Background signal suppression by opposite polarity subtraction for targeted DNP NMR spectroscopy on mixture samples.

A novel method for background signal suppression is introduced to improve the selectivity of dynamic nuclear polarization (DNP) NMR spectroscopy in the study of target molecules within complex mixtures. The method uses subtraction between positively and negatively enhanced DNP spectra, leading to an improved contrast factor, which is the ratio between the target and background signal intensities. The proposed approach was experimentally validated using a reverse-micelle system that confines the target molecules together with the polarizing agent, OX063 trityl. A substantial increase in the contrast factor was observed, and the contrast factor was optimized through careful selection of the DNP build-up time. A simulation study based on the experimental results provides insights into a strategy for choosing the appropriate DNP build-up time and the corresponding selectivity of the method. Further analysis revealed a broad applicability of the technique, encompassing studies from large biomolecules to surface-modified polymers, depending on the nuclear spin diffusion rate with a range of gyromagnetic ratios.

Optimal-Control-Based Cß Chemical Shift Encoding for Efficient Signal Assignment of Solid Proteins.

Fast magic-angle spinning (MAS) solid-state NMR spectroscopy is a powerful tool for gaining structural and dynamic information on solid proteins. To access such information site-specifically, the signal assignment process is unavoidable. In the assignment process, Cα and Cß chemical shifts are of paramount importance in identifying the type of amino acid residues. Conventionally, however, recording the Cß chemical shift of solid proteins with relatively short transverse relaxation time is often limited by the long delay required for the magnetization transfer to Cß spins and its evolution, that is, by the sensitivity drop. In this article, we propose a new method that encodes the Cß chemical shifts onto the intensities of the scalar-coupled Cα signals by combining an optimal control-based spin manipulation pulse and a spin-state filter. This reduces the total required transverse evolution to less than half of that for the previously proposed method, opening up the concept of the Cß-encoding nearest-neighbor NMR, for the first time, to solid proteins. Also, the total measurement time was shorter than that required for the explicit Cß shift evolution. We demonstrate the sequential signal assignment for microcrystalline protein GB1, and then discuss the prospects for more challenging proteins.

Mechanistic origins of methyl-driven Overhauser DNP.

The Overhauser effect in the dynamic nuclear polarization (DNP) of non-conducting solids has drawn much attention due to the potential for efficient high-field DNP as well as a general interest in the underlying principles that enable the Overhauser effect in small molecules. We recently reported the observation of 1H and 2H Overhauser effects in H3C- or D3C-functionalized Blatter radical analogs, which we presumed to be caused by methyl rotation. In this work, we look at the mechanism for methyl-driven Overhauser DNP in greater detail, considering methyl librations and tunneling in addition to classical rotation. We predict the temperature dependence of these mechanisms using density functional theory and spin dynamics simulations. Comparisons with results from ultralow-temperature magic angle spinning-DNP experiments revealed that cross-relaxation at temperatures above 60 K originates from both libration and rotation, while librations dominate at lower temperatures. Due to the zero-point vibrational nature of these motions, they are not quenched by very low temperatures, and methyl-driven Overhauser DNP is expected to increase in efficiency down to 0 K, predominantly due to increases in nuclear relaxation times.

Evolution of α-synuclein conformation ensemble toward amyloid fibril via liquid-liquid phase separation (LLPS) as investigated by dynamic nuclear polarization-enhanced solid-state MAS NMR.

Protein fibrillation and human neurodegenerative diseases, with a profound underlying connection suggested between them, have been the subject of intense investigations in the medical, biophysical and bio-engineering sciences. For gaining the molecular mechanistic insights into such connection, i.e., the cause and effect, atomic-resolution molecular structure information especially on the initial oligomeric states is of paramount importance, not only that on the mature amyloid fibrils. α-Synuclein (αSyn) and its amyloid fibril has a direct relevance to the Parkinson's disease and other synucleinopathies, but what triggers the fibrillation is still not entirely clear. We here describe the liquid-liquid phase separation (LLPS) of αSyn and investigate its conformational evolution from its monomeric state into oligomer state within the early-stage of the phase-separated droplets, mainly using solution and magic-angle spinning (MAS) solid-state nuclear magnetic resonance (NMR) spectroscopies, aided with optical and fluorescent microscopies and CD spectroscopy. Based on the analysis of the intricately broadened shapes of the MAS NMR peaks observed for isotopically 13C-labeled His-50 of αSyn, we show that the distribution of the αSyn conformation is skewed from the initial completely random state to a loose ß-rich ensembles at/around His-50 as early as day-3 (d3) within the droplet. This intra-droplet loose ß-rich assembly showed a very slow progression until d8, and eventually maturated into ThT-positive, long and unbranched amyloid fibrils after 8 weeks. The obtained information on the evolution of the distribution of the conformation ensemble is unique, and difficult to obtain with X-ray crystallography and cryo-electron microscopy (cryoEM). In particular, the sensitivity-enhanced MAS NMR based on the low-temperature dynamic nuclear polarization (DNP) technique was proven to be a key tool in characterizing the conformational ensemble with dilute protein samples such as the liquid-phase droplets.

