Vik Bajaj

Nuclear magnetic resonance (NMR) relaxometry and diffusometry are important tools for the characterization of heterogeneous materials and porous media, with applications including medical imaging, food characterization and oil-well logging. These methods can be extremely effective in applications where high-resolution NMR is either unnecessary, impractical, or both, as is the case in the emerging field of portable chemical characterization. Here, we present a proof-of-concept experiment… Show more

Nuclear magnetic resonance (NMR) relaxometry and diffusometry are important tools for the characterization of heterogeneous materials and porous media, with applications including medical imaging, food characterization and oil-well logging. These methods can be extremely effective in applications where high-resolution NMR is either unnecessary, impractical, or both, as is the case in the emerging field of portable chemical characterization. Here, we present a proof-of-concept experiment demonstrating the use of high-sensitivity optical magnetometers as detectors for ultra-low-field NMR relaxation and diffusion measurements. Show less

The application of magnetic resonance spectroscopy at progressively smaller length scales may eventually permit ‘chemical imaging’ of spins at the surfaces of materials and biological complexes. In particular, the negatively charged nitrogen-vacancy (NV−) centre in diamond has been exploited as an optical transducer for nanoscale nuclear magnetic resonance. However, the spectra of detected spins are generally broadened by their interaction with proximate paramagnetic NV− centres through… Show more

The application of magnetic resonance spectroscopy at progressively smaller length scales may eventually permit ‘chemical imaging’ of spins at the surfaces of materials and biological complexes. In particular, the negatively charged nitrogen-vacancy (NV−) centre in diamond has been exploited as an optical transducer for nanoscale nuclear magnetic resonance. However, the spectra of detected spins are generally broadened by their interaction with proximate paramagnetic NV− centres through coherent and incoherent mechanisms. Here we demonstrate a detection technique that can resolve the spectra of electron spins coupled to NV− centres, in this case, substitutional nitrogen and neutral nitrogen-vacancy centres in diamond, through optically detected cross-relaxation. The hyperfine spectra of these spins are a unique chemical identifier, suggesting the possibility, in combination with recent results in diamonds harbouring shallow NV− implants, that the spectra of spins external to the diamond can be similarly detected.
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We report sensitive detection of the nuclear quadrupolar interaction of the N14 nuclear spin of the nitrogen-vacancy (NV) center using the electron spin-echo envelope modulation technique. We applied a weak transverse magnetic field to the spin system so that certain forbidden transitions became weakly allowed due to second-order effects involving the nonsecular terms of the hyperfine interaction. The weak transitions cause modulation of the electron spin-echo signal, and a theoretical analysis… Show more

We report sensitive detection of the nuclear quadrupolar interaction of the N14 nuclear spin of the nitrogen-vacancy (NV) center using the electron spin-echo envelope modulation technique. We applied a weak transverse magnetic field to the spin system so that certain forbidden transitions became weakly allowed due to second-order effects involving the nonsecular terms of the hyperfine interaction. The weak transitions cause modulation of the electron spin-echo signal, and a theoretical analysis suggests that the modulation frequency is primarily determined by the nuclear quadrupolar frequency; numerical simulations confirm the analytical results and show excellent quantitative agreement with experiments. This is an experimentally simple method of detecting quadrupolar interactions, and it can be used to study spin systems with an energy structure similar to that of the NV center. Show less

Magnetic resonance imaging (MRI) enables high-resolution non-invasive observation of the anatomy and function of intact organisms. However, previous MRI reporters of key biological processes tied to gene expression have been limited by the inherently low molecular sensitivity of conventional 1H MRI. This limitation could be overcome through the use of hyperpolarized nuclei, such as in the noble gas xenon, but previous reporters acting on such nuclei have been synthetic. Here, we introduce the… Show more

Magnetic resonance imaging (MRI) enables high-resolution non-invasive observation of the anatomy and function of intact organisms. However, previous MRI reporters of key biological processes tied to gene expression have been limited by the inherently low molecular sensitivity of conventional 1H MRI. This limitation could be overcome through the use of hyperpolarized nuclei, such as in the noble gas xenon, but previous reporters acting on such nuclei have been synthetic. Here, we introduce the first genetically encoded reporters for hyperpolarized 129Xe MRI. These expressible reporters are based on gas vesicles (GVs), gas-binding protein nanostructures expressed by certain buoyant microorganisms. We show that GVs are capable of chemical exchange saturation transfer interactions with xenon, which enables chemically amplified GV detection at picomolar concentrations (a 100- to 10,000-fold improvement over comparable constructs for 1H MRI). We demonstrate the use of GVs as heterologously expressed indicators of gene expression and chemically targeted exogenous labels in MRI experiments performed on living cells. Show less

Optically hyperpolarized 129Xe gas has become a powerful contrast agent in nuclear magnetic resonance (NMR) spectroscopy and imaging, with applications ranging from studies of the human lung to the targeted detection of biomolecules. Equally attractive is its potential use to enhance the sensitivity of microfluidic NMR experiments, in which small sample volumes yield poor sensitivity. Unfortunately, most 129Xe polarization systems are large and non-portable. Here we present a microfabricated… Show more

Optically hyperpolarized 129Xe gas has become a powerful contrast agent in nuclear magnetic resonance (NMR) spectroscopy and imaging, with applications ranging from studies of the human lung to the targeted detection of biomolecules. Equally attractive is its potential use to enhance the sensitivity of microfluidic NMR experiments, in which small sample volumes yield poor sensitivity. Unfortunately, most 129Xe polarization systems are large and non-portable. Here we present a microfabricated chip that optically polarizes 129Xe gas. We have achieved 129Xe polarizations >0.5% at flow rates of several microlitres per second, compatible with typical microfluidic applications. We employ in situ optical magnetometry to sensitively detect and characterize the 129Xe polarization at magnetic fields of 1 μT. We construct the device using standard microfabrication techniques, which will facilitate its integration with existing microfluidic platforms. This device may enable the implementation of highly sensitive 129Xe NMR in compact, low-cost, portable devices.
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We demonstrate that the spin decoherence of nitrogen vacancy (NV) centers in diamond can be suppressed by a transverse magnetic field if the electron spin bath is the primary decoherence source. The NV spin coherence, created in “a decoherence-free subspace,” is protected by the transverse component of the zero-field splitting, increasing the spin-coherence time about twofold. The decoherence due to the electron spin bath is also suppressed at magnetic fields stronger than ∼25 G when applied… Show more

