Low-temperature Dynamic Nuclear Polarization Research Articles

Hydrogen bonding in duplex DNA probed by DNP enhanced solid-state NMR N-H bond length measurements.

Numerous biological processes and mechanisms depend on details of base pairing and hydrogen bonding in DNA. Hydrogen bonds are challenging to quantify by X-ray crystallography and cryo-EM due to difficulty of visualizing hydrogen atom locations but can be probed with site specificity by NMR spectroscopy in solution and the solid state with the latter particularly suited to large, slowly tumbling DNA complexes. Recently, we showed that low-temperature dynamic nuclear polarization (DNP) enhanced solid-state NMR is a valuable tool for distinguishing Hoogsteen base pairs (bps) from canonical Watson-Crick bps in various DNA systems under native-like conditions. Here, using a model 12-mer DNA duplex containing two central adenine-thymine (A-T) bps in either Watson-Crick or Hoogsteen confirmation, we demonstrate DNP solid-state NMR measurements of thymine N3-H3 bond lengths, which are sensitive to details of N-H···N hydrogen bonding and permit hydrogen bonds for the two bp conformers to be systematically compared within the same DNA sequence context. For this DNA duplex, effectively identical TN3-H3 bond lengths of 1.055 ± 0.011Å and 1.060 ± 0.011Å were found for Watson-Crick A-T and Hoogsteen A (syn)-T base pairs, respectively, relative to a reference amide bond length of 1.015 ± 0.010Å determined for N-acetyl-valine under comparable experimental conditions. Considering that prior quantum chemical calculations which account for zero-point motions predict a somewhat longer effective peptide N-H bond length of 1.041Å, in agreement with solution and solid-state NMR studies of peptides and proteins at ambient temperature, to facilitate direct comparisons with these earlier studies TN3-H3 bond lengths for the DNA samples can be readily scaled appropriately to yield 1.083Å and 1.087Å for Watson-Crick A-T and Hoogsteen A (syn)-T bps, respectively, relative to the 1.041Å reference peptide N-H bond length. Remarkably, in the context of the model DNA duplex, these results indicate that there are no significant differences in N-H···N A-T hydrogen bonds between Watson-Crick and Hoogsteen bp conformers. More generally, high precision measurements of N-H bond lengths by low-temperature DNP solid-state NMR based methods are expected to facilitate detailed comparative analysis of hydrogen bonding for a range of DNA complexes and base pairing environments.

Nitroxide-based triradical dopants for efficient low-temperature dynamic nuclear polarization in aqueous solutions over a broad pH range

Dynamic nuclear polarization (DNP) can provide substantial sensitivity enhancements in solid state nuclear magnetic resonance (ssNMR) measurements on frozen solutions, thereby enabling experiments that would otherwise be impractical. Previous work has shown that nitroxide-based triradical compounds are particularly effective as dopants in DNP-enhanced measurements at moderate magic-angle spinning frequencies and moderate magnetic field strengths, generally leading to a more rapid build-up of nuclear spin polarizations under microwave irradiation than the more common biradical dopants at the same electron spin concentrations. Here we report the synthesis and DNP performance at 25 K and 9.41 T for two new triradical compounds, sulfoacetyl-DOTOPA and PEG12-DOTOPA. Under our experimental conditions, these compounds exhibit ssNMR signal enhancements and DNP build-up times that are nearly identical to those of previously described triradical dopants. Moreover, these compounds have high solubility in aqueous buffers and water/glycerol mixtures at both acidic and basic pH values, making them useful in a wide variety of experiments on biomolecular systems.

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 β-rich ensembles at/around His-50 as early as day-3 (d3) within the droplet. This intra-droplet loose β-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.