Cryogenic signal amplification combined with helium-temperature MAS DNP toward ultimate NMR sensitivity at high field conditions.

The low sensitivity of NMR spectroscopy is of historical concern in the field, and various approaches have been developed to mitigate this limitation. On the shoulder of giants, today one can routinely implement, for example, the pulse/Fourier transform NMR with the cross polarization together with the ultra-low temperature MAS DNP under high-field conditions. We show in this work this current opportunity should further be augmented by combining them with the cryogenic signal amplification. Our presented MAS DNP probe operates with the closed-cycle helium MAS system, and cools the internal preamplifier-duplexer module with the "return" helium gas on its way back to the compressor in the loop. The signal-to-noise (S/N) gain relative to the room-temperature measurements of a factor of 4.6 and 2.4 was found for the measurement using the cold- and room-temperature preamplifier, respectively, at the sample temperature of T = 20 K at B0 = 16.4 T. The ratio of these factors reveals âˆ¼ two-fold sensitivity improvement that results purely from the introduction of the cold signal amplification, i.e., noise reduction. Together with the increase of the thermal Boltzmann polarization at low temperatures, the combined S/N gain of max. ∼70-fold is possible without DNP. The DNP enhancement factor of ∼40 as we found in this work for a microcrystalline MLF sample may be multiplied to this gain. We also demonstrated the sensitivity improvement with a 13C-detected 2D NCaCx spectrum, illustrating the generality of the S/N gain from combining DNP with the cold signal amplification.

Efficiency analysis of helium-cooled MAS DNP: case studies of surface-modified nanoparticles and homogeneous small-molecule solutions.

Despite the growing number of successful applications of dynamic nuclear polarization (DNP)-enhanced magic-angle spinning (MAS) NMR in structural biology and materials science, the nuclear polarizations achieved by current MAS DNP instrumentation are still considerably lower than the theoretical maximum. The method could be significantly strengthened if experiments were performed at temperatures much lower than those currently widely used (∼100 K). Recently, the prospects of helium (He)-cooled MAS DNP have been increased with the instrumental developments in MAS technology that uses cold helium gas for sample cooling. Despite the additional gains in sensitivity that have been observed with He-cooled MAS DNP, the performance of the technique has not been evaluated in the case of surfaces and interfaces that benefit the most from DNP. Herein, we studied the efficiency of DNP at temperatures between ∼30 K and ∼100 K for organically functionalized silica material and a homogeneous solution of small organic molecules at a magnetic field B0 = 16.4 T. We recorded the changes in signal enhancement, paramagnet-induced quenching and depolarization effects, DNP build-up rate, and Boltzmann polarization. For these samples, the increases in MAS-induced depolarization and DNP build-up times at around 30 K were not as severe as anticipated. In the case of the surface species, we determined that MAS DNP at 30 K provided ∼10 times higher sensitivity than MAS DNP at 90 K, which corresponds to the acceleration of experiments by multiplicative factors of up to 100.

Structural analysis of cross-linked poly(vinyl alcohol) using high-field DNP-NMR.