We demonstrate that the spin decoherence of nitrogen vacancy (NV) centers in diamond can be suppressed by a transverse magnetic field if the electron spin bath is the primary decoherence source. The NV spin coherence, created in “a decoherence-free subspace,” is protected by the transverse component of the zero-field splitting, increasing the spin-coherence time about twofold. The decoherence due to the electron spin bath is also suppressed at magnetic fields stronger than ∼25 G when applied parallel to the NV symmetry axis. Our method can be used to extend the spin-coherence time of similar spin systems for applications in quantum computing, field sensing, and other metrologies. Show less

The cross-β amyloid form of peptides and proteins represents an archetypal and widely accessible structure consisting of ordered arrays of β-sheet filaments. These complex aggregates have remarkable chemical and physical properties, and the conversion of normally soluble functional forms of proteins into amyloid structures is linked to many debilitating human diseases, including several common forms of age-related dementia. Despite their importance, however, cross-β amyloid fibrils have proved… Show more

The cross-β amyloid form of peptides and proteins represents an archetypal and widely accessible structure consisting of ordered arrays of β-sheet filaments. These complex aggregates have remarkable chemical and physical properties, and the conversion of normally soluble functional forms of proteins into amyloid structures is linked to many debilitating human diseases, including several common forms of age-related dementia. Despite their importance, however, cross-β amyloid fibrils have proved to be recalcitrant to detailed structural analysis. By combining structural constraints from a series of experimental techniques spanning five orders of magnitude in length scale--including magic angle spinning nuclear magnetic resonance spectroscopy, X-ray fiber diffraction, cryoelectron microscopy, scanning transmission electron microscopy, and atomic force microscopy--we report the atomic-resolution (0.5 Å) structures of three amyloid polymorphs formed by an 11-residue peptide. These structures reveal the details of the packing interactions by which the constituent β-strands are assembled hierarchically into protofilaments, filaments, and mature fibrils.
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Dynamic nuclear polarization, which transfers the spin polarization of electrons to nuclei, is routinely applied to enhance the sensitivity of nuclear magnetic resonance. This method is particularly useful when spin hyperpolarization can be produced and controlled optically or electrically. Here we show complete polarization of nuclei located near optically polarized nitrogen-vacancy centres in diamond. Close to the ground-state level anti-crossing condition of the nitrogen-vacancy electron… Show more

Dynamic nuclear polarization, which transfers the spin polarization of electrons to nuclei, is routinely applied to enhance the sensitivity of nuclear magnetic resonance. This method is particularly useful when spin hyperpolarization can be produced and controlled optically or electrically. Here we show complete polarization of nuclei located near optically polarized nitrogen-vacancy centres in diamond. Close to the ground-state level anti-crossing condition of the nitrogen-vacancy electron spins, (13)C nuclei in the first shell are polarized in a pattern that depends sensitively upon the magnetic field. Based on the anisotropy of the hyperfine coupling and of the optical polarization mechanism, we predict and observe a reversal of the nuclear spin polarization with only a few millitesla change in the magnetic field. This method of magnetic control of high nuclear polarization at room temperature can be applied in sensitivity enhanced nuclear magnetic resonance of bulk nuclei, nuclear-based spintronics, and quantum computation in diamond.
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The design and operation of microfluidic analytical devices depends critically on tools to probe microscale chemistry and flow dynamics. Magnetic resonance imaging (MRI) seems ideally suited to this task, but its sensitivity is compromised because the fluid-containing channels in "lab on a chip" devices occupy only a small fraction of the enclosing detector's volume; as a result, the few microfluidic applications of NMR have required custom-designed chips harboring many detectors at specific… Show more

The design and operation of microfluidic analytical devices depends critically on tools to probe microscale chemistry and flow dynamics. Magnetic resonance imaging (MRI) seems ideally suited to this task, but its sensitivity is compromised because the fluid-containing channels in "lab on a chip" devices occupy only a small fraction of the enclosing detector's volume; as a result, the few microfluidic applications of NMR have required custom-designed chips harboring many detectors at specific points of interest. To overcome this limitation, we have developed remotely detected microfluidic MRI, in which an MR image is stored in the phase and intensity of each analyte's NMR signal and sensitively detected by a single, volume-matched detector at the device outflow, and combined it with compressed sensing for rapid image acquisition. Here, we build upon our previous work and introduce a method that incorporates our prior knowledge of the microfluidic device geometry to further decrease acquisition times. We demonstrate its use in multidimensional velocimetric imaging of a microfluidic mixer, acquiring microscopically detailed images 128 times faster than is possible with conventional sampling. This prior information also informs our choice of sampling schedule, resulting in a scheme that is optimized for a specific flow geometry. Finally, we test our approach in synthetic data and explore potential reconstruction errors as a function of optimization and reconstruction parameters.
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Molecular imaging based on saturation transfer in exchanging systems is a tool for amplified and chemically specific magnetic resonance imaging. Xenon-based molecular sensors are a promising category of molecular imaging agents in which chemical exchange of dissolved xenon between its bulk and agent-bound phases has been use to achieve sub-picomolar detection sensitivity. Control over the saturation transfer dynamics, particularly when multiple exchanging resonances are present in the spectra… Show more