Succinyl-DOTOPA: An effective triradical dopant for low-temperature dynamic nuclear polarization with high solubility in aqueous solvent mixtures at neutral pH

We report the synthesis of the nitroxide-based triradical compound succinyl-DOTOPA and the characterization of its performance as a dopant for dynamic nuclear polarization (DNP) experiments in frozen solutions at low temperatures. Compared with previously described DOTOPA derivatives, succinyl-DOTOPA has substantially greater solubility in glycerol/water mixtures with pH > 4 and therefore has wider applicability. Solid state nuclear magnetic resonance (ssNMR) measurements at 9.39 T and 25 K, with magic-angle spinning at 7.00 kHz, show that build-up times of DNP-enhanced, cross-polarized 13C ssNMR signals are shorter and that signal amplitudes are larger for glycerol/water solutions of L-proline containing succinyl-DOTOPA than for solutions containing the biradical AMUPol, with electron spin concentrations of 15 mM or 30 mM, resulting in greater net sensitivity gains from DNP. In similar measurements at 90 K, AMUPol yields greater net sensitivity, apparently due to its longer electron spin-lattice and spin-spin relaxation times. One- and two-dimensional 13C ssNMR measurements at 25 K on the complex of the 27-residue peptide M13 with the calcium-sensing protein calmodulin, in glycerol/water with 10 mM succinyl-DOTOPA, demonstrate the utility of this compound in DNP-enhanced ssNMR studies of biomolecular systems.

An efficient liquid helium / gas-gap switch allowing rapidly servicing low-temperature dynamic nuclear polarization systems

The authors present a novel design for servicing inserts for Hyperpolarising or similar systems operating at temperatures below 4 K. Sample space sleeves that accommodate variable temperature inserts are usually deeply integrated in the cryostat structure and are impossible to remove or to warm up in case of service needs. We present experimental results that show the performance of a liquid helium / gas-gap switch that will be used together with a sample space insert which is in direct contact to a cold plate. This sample space sleeve can be serviced by thermally disconnecting the switch from the cold plate. Furthermore, the additively manufactured heat switch features a novel cold plate interposer for heat transfer that allows the physical separation of the sample sleeve tube from the cold plate enabling a room temperature warm up or complete sleeve removal, if required.

Determining Cholesterol Binding to Membrane Proteins by Cholesterol 13C Labeling in Yeast and Dynamic Nuclear Polarization NMR.

We present a general strategy for determining the cholesterol-binding site of eukaryotic membrane proteins in native-like lipid membranes by NMR spectroscopy. The strategy combines yeast biosynthetic 13C enrichment of cholesterol with detection of protein-cholesterol 13C-13C cross peaks in 2D correlation NMR spectra under the dynamic nuclear polarization (DNP) condition. Low-temperature DNP not only allows high-sensitivity detection of weak protein-cholesterol cross peaks in 2D spectra but also immobilizes cholesterol and protein to enable intermolecular distance measurements. We demonstrate this approach on the influenza M2 protein, which utilizes cholesterol to conduct membrane scission in the last step of virus budding and release from the host cell plasma membrane. A 13C-13C double-quantum filter was employed to significantly simplify the 2D 13C-13C correlation spectra and facilitate the identification of protein-cholesterol cross peaks. A number of cross peaks between the M2 transmembrane residues' side chains and the cholesterol sterol group were detected, which complement recently measured protein contacts to the isooctyl tail of cholesterol to define an extended binding interface. These results provide atomic-level evidence of M2-cholesterol interaction to cause membrane curvature and scission, and the approach is generally applicable to other eukaryotic membrane proteins for understanding the influence of cholesterol on membrane protein function.

Chemisorption of Water on the Surface of Silicon Microparticles Measured by Dynamic Nuclear Polarization Enhanced NMR

We use dynamic nuclear polarization (DNP) enhanced nuclear magnetic resonance (NMR) at liquid helium temperatures to directly detect hydrogen attached to the surface of silicon microparticles. The proton NMR spectrum from a dry sample of polycrystalline silicon powder (1-5 $\mu$m) shows a distinctively narrow Lorentzian-shaped resonance with a width of 6.2 kHz, indicative of a very sparse distribution of protons attached to the silicon surface. These protons are within a few atomic monolayers of the silicon surface. The high sensitivity NMR detection of surface protons from low surface area ($0.26 - 1.3 \: m^2/g$) particles is enabled by an overall signal enhancement of 4150 over the room temperature NMR signal at the same field. When the particles were suspended in a solvent with 80% H2O and 20% D2O, the narrow peak was observed to grow in intensity over time, indicating growth of the sparse surface proton layer. However, when the particles were suspended in a solvent with 20% H2O and 80% D2O, the narrow bound-proton peak was observed to shrink due to exchange between the surface protons and the deuterium in solution. This decrease was accompanied by a concomitant growth in the intensity of the frozen solvent peak, as the relative proton concentration of the solvent increased. When the particles were suspended in the organic solvent hexane, the proton NMR spectra remained unchanged over time. These results are consistent with the known chemisorption of water on the silicon surface resulting in the formation of hydride and hydroxyl species. Low-temperature DNP NMR can thus be used as a non-destructive probe of surface corrosion for silicon in aqueous environments. This is important in the context of using silicon MEMS and bioMEMS devices in such environments, for silicon micro- and nano-particle MRI imaging agents, and the use of nanosilicon for splitting water in fuel cells.