Poly(vinyl alcohol) (PVOH) is a water-soluble synthetic polymer, widely used in materials for functional films and moldings, fiber fabric sizing agents, paper coating resins, and adhesives. PVOH is mainly applied in the form of an aqueous solution, yet after its application, insolubility (water resistance) is required. To achieve this, additives are introduced. These additives used with PVOH are cross-linking agents which react with the hydroxyl groups and modified functional groups in PVOH. Because of the poor reactivity of unmodified PVOH, it does not react with cross-linking agents that have functional reactive groups. Therefore, modified PVOH that reacts with a cross-linking agent more successfully is required. These chemical bonding sites are so low in abundance that it is difficult to characterize the cross-linking structure. Solid-state 13C NMR is a powerful technique that can be used for the structural analysis of a polymer material. However, its sensitivity is low, hence it is difficult to determine crosslinking in a polymer, as it makes up only a small proportion of the product. Therefore, solid-state 13C NMR sensitivity can be enhanced by high-field dynamic nuclear polarization (DNP) using strong electron polarization. In this study, the reaction of acetoacetylated PVOH with a cross-linking agent, adipic dihydrazide, was analyzed. This crosslinked PVOH is the most popular vinyl alcohol polymer on the commercial market. The sensitivity enhanced 13C NMR spectra reveal that the carbonyl of the acetoacetyl group of PVOH crosslinks with adipic hydrazide by forming an imine bond (>C[double bond, length as m-dash]N-) this study also shows that the product has only seven crosslinking sites per molecular chain with a polymerization degree of 1000 and is water resistant.

Absolute 1H polarization measurement with a spin-correlated component of magnetization by hyperpolarized MAS-DNP solid-state NMR.

Sensitivity of magic-angle spinning (MAS) NMR spectroscopy has been dramatically improved by the advent of high-field dynamic nuclear polarization (DNP) technique and its rapid advances over the past decades. In this course, discussions on ways to improve the DNP enhancement factor or the overall sensitivity gain have been numerous, and led to a number of methodological and instrumental breakthroughs. Beyond the sensitivity gain, however, discussions on accurate quantification of the 1H polarization amplitude achievable in a sample with DNP have been relatively rare. Here, we propose a new method for quantifying the local 1H hyperpolarization amplitude, which is applicable to un-oriented/powdered solid samples under MAS NMR conditions. The method is based on the ability to observe the high-order spin-correlated term (2IzSz) intrinsic to a hyperpolarized IS two-spin state, separately from the lowest-order Zeeman term (Sz) in quasi-equilibrium magnetization. The quantification procedure does not require evaluation of signal amplitudes for a "microwave-off" condition and for an un-doped reference sample, and thus enables quick and accurate quantification unaffected by the effects of the paramagnetic quenching and the MAS-induced depolarization. The method is also shown to elucidate spatial polarization distribution through the 2IzSz term prepared domain-selectively. As a potential application, we also demonstrate 2D DQ-SQ spectroscopy utilizing the 2IzSz term that is generated in a spatially selective manner without using IS dipolar or J coupling. These salient features may be evolved into a way for characterizing mesoscopic molecular assemblies of medical/biological importance.

Advanced instrumentation for DNP-enhanced MAS NMR for higher magnetic fields and lower temperatures.

Sensitivity enhancement of MAS NMR using dynamic nuclear polarization (DNP) is gaining importance at moderate fields (B0<9T) and temperatures (T>90K) with potential applications in chemistry and material sciences. However, considering the ever-increasing size and complexity of the systems to be studied, it is crucial to establish DNP under higher field conditions, where the spectral resolution and the basic NMR sensitivity tend to improve. In this perspective, we overview our recent efforts on hardware developments, specifically targeted on improving DNP MAS NMR at high fields. It includes the development of gyrotrons that enable continuous frequency tuning and rapid frequency modulation for our 395 GHz-600 MHz and 460 GHz-700 MHz DNP NMR spectrometers. The latter 700 MHz system involves two gyrotrons and a quasi-optical transmission system that combines two independent sub-millimeter waves into a single dichromic wave. We also describe two cryogenic MAS NMR probe systems operating, respectively, at T ∼ 100K and ∼ 30K. The latter system utilizes a novel closed-loop helium recirculation mechanism, achieving cryogenic MAS without consuming any cryogen. These instruments altogether should promote high-field DNP toward more efficient, reliable and affordable technology. Some experimental DNP results obtained with these instruments are presented.

Closed-cycle cold helium magic-angle spinning for sensitivity-enhanced multi-dimensional solid-state NMR.