Molecular imaging based on saturation transfer in exchanging systems is a tool for amplified and chemically specific magnetic resonance imaging. Xenon-based molecular sensors are a promising category of molecular imaging agents in which chemical exchange of dissolved xenon between its bulk and agent-bound phases has been use to achieve sub-picomolar detection sensitivity. Control over the saturation transfer dynamics, particularly when multiple exchanging resonances are present in the spectra, requires saturation fields of limited bandwidth and is generally accomplished by continuous wave irradiation. We demonstrate instead how band-selective saturation sequences based on multiple pulse inversion elements can yield saturation bandwidth tuneable over a wide range, while depositing less RF power in the sample. We show how these sequences can be used in imaging experiments that require spatial-spectral and multispectral saturation. The results should be applicable to all CEST experiments and, in particular, will provide the spectroscopic control required for applications of arrays of xenon chemical sensors in microfluidic chemical analysis devices.
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An application of remotely detected magnetic resonance imaging is demonstrated for the characterization of flow and the detection of fast, small molecule separations within hypercrosslinked polymer monoliths. The hyper-cross-linked monoliths exhibited excellent ruggedness, with a transit time relative standard deviation of less than 2.1%, even after more than 300 column volumes were pumped through at high pressure and flow. Magnetic resonance imaging enabled high-resolution intensity and… Show more

An application of remotely detected magnetic resonance imaging is demonstrated for the characterization of flow and the detection of fast, small molecule separations within hypercrosslinked polymer monoliths. The hyper-cross-linked monoliths exhibited excellent ruggedness, with a transit time relative standard deviation of less than 2.1%, even after more than 300 column volumes were pumped through at high pressure and flow. Magnetic resonance imaging enabled high-resolution intensity and velocity-encoded images of mobile phase flow through the monolith. The images confirm that the presence of a polymer monolith within the capillary disrupts the parabolic laminar flow profile that is characteristic of mobile phase flow within an open tube. As a result, the mobile phase and analytes are equally distributed in the radial direction throughout the monolith. Also, in-line monitoring of chromatographic separations of small molecules at high flow rates is shown. The coupling of monolithic chromatography columns and NMR provides both real-time peak detection and chemical shift information for small aromatic molecules. These experiments demonstrate the unique power of magnetic resonance, both direct and remote, in studying chromatographic processes.
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Many NMR and MRI methods probe fluid dynamics within macro- and mesoporous materials, but with few exceptions, they report on its macroscopically averaged properties. MRI methods are generally unable to localize microscopic features of flow within macroscopic samples because the fraction of the enclosing detector volume occupied by these features is so small. We have recently overcome this problem using remotely detected MRI velocimetry, a technique in which spatial, chemical, and velocity… Show more

Many NMR and MRI methods probe fluid dynamics within macro- and mesoporous materials, but with few exceptions, they report on its macroscopically averaged properties. MRI methods are generally unable to localize microscopic features of flow within macroscopic samples because the fraction of the enclosing detector volume occupied by these features is so small. We have recently overcome this problem using remotely detected MRI velocimetry, a technique in which spatial, chemical, and velocity information about elements of the flow is encoded with a conventional NMR coil and detected sensitively at the sample outflow by a volume-matched microdetector. Here, we apply this method to microporous model systems, recording MRI images that correlate local velocity, spin relaxation, and time-of-flight in microscopic resolution and three spatial dimensions. Our results illustrate that remotely detected MRI is an effective approach to elucidate flow dynamics in porous materials including bead pack microreactors and chromatography columns.
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Magnetic resonance imaging (MRI) can elucidate the interior structure of an optically opaque object in unparalleled detail but is ultimately limited by the need to enclose the object within a detection coil; acquiring the image with increasingly smaller pixels reduces the sensitivity, because each pixel occupies a proportionately smaller fraction of the detector's volume. We developed a technique that overcomes this limitation by means of remotely detected MRI. Images of fluids flowing in… Show more

Magnetic resonance imaging (MRI) can elucidate the interior structure of an optically opaque object in unparalleled detail but is ultimately limited by the need to enclose the object within a detection coil; acquiring the image with increasingly smaller pixels reduces the sensitivity, because each pixel occupies a proportionately smaller fraction of the detector's volume. We developed a technique that overcomes this limitation by means of remotely detected MRI. Images of fluids flowing in channel assemblies are encoded into the phase and intensity of the constituent molecules' nuclear magnetic resonance signals and then decoded by a volume-matched detector after the fluids flow out of the sample. In combination with compressive sampling, we thus obtain microscopic images of flow and velocity distributions ~10(6) times faster than is possible with conventional MRI on this hardware. Our results illustrate the facile integration of MRI with microfluidic assays and suggest generalizations to other systems involving microscopic flow.
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Amyloid fibrils are structurally ordered aggregates of proteins whose formation is associated with many neurodegenerative and other diseases. For that reason, their high-resolution structures are of considerable interest and have been studied using a wide range of techniques, notably electron microscopy, X-ray diffraction, and magic angle spinning (MAS) NMR. Because of the excellent resolution in the spectra, MAS NMR is uniquely capable of delivering site-specific, atomic resolution information… Show more

Amyloid fibrils are structurally ordered aggregates of proteins whose formation is associated with many neurodegenerative and other diseases. For that reason, their high-resolution structures are of considerable interest and have been studied using a wide range of techniques, notably electron microscopy, X-ray diffraction, and magic angle spinning (MAS) NMR. Because of the excellent resolution in the spectra, MAS NMR is uniquely capable of delivering site-specific, atomic resolution information about all levels of amyloid structure: (1) the monomer, which packs into several (2) protofilaments that in turn associate to form a (3) fibril. Building upon our high-resolution structure of the monomer of an amyloid-forming peptide from transthyretin (TTR(105-115)), we introduce single 1-(13)C labeled amino acids at seven different sites in the peptide and measure intermolecular carbonyl-carbonyl distances with an accuracy of ~0.11 A. Our results conclusively establish a parallel, in register, topology for the packing of this peptide into a β-sheet and provide constraints essential for the determination of an atomic resolution structure of the fibril. Furthermore, the approach we employ, based on a combination of a double-quantum filtered variant of the DRAWS recoupling sequence and multispin numerical simulations in SPINEVOLUTION, is general and should be applicable to a wide range of systems.
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NMR and MRI can yield detailed chemical and dynamic information about flow at microscopic resolutions, but suffer from low signal to noise relative to alternative techniques for flow measurements. In porous media and microfluidic devices, this sensitivity problem is further exacerbated by magnetic susceptibility broadening and low coil filling factor. Fortunately, remote detection can mitigate these issues by physically separating signal detection from the other steps of the experiment. The… Show more