Low-temperature dynamic nuclear polarization with helium-cooled samples and nitrogen-driven magic-angle spinning

We describe novel instrumentation for low-temperature solid state nuclear magnetic resonance (NMR) with dynamic nuclear polarization (DNP) and magic-angle spinning (MAS), focusing on aspects of this instrumentation that have not been described in detail in previous publications. We characterize the performance of an extended interaction oscillator (EIO) microwave source, operating near 264GHz with 1.5W output power, which we use in conjunction with a quasi-optical microwave polarizing system and a MAS NMR probe that employs liquid helium for sample cooling and nitrogen gas for sample spinning. Enhancement factors for cross-polarized 13C NMR signals in the 100–200 range are demonstrated with DNP at 25K. The dependences of signal amplitudes on sample temperature, as well as microwave power, polarization, and frequency, are presented. We show that sample temperatures below 30K can be achieved with helium consumption rates below 1.3l/h. To illustrate potential applications of this instrumentation in structural studies of biochemical systems, we compare results from low-temperature DNP experiments on a calmodulin-binding peptide in its free and bound states.

Efficient DNP NMR of membrane proteins: sample preparation protocols, sensitivity, and radical location.

Although dynamic nuclear polarization (DNP) has dramatically enhanced solid-state NMR spectral sensitivities of many synthetic materials and some biological macromolecules, recent studies of membrane-protein DNP using exogenously doped paramagnetic radicals as polarizing agents have reported varied and sometimes surprisingly limited enhancement factors. This motivated us to carry out a systematic evaluation of sample preparation protocols for optimizing the sensitivity of DNP NMR spectra of membrane-bound peptides and proteins at cryogenic temperatures of ~110 K. We show that mixing the radical with the membrane by direct titration instead of centrifugation gives a significant boost to DNP enhancement. We quantify the relative sensitivity enhancement between AMUPol and TOTAPOL, two commonly used radicals, and between deuterated and protonated lipid membranes. AMUPol shows ~fourfold higher sensitivity enhancement than TOTAPOL, while deuterated lipid membrane does not give net higher sensitivity for the membrane peptides than protonated membrane. Overall, a ~100 fold enhancement between the microwave-on and microwave-off spectra can be achieved on lipid-rich membranes containing conformationally disordered peptides, and absolute sensitivity gains of 105-160 can be obtained between low-temperature DNP spectra and high-temperature non-DNP spectra. We also measured the paramagnetic relaxation enhancement of lipid signals by TOTAPOL and AMUPol, to determine the depths of these two radicals in the lipid bilayer. Our data indicate a bimodal distribution of both radicals, a surface-bound fraction and a membrane-bound fraction where the nitroxides lie at ~10 Å from the membrane surface. TOTAPOL appears to have a higher membrane-embedded fraction than AMUPol. These results should be useful for membrane-protein solid-state NMR studies under DNP conditions and provide insights into how biradicals interact with phospholipid membranes.

Synthesis and evaluation of nitroxide-based oligoradicals for low-temperature dynamic nuclear polarization in solid state NMR

We describe the synthesis of new nitroxide-based biradical, triradical, and tetraradical compounds and the evaluation of their performance as paramagnetic dopants in dynamic nuclear polarization (DNP) experiments in solid state nuclear magnetic resonance (NMR) spectroscopy with magic-angle spinning (MAS). Under our experimental conditions, which include temperatures in the 25–30K range, a 9.4T magnetic field, MAS frequencies of 6.2–6.8kHz, and microwave irradiation at 264.0GHz from a 800mW extended interaction oscillator source, the most effective compounds are triradicals that are related to the previously-described compound DOTOPA-TEMPO (see Thurber et al., 2010), but have improved solubility in glycerol/water solvent near neutral pH. Using these compounds at 30mM total nitroxide concentration, we observe DNP enhancement factors of 92–128 for cross-polarized 13C NMR signals from 15N,13C-labeled melittin in partially protonated glycerol/water, and build-up times of 2.6–3.8s for 1H spin polarizations. Net sensitivity enhancements with biradical and tetraradical dopants, taking into account absolute 13C NMR signal amplitudes and build-up times, are approximately 2–4 times lower than with the best triradicals.