Magic-angle spinning (MAS) NMR is a powerful tool for studying molecular structure and dynamics, but suffers from its low sensitivity. Here, we developed a novel helium-cooling MAS NMR probe system adopting a closed-loop gas recirculation mechanism. In addition to the sensitivity gain due to low temperature, the present system has enabled highly stable MAS (vR=4-12 kHz) at cryogenic temperatures (T=35-120 K) for over a week without consuming helium at a cost for electricity of 16 kW/h. High-resolution 1D and 2D data were recorded for a crystalline tri-peptide sample at T=40 K and B0=16.4 T, where an order of magnitude of sensitivity gain was demonstrated versus room temperature measurement. The low-cost and long-term stable MAS strongly promotes broader application of the brute-force sensitivity-enhanced multi-dimensional MAS NMR, as well as dynamic nuclear polarization (DNP)-enhanced NMR in a temperature range lower than 100 K.

Helium-cooling and -spinning dynamic nuclear polarization for sensitivity-enhanced solid-state NMR at 14 T and 30 K.

We describe a (1)H polarization enhancement via dynamic nuclear polarization (DNP) at very low sample temperature T≈30 K under magic-angle spinning (MAS) conditions for sensitivity-enhanced solid-state NMR measurement. Experiments were conducted at a high external field strength of 14.1 T. For MAS DNP experiments at T<<90 K, a new probe system using cold helium gas for both sample-cooling and -spinning was developed. The novel system can sustain a low sample temperature between 30 and 90K for a period of time >10 h under MAS at ν(R)≈3 kHz with liquid He consumption of ≈6 L/h. As a microwave source, we employed a high-power, continuously frequency-tunable gyrotron. At T≈34 K, (1)H DNP enhancement factors of 47 and 23 were observed with and without MAS, respectively. On the basis of these observations, a discussion on the total NMR sensitivity that takes into account the effect of sample temperature and external field strength used in DNP experiments is presented. It was determined that the use of low sample temperature and high external field is generally rewarding for the total sensitivity, in spite of the slower polarization buildup at lower temperature and lower DNP efficiency at higher field. These findings highlight the potential of the current continuous-wave DNP technique also at very high field conditions suitable to analyze large and complex systems, such as biological macromolecules.

Boosting protein dynamics studies using quantitative nonuniform sampling NMR spectroscopy.

NMR spectroscopy is uniquely suited to study protein dynamics over a wide range of time scales at atomic resolution. However, existing NMR relaxation methods require highly serial, lengthy data collection, ultimately limiting their application to short-lived samples, such as proteins in living cells. In recent years, the utility of nonuniform sampling (NUS) NMR methodologies has been increasingly recognized, but their application has been rare in relaxation measurements where highly accurate spectral quantification is demanded. Recently, Matsuki et al. developed a new NUS-processing method, SIFT (Spectroscopy by Integration of Frequency and Time domain information), which is highly robust and faithful in reproducing signals. In this work, we demonstrate the gains that are possible with more aggressive use of frequency domain information than was employed previously. This improvement is crucial for SIFT to be used in accelerating relaxation measurements while preserving full analytical accuracy. By taking the KIX domain of mouse CREB-binding protein (CBP) as an example, we demonstrate that this quantitative NUS processing method enables total 10-fold expedition of the R(2) relaxation dispersion measurements. The advanced SIFT processing should be equally useful for other NMR relaxation measurements.

Molecular structure of humin and melanoidin via solid state NMR.

Sugar-derived humins and melanoidins figure significantly in food chemistry, agricultural chemistry, biochemistry, and prebiotic chemistry. Despite wide interest and significant experimental attention, the amorphous and insoluble nature of the polymers has made them resistant to conventional structural characterization. Here we make use of solid-state NMR methods, including selective (13)C substitution, (1)H-dephasing, and double quantum filtration. The spectra, and their interpretation, are simplified by relying exclusively on hydronium for catalysis. The results for polymers derived from ribose, deoxyribose, and fructose indicate diverse pathways to furans, suggest a simple route to pyrroles in the presence of amines, and reveal a heterogeneous network-type polymer in which sugar molecules cross-link the heterocycles.

Microwave field distribution in a magic angle spinning dynamic nuclear polarization NMR probe.