NMR and MRI can yield detailed chemical and dynamic information about flow at microscopic resolutions, but suffer from low signal to noise relative to alternative techniques for flow measurements. In porous media and microfluidic devices, this sensitivity problem is further exacerbated by magnetic susceptibility broadening and low coil filling factor. Fortunately, remote detection can mitigate these issues by physically separating signal detection from the other steps of the experiment. The technique requires, however, that any measured interactions be encoded in indirectly sampled dimensions, leading to experiments of high dimensionality and correspondingly long acquisition times. We have applied compressed sensing, a reconstruction technique used in MRI, to dramatically reduce these experiment times by 8-64x through partial sampling (sub-sampling) of k-space, allowing for the collection of images with significantly higher resolutions in reasonable amounts of time. Here, we demonstrate this reconstruction technique to remotely detected flow measurements in a serpentine mixing chip and in a microfluidic channel harboring a constriction. We find that compressed sensing allows for significantly higher resolution images to be collected in a practical amount of time, thus significantly enhancing the applicability of remote detection to flow imaging.
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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… Show more

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.
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Functional MRI has become an important tool of researchers and clinicians who seek to understand patterns of neuronal activation that accompany sensory and cognitive processes. However, the interpretation of fMRI images rests on assumptions about the relationship between neuronal firing and hemodynamic response that are not firmly grounded in rigorous theory or experimental evidence. Further, the blood-oxygen-level-dependent effect, which correlates an MRI observable to neuronal firing, evolves… Show more

Functional MRI has become an important tool of researchers and clinicians who seek to understand patterns of neuronal activation that accompany sensory and cognitive processes. However, the interpretation of fMRI images rests on assumptions about the relationship between neuronal firing and hemodynamic response that are not firmly grounded in rigorous theory or experimental evidence. Further, the blood-oxygen-level-dependent effect, which correlates an MRI observable to neuronal firing, evolves over a period that is 2 orders of magnitude longer than the underlying processes that are thought to cause it. Here, we instead demonstrate experiments to directly image oscillating currents by MRI. The approach rests on a resonant interaction between an applied rf field and an oscillating magnetic field in the sample and, as such, permits quantitative, frequency-selective measurements of current density without spatial or temporal cancellation. We apply this method in a current loop phantom, mapping its magnetic field and achieving a detection sensitivity near the threshold required for the detection of neuronal currents. Because the contrast mechanism is under spectroscopic control, we are able to demonstrate how ramped and phase-modulated spin-lock radiation can enhance the sensitivity and robustness of the experiment. We further demonstrate the combination of these methods with remote detection, a technique in which the encoding and detection of an MRI experiment are separated by sample flow or translation. We illustrate that remotely detected MRI permits the measurement of currents in small volumes of flowing water with high sensitivity and spatial resolution.
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In MRI, anatomical structures are most often differentiated by variations in their bulk magnetic properties. Alternatively, exogenous contrast agents can be attached to chemical moieties that confer affinity to molecular targets; the distribution of such contrast agents can be imaged by magnetic resonance. Xenon-based molecular sensors are molecular imaging agents that rely on the reversible exchange of hyperpolarized xenon between the bulk and a specifically targeted host-guest complex. We… Show more

In MRI, anatomical structures are most often differentiated by variations in their bulk magnetic properties. Alternatively, exogenous contrast agents can be attached to chemical moieties that confer affinity to molecular targets; the distribution of such contrast agents can be imaged by magnetic resonance. Xenon-based molecular sensors are molecular imaging agents that rely on the reversible exchange of hyperpolarized xenon between the bulk and a specifically targeted host-guest complex. We have incorporated approximately 125 xenon sensor molecules in the interior of an MS2 viral capsid, conferring multivalency and other properties of the viral capsid to the sensor molecule. The resulting signal amplification facilitates the detection of sensor at 0.7 pM, the lowest to date for any molecular imaging agent used in magnetic resonance. This amplification promises the detection of chemical targets at much lower concentrations than would be possible without the capsid scaffold.
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Since experimental measurements of NMR chemical shifts provide time and ensemble averaged values, we investigated how these effects should be included when chemical shifts are computed using density functional theory (DFT). We measured the chemical shifts of the N-formyl-L-methionyl-L-leucyl-L-phenylalanine-OMe (MLF) peptide in the solid state, and then used the X-ray structure to calculate the (13)C chemical shifts using the gauge including projector augmented wave (GIPAW) method, which… Show more

Since experimental measurements of NMR chemical shifts provide time and ensemble averaged values, we investigated how these effects should be included when chemical shifts are computed using density functional theory (DFT). We measured the chemical shifts of the N-formyl-L-methionyl-L-leucyl-L-phenylalanine-OMe (MLF) peptide in the solid state, and then used the X-ray structure to calculate the (13)C chemical shifts using the gauge including projector augmented wave (GIPAW) method, which accounts for the periodic nature of the crystal structure, obtaining an overall accuracy of 4.2 ppm. In order to understand the origin of the difference between experimental and calculated chemical shifts, we carried out first-principles molecular dynamics simulations to characterize the molecular motion of the MLF peptide on the picosecond time scale. We found that (13)C chemical shifts experience very rapid fluctuations of more than 20 ppm that are averaged out over less than 200 fs. Taking account of these fluctuations in the calculation of the chemical shifts resulted in an accuracy of 3.3 ppm. To investigate the effects of averaging over longer time scales we sampled the rotameric states populated by the MLF peptides in the solid state by performing a total of 5 micros classical molecular dynamics simulations. By averaging the chemical shifts over these rotameric states, we increased the accuracy of the chemical shift calculations to 3.0 ppm, with less than 1 ppm error in 10 out of 22 cases. These results suggests that better DFT-based predictions of chemical shifts of peptides and proteins will be achieved by developing improved computational strategies capable of taking into account the averaging process up to the millisecond time scale on which the chemical shift measurements report.
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We describe a new approach to multiple (13)C-(15)N distance measurements in uniformly labeled solids, frequency-selective (FS) TEDOR. The method shares features with FS-REDOR and ZF- and BASE-TEDOR, which also provide quantitative (15)N-(13)C spectral assignments and distance measurements in U-[(13)C,(15)N] samples. To demonstrate the validity of the FS-TEDOR sequence, we measured distances in [U-(13)C,(15)N]-asparagine which are in good agreement with other methods. In addition, we integrate… Show more