Synthesis of long T₁ silicon nanoparticles for hyperpolarized ²⁹Si magnetic resonance imaging.

We describe the synthesis, materials characterization, and dynamic nuclear polarization (DNP) of amorphous and crystalline silicon nanoparticles for use as hyperpolarized magnetic resonance imaging (MRI) agents. The particles were synthesized by means of a metathesis reaction between sodium silicide (Na₄Si₄) and silicon tetrachloride (SiCl₄) and were surface functionalized with a variety of passivating ligands. The synthesis scheme results in particles of diameter ∼10 nm with long size-adjusted ²⁹Si spin-lattice relaxation (T₁) times (>600 s), which are retained after hyperpolarization by low-temperature DNP.

The Different Magnetic Resonance Communities Join Forces for Progress in DNP: an Editorial for the Special Issue on DNP

This special issue of Applied Magnetic Resonance is the result of an attempt to show the very broad range of current research activities revolving around the central theme of dynamic nuclear polarization (DNP) and the impact DNP is currently making in many different scientific disciplines. It is fascinating to see how an idea that is almost as old as the whole field of nuclear magnetic resonance (NMR) is now revolutionizing the way people think about the maximal sensitivity that can be achieved in NMR experiments. Nowadays, scientists try to produce fully polarized nuclear spin systems for use as highly sensitive probes in medical diagnostic imaging or to generate high spin polarization on surfaces to investigate conformation of molecules in the boundary layers. In particular, two developments have tremendously stimulated the recent progress in DNP research. First, through the continuous and persistent efforts of Prof. R Griffin at the Massachusetts Institute of Technology, Boston US, robust hardware and experimental strategies are now available for solid-state magic angle spinning NMR experiments in conjunction with DNP. The second development was the use of low-temperature DNP followed by a fast rise in temperature to produce solutions containing highly polarized spin systems. This technique is now commonly referred to as dissolution DNP and has been conceived and developed by Prof. K Golman and Prof. J.-H. ArdenkjaerLarsen in Malmo, Sweden. The impact that these two major achievements are currently generating on the design of novel strategies for NMR spectroscopy and MR imaging and on increasing the range of applications cannot be rated highly enough. On the other hand, it is clear that the idea of using the electrons to generate

In Vivo Clinical Measures of Intermediary Metabolism Are Inadequate

The heart derives most of its energy via mitochondrial oxidative metabolism. The heart's fuels or energy substrates include fatty acids, lactate, glucose, and ketones. Substrate concentrations vary during the day, driven mainly by postprandial status. Entry into mitochondrial oxidation requires metabolism to acetyl-coenzyme A (CoA), the fuel of the tricarboxylic acid (TCA) cycle. Acetyl-CoA oxidation generates reduced Nicotinamide adenine dinucleotide (NADH), which drives oxidative phosphorylation, the reduction of O2 to H2O, and, ultimately, the synthesis of ATP, which is essential for myocardial mechanics. The heart uses this highly regulated series of enzyme-catalyzed reactions to convert chemical energy into mechanical energy.1 Metabolism and function in the heart are inextricably linked. Article see p 201 The pathological states of the heart, such as ischemia, hypertrophy, and failure, modify metabolism. Available technologies to measure intermediary metabolism have been inadequate. Magnetic resonance spectroscopy (MRS) can be used to spatially map or at least locally measure metabolism in vivo. Measurement of endogenous triglycerides in patients using 1H MRS has demonstrated that cardiac steatosis precedes the onset of type 2 diabetes mellitus and left ventricular systolic dysfunction.2 In the failing human heart and in preclinical heart failure models, 31P MRS has shown that flux through the creatinine kinase reaction is altered.3,4 Also, ATP and phosphocreatine are reduced after myocardial infarction due to myocyte loss.5 Myocardial energy substrate can be measured with 13C MRS. It has the chemical specificity, but its inherently low sensitivity limits the spatial and temporal resolution necessary for clinical application. In isolated hearts, 13C MRS has to be used with labeled substrate molecules to define TCA cycle kinetics. The metabolites of the TCA cycle have not been measured directly in these experiments. The content of the TCA cycle metabolites is …

