silica-based mesostructured materials impregnated with 1,1,2,2-tetrachloroethane radical containing solutions, as well as in frozen bulk solutions, yielding DNP enhancement factors (ε) of over 100 at a magnetic field of 9.4 T and sample temperatures of ~100 K. The effects of radical concentration on the DNP enhancement factors and on the overall sensitivity enhancements (Σ(†)) are reported. The relatively high DNP efficiency of the biradical is attributed to an increased T(1e), which enables more effective saturation of the electron resonance. This new biradical is shown to outperform the polarizing agents used so far in DNP surface-enhanced NMR spectroscopy of materials, yielding a 113-fold increase in overall sensitivity for silicon-29 CPMAS spectra as compared to conventional NMR experiments at room temperature. This results in a reduction in experimental times by a factor >12,700, making the acquisition of (13)C and (15)N one- and two-dimensional NMR spectra at natural isotopic abundance rapid (hours). It has been used here to monitor a series of chemical reactions carried out on the surface functionalities of a hybrid organic-silica material.

Intermolecular structure determination of amyloid fibrils with magic-angle spinning and dynamic nuclear polarization NMR.

We describe magic-angle spinning NMR experiments designed to elucidate the interstrand architecture of amyloid fibrils. Three methods are introduced for this purpose, two being based on the analysis of long-range (13)C-(13)C correlation spectra and the third based on the identification of intermolecular interactions in (13)C-(15)N spectra. We show, in studies of fibrils formed by the 86-residue SH3 domain of PI3 kinase (PI3-SH3 or PI3K-SH3), that efficient (13)C-(13)C correlation spectra display a resonance degeneracy that establishes a parallel, in-register alignment of the proteins in the amyloid fibrils. In addition, this degeneracy can be circumvented to yield direct intermolecular constraints. The (13)C-(13)C experiments are corroborated by (15)N-(13)C correlation spectra obtained from a mixed [(15)N,(12)C]/[(14)N,(13)C] sample which directly quantify interstrand distances. Furthermore, when the spectra are recorded with signal enhancement provided by dynamic nuclear polarization (DNP) at 100 K, we demonstrate a dramatic increase (from 23 to 52) in the number of intermolecular (15)N-(13)C constraints detectable in the spectra. The increase in the information content is due to the enhanced signal intensities and to the fact that dynamic processes, leading to spectral intensity losses, are quenched at low temperatures. Thus, acquisition of low temperature spectra addresses a problem that is frequently encountered in MAS spectra of proteins. In total, the experiments provide 111 intermolecular (13)C-(13)C and (15)N-(13)C constraints that establish that the PI3-SH3 protein strands are aligned in a parallel, in-register arrangement within the amyloid fibril.

Solid-state NMR spectroscopy of oriented membrane polypeptides at 100 K with signal enhancement by dynamic nuclear polarization.

Oriented membrane samples encompassing the biradical bTbK and a transmembrane peptide carrying a single (15)N labeled residue have been prepared on polymer sheets with sample geometries that fit into a 3.2 mm MAS rotor. The proton-decoupled (15)N cross-polarization spectra of the peptide were characterized by a single line at fast magic angle spinning speeds of approximately 8 kHz. Irradiating these samples with mu-waves resulted in Dynamic Nuclear Polarization and a concomitant 18-fold signal enhancement which considerably shortened the NMR acquisition times. Furthermore, the sideband patterns of magic angle oriented sample spinning (MAOSS) solid-state NMR spectra at slow spinning speeds (approximately 1 kHz) are indicative that the lipids and peptides form well-oriented bilayers at 100 K despite the narrow inner diameter of the rotor (2.2 mm) and the presence of considerable amounts of biradicals. The DNP signal enhancement opens up enhanced possibilities for multidimensional solid-state NMR investigation of oriented membrane polypeptides.

Dynamic nuclear polarization-enhanced solid-state NMR spectroscopy of GNNQQNY nanocrystals and amyloid fibrils.

Dynamic nuclear polarization (DNP) utilizes the inherently larger polarization of electrons to enhance the sensitivity of conventional solid-state NMR experiments at low temperature. Recent advances in instrumentation development and sample preparation have transformed this field and have opened up new opportunities for its application to biological systems. Here, we present DNP-enhanced (13)C-(13)C and (15)N-(13)C correlation experiments on GNNQQNY nanocrystals and amyloid fibrils acquired at 9.4 T and 100 K and demonstrate that DNP can be used to obtain assignments and site-specific structural information very efficiently. We investigate the influence of temperature on the resolution, molecular conformation, structural integrity and dynamics in these two systems. In addition, we assess the low-temperature performance of two commonly used solid-state NMR experiments, proton-driven spin diffusion (PDSD) and transferred echo double resonance (TEDOR), and discuss their potential as tools for measurement of structurally relevant distances at low temperature in combination with DNP.

Solid-state dynamic nuclear polarization at 263 GHz: spectrometer design and experimental results.

Dynamic Nuclear Polarization (DNP) experiments transfer polarization from electron spins to nuclear spins with microwave irradiation of the electron spins for enhanced sensitivity in nuclear magnetic resonance (NMR) spectroscopy. Design and testing of a spectrometer for magic angle spinning (MAS) DNP experiments at 263 GHz microwave frequency, 400 MHz (1)H frequency is described. Microwaves are generated by a novel continuous-wave gyrotron, transmitted to the NMR probe via a transmission line, and irradiated on a 3.2 mm rotor for MAS DNP experiments. DNP signal enhancements of up to 80 have been measured at 95 K on urea and proline in water-glycerol with the biradical polarizing agent TOTAPOL. We characterize the experimental parameters affecting the DNP efficiency: the magnetic field dependence, temperature dependence and polarization build-up times, microwave power dependence, sample heating effects, and spinning frequency dependence of the DNP signal enhancement. Stable system operation, including DNP performance, is also demonstrated over a 36 h period.