We describe a new approach to multiple (13)C-(15)N distance measurements in uniformly labeled solids, frequency-selective (FS) TEDOR. The method shares features with FS-REDOR and ZF- and BASE-TEDOR, which also provide quantitative (15)N-(13)C spectral assignments and distance measurements in U-[(13)C,(15)N] samples. To demonstrate the validity of the FS-TEDOR sequence, we measured distances in [U-(13)C,(15)N]-asparagine which are in good agreement with other methods. In addition, we integrate high frequency dynamic nuclear polarization (DNP) into the experimental protocol and use FS-TEDOR to record a resolved correlation spectrum of the Arg-(13)C(gamma)-(15)N(epsilon) region in [U-(13)C,(15)N]-bacteriorhodopsin. We resolve six of the seven cross-peaks expected based on the primary sequence of this membrane protein.
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Observation and structural studies of reaction intermediates of proteins are challenging because of the mixtures of states usually present at low concentrations. Here, we use a 250 GHz gyrotron (cyclotron resonance maser) and cryogenic temperatures to perform high-frequency dynamic nuclear polarization (DNP) NMR experiments that enhance sensitivity in magic-angle spinning NMR spectra of cryo-trapped photocycle intermediates of bacteriorhodopsin (bR) by a factor of approximately 90… Show more

Observation and structural studies of reaction intermediates of proteins are challenging because of the mixtures of states usually present at low concentrations. Here, we use a 250 GHz gyrotron (cyclotron resonance maser) and cryogenic temperatures to perform high-frequency dynamic nuclear polarization (DNP) NMR experiments that enhance sensitivity in magic-angle spinning NMR spectra of cryo-trapped photocycle intermediates of bacteriorhodopsin (bR) by a factor of approximately 90. Multidimensional spectroscopy of U-(13)C,(15)N-labeled samples resolved coexisting states and allowed chemical shift assignments in the retinylidene chromophore for several intermediates not observed previously. The correlation spectra reveal unexpected heterogeneity in dark-adapted bR, distortion in the K state, and, most importantly, 4 discrete L substates. Thermal relaxation of the mixture of L's showed that 3 of these substates revert to bR(568) and that only the 1 substate with both the strongest counterion and a fully relaxed 13-cis bond is functional. These definitive observations of functional and shunt states in the bR photocycle provide a preview of the mechanistic insights that will be accessible in membrane proteins via sensitivity-enhanced DNP NMR. These observations would have not been possible absent the signal enhancement available from DNP.
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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… Show more

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.
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At reduced temperatures, proteins and other biomolecules are generally found to exhibit dynamic as well as structural transitions. This includes a so-called protein glass transition that is universally observed in systems cooled between 200 and 230 K, and which is generally attributed to interactions between hydrating solvent molecules and protein side chains. However, there is also experimental and theoretical evidence for a low-temperature transition in the intrinsic dynamics of the protein… Show more

At reduced temperatures, proteins and other biomolecules are generally found to exhibit dynamic as well as structural transitions. This includes a so-called protein glass transition that is universally observed in systems cooled between 200 and 230 K, and which is generally attributed to interactions between hydrating solvent molecules and protein side chains. However, there is also experimental and theoretical evidence for a low-temperature transition in the intrinsic dynamics of the protein itself, absent any solvent. Here, we use low-temperature solid-state NMR to examine site-specific fluctuations in atomic structure and dynamics in the absence of solvents. In particular, we employ magic angle spinning NMR to examine a structural phase transition associated with dynamic processes in a solvent-free polypeptide, N-f-MLF-OH, lattice at temperatures as low as 90 K. This transition is characterized by the appearance of an extra set of lines in 1D (15)N spectra as well as additional cross peaks in 2D (13)C-(13)C and (13)C-(15)N spectra. Interestingly, the gradual, temperature-dependent appearance of the new spectral component is not accompanied by the line broadening typical of dynamic transitions. A direct comparison between the spectra of N-f-MLF-OH and the analog N-f-MLF-OMe, which does not display this transition, indicates a correlation of the structural transition to the temperature dependent motion of the aromatic phenylalanine side chain. Several quantitative solid state NMR experiments were employed to provide site-specific measurements of structural and motional features of the observed transition.
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Dynamic nuclear polarization (DNP) results in a substantial nuclear polarization enhancement through a transfer of the magnetization from electrons to nuclei. Recent years have seen considerable progress in the development of DNP experiments directed towards enhancing sensitivity in biological nuclear magnetic resonance (NMR). This review covers the applications, hardware, polarizing agents, and theoretical descriptions that were developed at the Francis Bitter Magnet Laboratory at… Show more

Dynamic nuclear polarization (DNP) results in a substantial nuclear polarization enhancement through a transfer of the magnetization from electrons to nuclei. Recent years have seen considerable progress in the development of DNP experiments directed towards enhancing sensitivity in biological nuclear magnetic resonance (NMR). This review covers the applications, hardware, polarizing agents, and theoretical descriptions that were developed at the Francis Bitter Magnet Laboratory at Massachusetts Institute of Technology for high-field DNP experiments. In frozen dielectrics, the enhanced nuclear polarization developed in the vicinity of the polarizing agent can be efficiently dispersed to the bulk of the sample via (1)H spin diffusion. This strategy has been proven effective in polarizing biologically interesting systems, such as nanocrystalline peptides and membrane proteins, without leading to paramagnetic broadening of the NMR signals. Gyrotrons have been used as a source of high-power (5-10 W) microwaves up to 460 GHz as required for the DNP experiments. Other hardware has also been developed allowing in situ microwave irradiation integrated with cryogenic magic-angle-spinning solid-state NMR. Advances in the quantum mechanical treatment are successful in describing the mechanism by which new biradical polarizing agents yield larger enhancements at higher magnetic fields. Finally, pulsed methods and solution experiments should play a prominent role in the future of DNP.
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Dynamic nuclear polarization (DNP) is a method that permits NMR signal intensities of solids and liquids to be enhanced significantly, and is therefore potentially an important tool in structural and mechanistic studies of biologically relevant molecules. During a DNP experiment, the large polarization of an exogeneous or endogeneous unpaired electron is transferred to the nuclei of interest (I) by microwave (microw) irradiation of the sample. The maximum theoretical enhancement achievable is… Show more