We present a calculation of the microwave field distribution in a magic angle spinning (MAS) probe utilized in dynamic nuclear polarization (DNP) experiments. The microwave magnetic field (B(1S)) profile was obtained from simulations performed with the High Frequency Structure Simulator (HFSS) software suite, using a model that includes the launching antenna, the outer Kel-F stator housing coated with Ag, the RF coil, and the 4mm diameter sapphire rotor containing the sample. The predicted average B(1S) field is 13µT/W(1/2), where S denotes the electron spin. For a routinely achievable input power of 5W the corresponding value is γ(S)B(1S)=0.84MHz. The calculations provide insights into the coupling of the microwave power to the sample, including reflections from the RF coil and diffraction of the power transmitted through the coil. The variation of enhancement with rotor wall thickness was also successfully simulated. A second, simplified calculation was performed using a single pass model based on Gaussian beam propagation and Fresnel diffraction. This model provided additional physical insight and was in good agreement with the full HFSS simulation. These calculations indicate approaches to increasing the coupling of the microwave power to the sample, including the use of a converging lens and fine adjustment of the spacing of the windings of the RF coil. The present results should prove useful in optimizing the coupling of microwave power to the sample in future DNP experiments. Finally, the results of the simulation were used to predict the cross effect DNP enhancement (ϵ) vs. ω(1S)/(2π) for a sample of (13)C-urea dissolved in a 60:40 glycerol/water mixture containing the polarizing agent TOTAPOL; very good agreement was obtained between theory and experiment.

Dynamic nuclear polarization experiments at 14.1 T for solid-state NMR.

Instrumentation for high-field dynamic nuclear polarization (DNP) at 14.1 T was developed to enhance the nuclear polarization for NMR of solids. The gyrotron generated 394.5 GHz submillimeter (sub-mm) wave with a power of 40 W in the second harmonic TE(0,6) mode. The sub-mm wave with a power of 0.5-3 W was transmitted to the sample in a low-temperature DNP-NMR probe with a smooth-wall circular waveguide system. The (1)H polarization enhancement factor of up to about 10 was observed for a (13)C-labeled compound with nitroxyl biradical TOTAPOL. The DNP enhancement was confirmed by the static magnetic field dependence of the NMR signal amplitude at 90 K. Improvements of the high-field DNP experiments are discussed.

Resolution and polarization distribution in cryogenic DNP/MAS experiments.

This contribution addresses four potential misconceptions associated with high-resolution dynamic nuclear polarization/magic angle spinning (DNP/MAS) experiments. First, spectral resolution is not generally compromised at the cryogenic temperatures at which DNP experiments are performed. As we demonstrate at a modest field of 9 T (380 MHz (1)H), 1 ppm linewidths are observed in DNP/MAS spectra of a membrane protein in its native lipid bilayer, and <0.4 ppm linewidths are reported in a crystalline peptide at 85 K. Second, we address the concerns about paramagnetic broadening in DNP/MAS spectra of proteins by demonstrating that the exogenous radical polarizing agents utilized for DNP are distributed in the sample in such a manner as to avoid paramagnetic broadening and thus maintain full spectral resolution. Third, the enhanced polarization is not localized around the polarizing agent, but rather is effectively and uniformly dispersed throughout the sample, even in the case of membrane proteins. Fourth, the distribution of polarization from the electron spins mediated via spin diffusion between (1)H-(1)H strongly dipolar coupled spins is so rapid that shorter magnetization recovery periods between signal averaging transients can be utilized in DNP/MAS experiments than in typical experiments performed at ambient temperature.

Dynamic nuclear polarization with a rigid biradical.

A new polarizing agent with superior performance in dynamic nuclear polarization experiments is introduced, and utilizes two TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl) moieties connected through a rigid spiro tether (see structure). The observed NMR signal intensities were enhanced by a factor of 1.4 compared to those of TOTAPOL, a previously described TEMPO-based biradical with a flexible tether.

Cryogenic sample exchange NMR probe for magic angle spinning dynamic nuclear polarization.

We describe a cryogenic sample exchange system that dramatically improves the efficiency of magic angle spinning (MAS) dynamic nuclear polarization (DNP) experiments by reducing the time required to change samples and by improving long-term instrument stability. Changing samples in conventional cryogenic MAS DNP/NMR experiments involves warming the probe to room temperature, detaching all cryogenic, RF, and microwave connections, removing the probe from the magnet, replacing the sample, and reversing all the previous steps, with the entire cycle requiring a few hours. The sample exchange system described here-which relies on an eject pipe attached to the front of the MAS stator and a vacuum jacketed dewar with a bellowed hole-circumvents these procedures. To demonstrate the excellent sensitivity, resolution, and stability achieved with this quadruple resonance sample exchange probe, we have performed high precision distance measurements on the active site of the membrane protein bacteriorhodopsin. We also include a spectrum of the tripeptide N-f-MLF-OH at 100K which shows 30 Hz linewidths.