Hyperpolarizing Gases via Dynamic Nuclear Polarization and Sublimation

A high throughput method was designed to produce hyperpolarized gases by combining low-temperature dynamic nuclear polarization with a sublimation procedure. It is illustrated by applications to 129Xe nuclear magnetic resonance in xenon gas, leading to a signal enhancement of 3 to 4 orders of magnitude compared to the room-temperature thermal equilibrium signal at 7.05T.

Quantum rotor induced hyperpolarization

Despite its broad applicability NMR has always been limited by its inherently low sensitivity. Hyperpolarization methods have the potential to overcome this limitation and, in the case of ex situ dynamic nuclear polarization (DNP), large enhancement factors have been achieved. Although many other polarization methods have been described in the past, including chemically and parahydrogen-induced polarization and optical pumping, DNP has recently been the most popular. Here we present an additional polarization mechanism arising from quantum rotor effects in methyl groups, which generates polarizations at temperatures < 1.5 K and interferes with DNP at such temperatures. The polarization generated by this mechanism is efficiently transferred via carbon bound protons. Although quantum rotor polarizations have been studied for a small range of molecules in great detail, we observe such effects for a much broader range of substances with very different polarization rates at temperatures < 1.5 K. Moreover, we report transfer of quantum rotor polarization across a chain of protons. The observed effect not only influences the polarization in low-temperature DNP experiments but also opens a new independent avenue to generate enhanced sensitivity for NMR.

Low-temperature dynamic nuclear polarization at 9.4 T with a 30 mW microwave source

Dynamic nuclear polarization (DNP) can provide large signal enhancements in nuclear magnetic resonance (NMR) by transfer of polarization from electron spins to nuclear spins. We discuss several aspects of DNP experiments at 9.4 T (400 MHz resonant frequency for 1H, 264 GHz for electron spins in organic radicals) in the 7–80 K temperature range, using a 30 mW, frequency-tunable microwave source and a quasi-optical microwave bridge for polarization control and low-loss microwave transmission. In experiments on frozen glycerol/water doped with nitroxide radicals, DNP signal enhancements up to a factor of 80 are observed (relative to 1H NMR signals with thermal equilibrium spin polarization). The largest sensitivity enhancements are observed with a new triradical dopant, DOTOPA-TEMPO. Field modulation with a 10 G root-mean-squared amplitude during DNP increases the nuclear spin polarizations by up to 135%. Dependencies of 1H NMR signal amplitudes, nuclear spin relaxation times, and DNP build-up times on the dopant and its concentration, temperature, microwave power, and modulation frequency are reported and discussed. The benefits of low-temperature DNP can be dramatic: the 1H spin polarization is increased approximately 1000-fold at 7 K with DNP, relative to thermal polarization at 80 K.

Prospects for sub-micron solid state nuclear magnetic resonance imaging with low-temperature dynamic nuclear polarization.

We evaluate the feasibility of (1)H nuclear magnetic resonance (NMR) imaging with sub-micron voxel dimensions using a combination of low temperatures and dynamic nuclear polarization (DNP). Experiments are performed on nitroxide-doped glycerol-water at 9.4 T and temperatures below 40 K, using a 30 mW tunable microwave source for DNP. With DNP at 7 K, a 0.5 microL sample yields a (1)H NMR signal-to-noise ratio of 770 in two scans with pulsed spin-lock detection and after 80 db signal attenuation. With reasonable extrapolations, we infer that (1)H NMR signals from 1 microm(3) voxel volumes should be readily detectable, and voxels as small as 0.03 microm(3) may eventually be detectable. Through homonuclear decoupling with a frequency-switched Lee-Goldburg spin echo technique, we obtain 830 Hz (1)H NMR linewidths at low temperatures, implying that pulsed field gradients equal to 0.4 G/d or less would be required during spatial encoding dimensions of an imaging sequence, where d is the resolution in each dimension.