Dynamic nuclear polarization (DNP) is a method that permits NMR signal intensities of solids and liquids to be enhanced significantly, and is therefore potentially an important tool in structural and mechanistic studies of biologically relevant molecules. During a DNP experiment, the large polarization of an exogeneous or endogeneous unpaired electron is transferred to the nuclei of interest (I) by microwave (microw) irradiation of the sample. The maximum theoretical enhancement achievable is given by the gyromagnetic ratios (gamma(e)gamma(l)), being approximately 660 for protons. In the early 1950s, the DNP phenomenon was demonstrated experimentally, and intensively investigated in the following four decades, primarily at low magnetic fields. This review focuses on recent developments in the field of DNP with a special emphasis on work done at high magnetic fields (> or =5 T), the regime where contemporary NMR experiments are performed. After a brief historical survey, we present a review of the classical continuous wave (cw) DNP mechanisms-the Overhauser effect, the solid effect, the cross effect, and thermal mixing. A special section is devoted to the theory of coherent polarization transfer mechanisms, since they are potentially more efficient at high fields than classical polarization schemes. The implementation of DNP at high magnetic fields has required the development and improvement of new and existing instrumentation. Therefore, we also review some recent developments in microw and probe technology, followed by an overview of DNP applications in biological solids and liquids. Finally, we outline some possible areas for future developments.
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By exploiting dynamic nuclear polarization (DNP) at 90 K, we observe the first NMR spectrum of the K intermediate in the ion-motive photocycle of bacteriorhodopsin. The intermediate is identified by its reversion to the resting state of the protein in red light and by its thermal decay to the L intermediate. The (15)N chemical shift of the Schiff base in K indicates that contact has been lost with its counterion. Under these circumstances, the visible absorption of K is expected to be more… Show more

By exploiting dynamic nuclear polarization (DNP) at 90 K, we observe the first NMR spectrum of the K intermediate in the ion-motive photocycle of bacteriorhodopsin. The intermediate is identified by its reversion to the resting state of the protein in red light and by its thermal decay to the L intermediate. The (15)N chemical shift of the Schiff base in K indicates that contact has been lost with its counterion. Under these circumstances, the visible absorption of K is expected to be more red-shifted than is observed and this suggests torsion around single bonds of the retinylidene chromophore. This is in contrast to the development of a strong counterion interaction and double bond torsion in L. Thus, photon energy is stored in electrostatic modes in K and is transferred to torsional modes in L. This transfer is facilitated by the reduction in bond alternation that occurs with the initial loss of the counterion interaction, and is driven by the attraction of the Schiff base to a new counterion. Nevertheless, the process appears to be difficult, as judged by the multiple L substates, with weaker counterion interactions, that are trapped at lower temperatures. The double-bond torsion ultimately developed in the first half of the photocycle is probably responsible for enforcing vectoriality in the pump by causing a decisive switch in the connectivity of the active site once the Schiff base and its counterion are neutralized by proton transfer.
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In this paper, we describe a 250 GHz gyrotron oscillator, a critical component of an integrated system for magic angle spinning (MAS) dynamic nuclear polarization (DNP) experiments at 9T, corresponding to 380 MHz (1)H frequency. The 250 GHz gyrotron is the first gyro-device designed with the goal of seamless integration with an NMR spectrometer for routine DNP enhanced NMR spectroscopy and has operated under computer control for periods of up to 21 days with a 100% duty cycle. Following a brief… Show more

In this paper, we describe a 250 GHz gyrotron oscillator, a critical component of an integrated system for magic angle spinning (MAS) dynamic nuclear polarization (DNP) experiments at 9T, corresponding to 380 MHz (1)H frequency. The 250 GHz gyrotron is the first gyro-device designed with the goal of seamless integration with an NMR spectrometer for routine DNP enhanced NMR spectroscopy and has operated under computer control for periods of up to 21 days with a 100% duty cycle. Following a brief historical review of the field, we present studies of the membrane protein bacteriorhodopsin (bR) using DNP enhanced multidimensional NMR. These results include assignment of active site resonances in [U-(13)C, (15)N]-bR and demonstrate the utility of DNP for studies of membrane proteins. Next, we review the theory of gyro-devices from quantum mechanical and classical viewpoints and discuss the unique considerations that apply to gyrotron oscillators designed for DNP experiments. We then characterize the operation of the 250 GHz gyrotron in detail, including its long-term stability and controllability. We have measured the spectral purity of the gyrotron emission using both homodyne and heterodyne techniques. Radiation intensity patterns from the corrugated waveguide that delivers power to the NMR probe were measured using two new techniques to confirm pure mode content:..... Show less

The gyrotron oscillator is a source of high average power millimeter-wave through terahertz radiation. In this paper, we report low beam power and high-efficiency operation of a tunable gyrotron oscillator at 233 GHz. The low-voltage operating mode provides a path to further miniaturization of the gyrotron through reduction in the size of the electron gun, power supply, collector, and cooling system, which will benefit industrial and scientific applications requiring portability. Detailed… Show more

The gyrotron oscillator is a source of high average power millimeter-wave through terahertz radiation. In this paper, we report low beam power and high-efficiency operation of a tunable gyrotron oscillator at 233 GHz. The low-voltage operating mode provides a path to further miniaturization of the gyrotron through reduction in the size of the electron gun, power supply, collector, and cooling system, which will benefit industrial and scientific applications requiring portability. Detailed studies of low-voltage operation in the TE(2) (,) (3) (,) (1) mode reveal that the mode can be excited with less than 7 W of beam power at 3.5 kV. During CW operation with 3.5-kV beam voltage and 50-mA beam current, the gyrotron generates 12 W of RF power at 233.2 GHz. The EGUN electron optics code describes the low-voltage operation of the electron gun. Using gun-operating parameters derived from EGUN simulations, we show that a linear theory adequately predicts the low experimental starting currents.
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In a previous communication [Hu et al., J. Am. Chem. Soc. 126, 10844 (2004)], an approach was demonstrated that improves the efficiency of the cross-effect polarization mechanism employed in high field dynamic nuclear polarization (DNP) experiments. Specifically, it was shown that tethering two TEMPO (2,2,6,6-tetramethylpiperidin-1-oxyl) radicals increases the electron-electron dipole coupling from approximately 1 MHz in solutions of monomeric TEMPO to approximately 25 MHz in a tethered… Show more

In a previous communication [Hu et al., J. Am. Chem. Soc. 126, 10844 (2004)], an approach was demonstrated that improves the efficiency of the cross-effect polarization mechanism employed in high field dynamic nuclear polarization (DNP) experiments. Specifically, it was shown that tethering two TEMPO (2,2,6,6-tetramethylpiperidin-1-oxyl) radicals increases the electron-electron dipole coupling from approximately 1 MHz in solutions of monomeric TEMPO to approximately 25 MHz in a tethered biradical. The larger coupling resulted in an increase in the DNP enhancements by a factor of approximately 3-4, from 45-50 to approximately 165. Here, a second approach to improving the efficiency of the polarization process is described that involves approximately satisfying the matching condition |omega(2e)-omega(1e)|=omega(n), where omega(2e) and omega(1e) are two frequencies in the electron paramagnetic resonance (EPR) spectrum and omega(n) is the Larmor frequency of the nuclear spins being polarized. Specifically, in a mixture of TEMPO and trityl [tris (8-carboxy-2,2,6,6-tetramethyl(-d3)-benzo[1,2d:4,5-d']bis(1,3)dithiol-4-yl) methyl] radicals, the intensity maxima in the EPR spectra of these two species are approximately separated by the (1)H NMR frequency. In this case the frequency difference between the g(yy) value of TEMPO and the narrow pseudo-isotropic g-value of trityl is approximately 224 MHz and the (1)H Larmor frequency is 211 MHz. The optimal magnetic field for DNP using the mixtures was found to coincide with the trityl EPR resonance. At 90 K and 5 T, a mixture of 20 mM TEMPO and 20 mM trityl enhanced the (1)H polarization by a factor of approximately 160, an improvement over the enhancement of approximately 50 with 40 mM TEMPO. The reasons for the improvement are discussed and evidence is presented suggesting that DNP enhancement can be improved further by tethering TEMPO and trityl or two similar radicals.
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We report the regulated continuous-wave (CW) operation of a second harmonic gyrotron oscillator at output power levels of over 8 W (12.4 kV and 135 mA beam voltage and current) in the TE(0,6,1) mode near 460 GHz. The gyrotron also operates in the second harmonic TE(2,6,1) mode at 456 GHz and in the TE(2,3,1) fundamental mode at 233 GHz. CW operation was demonstrated for a one-hour period in the TE(0,6,1) mode with better than 1% power stability, where the power was regulated using feedback… Show more

We report the regulated continuous-wave (CW) operation of a second harmonic gyrotron oscillator at output power levels of over 8 W (12.4 kV and 135 mA beam voltage and current) in the TE(0,6,1) mode near 460 GHz. The gyrotron also operates in the second harmonic TE(2,6,1) mode at 456 GHz and in the TE(2,3,1) fundamental mode at 233 GHz. CW operation was demonstrated for a one-hour period in the TE(0,6,1) mode with better than 1% power stability, where the power was regulated using feedback control. Nonlinear simulations of the gyrotron operation agree with the experimentally measured output power and radio-frequency (RF) efficiency when cavity ohmic losses are included in the analysis. The output radiation pattern was measured using a pyroelectric camera and is highly Gaussian, with an ellipticity of 4%. The 460-GHz gyrotron will serve as a millimeter-wave source for sensitivity-enhanced nuclear magnetic resonance (dynamic nuclear polarization) experiments at a magnetic field of 16.4 T.
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A 250-GHz corrugated transmission line with a directional coupler for forward and backward power monitoring has been constructed and tested for use with a 25-W continuous-wave gyrotron for dynamic nuclear polarization (DNP) experiments. The main corrugated line (22-mm internal diameter, 2.4-m long) connects the gyrotron output to the DNP probe input. The directional coupler, inserted approximately midway, is a four-port crossed waveguide beamsplitter design. Two beamsplitters, a quartz plate… Show more

A 250-GHz corrugated transmission line with a directional coupler for forward and backward power monitoring has been constructed and tested for use with a 25-W continuous-wave gyrotron for dynamic nuclear polarization (DNP) experiments. The main corrugated line (22-mm internal diameter, 2.4-m long) connects the gyrotron output to the DNP probe input. The directional coupler, inserted approximately midway, is a four-port crossed waveguide beamsplitter design. Two beamsplitters, a quartz plate and ten-wire array, were tested with output coupling of 2.5% (-16 dB) at 250.6 GHz and 1.6% (-18 dB), respectively. A pair of mirrors in the DNP probe transferred the gyrotron beam from the 22-mm waveguide to an 8-mm helically corrugated waveguide for transmission through the final 0.58-m distance inside the NMR magnet to the sample. The transmission-line components were all cold tested with a 248 ± 4-GHz radiometer. A total insertion loss of 0.8 dB was achieved for HE(11) -mode propagation from the gyrotron to the sample with only 1% insertion loss for the 22-mm-diameter waveguide. A clean Gaussian gyrotron beam at the waveguide output and reliable forward power monitoring were achieved for many hours of continuous operation.
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A formal theory for heteronuclear decoupling in solid-state magic angle spinning (MAS) nuclear magnetic resonance experiments is presented as a first application of multipole-multimode Floquet theory. The method permits a straightforward construction of the multispin basis and describes the spin dynamics via effective Floquet Hamiltonians obtained using the van Vleck transformation method in the Floquet-Liouville space. As a test case, we consider a model three-spin system (I2S) under… Show more

A formal theory for heteronuclear decoupling in solid-state magic angle spinning (MAS) nuclear magnetic resonance experiments is presented as a first application of multipole-multimode Floquet theory. The method permits a straightforward construction of the multispin basis and describes the spin dynamics via effective Floquet Hamiltonians obtained using the van Vleck transformation method in the Floquet-Liouville space. As a test case, we consider a model three-spin system (I2S) under asynchronous time modulations (both MAS and rf irradiation) and derive effective Hamiltonians for describing the spin dynamics in the Floquet-Liouville space during heteronuclear decoupling. Furthermore, we describe and evaluate the origin of cross terms between the various anisotropic interactions and illustrate their exact contributions to the spin dynamics. The theory presented herein should be applicable to the design and understanding of pulse sequences for heteronuclear and homonuclear recoupling and decoupling.
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Amyloid fibrils are self-assembled filamentous structures associated with protein deposition conditions including Alzheimer's disease and the transmissible spongiform encephalopathies. Despite the immense medical importance of amyloid fibrils, no atomic-resolution structures are available for these materials, because the intact fibrils are insoluble and do not form diffraction-quality 3D crystals. Here we report the high-resolution structure of a peptide fragment of the amyloidogenic protein… Show more

Amyloid fibrils are self-assembled filamentous structures associated with protein deposition conditions including Alzheimer's disease and the transmissible spongiform encephalopathies. Despite the immense medical importance of amyloid fibrils, no atomic-resolution structures are available for these materials, because the intact fibrils are insoluble and do not form diffraction-quality 3D crystals. Here we report the high-resolution structure of a peptide fragment of the amyloidogenic protein transthyretin, TTR(105-115), in its fibrillar form, determined by magic angle spinning NMR spectroscopy. The structure resolves not only the backbone fold but also the precise conformation of the side chains. Nearly complete (13)C and (15)N resonance assignments for TTR(105-115) formed the basis for the extraction of a set of distance and dihedral angle restraints. A total of 76 self-consistent experimental measurements, including 41 restraints on 19 backbone dihedral angles and 35 (13)C-(15)N distances between 3 and 6 A were obtained from 2D and 3D NMR spectra recorded on three fibril samples uniformly (13)C, (15)N-labeled in consecutive stretches of four amino acids and used to calculate an ensemble of peptide structures. Our results indicate that TTR(105-115) adopts an extended beta-strand conformation in the amyloid fibrils such that both the main- and side-chain torsion angles are close to their optimal values. Moreover, the structure of this peptide in the fibrillar form has a degree of long-range order that is generally associated only with crystalline materials. These findings provide an explanation of the unusual stability and characteristic properties of this form of polypeptide assembly.
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The rotational resonance width (R2W) experiment is a constant-time version of the rotational resonance (R2) experiment, in which the magnetization exchange is measured as a function of sample spinning frequency rather than the mixing time. The significant advantage of this experiment over conventional R2 is that both the dipolar coupling and the relaxation parameters can be independently and unambiguously extracted from the magnetization exchange profile. In this paper, we combine R2W with… Show more

The rotational resonance width (R2W) experiment is a constant-time version of the rotational resonance (R2) experiment, in which the magnetization exchange is measured as a function of sample spinning frequency rather than the mixing time. The significant advantage of this experiment over conventional R2 is that both the dipolar coupling and the relaxation parameters can be independently and unambiguously extracted from the magnetization exchange profile. In this paper, we combine R2W with two-dimensional 13C-13C chemical shift correlation spectroscopy and demonstrate the utility of this technique for the site-specific measurement of multiple 13C-13C distances in uniformly labeled solids. The dipolar truncation effects, usually associated with distance measurements in uniformly labeled solids, are considerably attenuated in R2W experiments. Thus, R2W experiments are applicable to uniformly labeled biological systems. To validate this statement, multiple 13C-13C distances (in the range of 3-6 A) were determined in N-acetyl-[U-13C,15N]l-Val-l-Leu with an average precision of +/-0.5 A. Furthermore, the distance constraints extracted using a two-spin model agree well with the X-ray crystallographic data.
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In this communication, we report enhancements of nuclear spin polarization by dynamic nuclear polarization (DNP) in static and spinning solids at a magnetic field strength of 9T (250 GHz for g=2 electrons, 380 MHz for 1H). In these experiments, 1H enhancements of up to 170+/-50 have been observed in 1-13C-glycine dispersed in a 60:40 glycerol/water matrix at temperatures of 20K; in addition, we have observed significant enhancements in 15N spectra of unoriented pf1-bacteriophage. Finally… Show more

In this communication, we report enhancements of nuclear spin polarization by dynamic nuclear polarization (DNP) in static and spinning solids at a magnetic field strength of 9T (250 GHz for g=2 electrons, 380 MHz for 1H). In these experiments, 1H enhancements of up to 170+/-50 have been observed in 1-13C-glycine dispersed in a 60:40 glycerol/water matrix at temperatures of 20K; in addition, we have observed significant enhancements in 15N spectra of unoriented pf1-bacteriophage. Finally, enhancements of approximately 17 have been obtained in two-dimensional 13C-13C chemical shift correlation spectra of the amino acid U-13C, 15N-proline during magic angle spinning (MAS), demonstrating the stability of the DNP experiment for sustained acquisition and for quantitative experiments incorporating dipolar recoupling. In all cases, we have exploited the thermal mixing DNP mechanism with the nitroxide radical 4-amino-TEMPO as the paramagnetic dopant. These are the highest frequency DNP experiments performed to date and indicate that significant signal enhancements can be realized using the thermal mixing mechanism even at elevated magnetic fields. In large measure, this is due to the high microwave power output of the 250 GHz gyrotron oscillator used in these experiments.
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