Application of Counter-wound Multi-arm Spirals in HTS Resonator Design.

Significant sensitivity improvements have been achieved by utilizing high temperature superconducting (HTS) resonators in nuclear magnetic resonance (NMR) probes. Many nuclei such as 13C benefit from strong excitation fields which cannot be produced by traditional HTS resonator designs. We investigate the use of double-sided, counter-wound multi-arm spiral HTS resonators with the aim of increasing the excitation field at the required nuclear Larmor frequency for 13C. When compared to double-sided, counter-wound spiral resonators with similar geometry, simulations indicate that the multi-arm spiral version develops a more uniform current distribution. Preliminary tests of a two-arm resonator indicate that it may produce a stronger excitation field.

Progress Towards a Higher Sensitivity 13C-Optimized 1.5 mm HTS NMR Probe.

Nuclear magnetic resonance (NMR) probes using thin-film high temperature superconducting (HTS) resonators offer high sensitivity and are particularly suitable for small-sample applications. We are developing an improved 1.5 mm HTS NMR probe designed for operation at 14.1 T and optimized for 13C detection. The total sample volume is about 35 µL and the active sample volume is 20 µL. The probe employs HTS resonators for 13C and 1H transmission and detection and the 2H lock. We examine the interactions of multiple superconducting resonators and normal metal tuning loops on coil resonance frequency and probe sensitivity. We test a recently introduced 13C resonator design, engineered to significantly increase 13C detection sensitivity over previous all-HTS probes. At zero field, we observe a 13C quality factor of 6000 which is several times higher than previous resonators. In this work the coil design considerations and probe build-out procedure are discussed.

Time and Frequency Domain Response of HTS Resonators for Use as NMR Transmit Coils.

Replacing normal metal NMR coils with thin-film high-temperature superconductor (HTS) resonators can significantly improve the sensitivity of analytical NMR. To study the use of these resonators for excitation as well as detection, we investigated the radio frequency properties of the HTS NMR coils in both frequency and time domain at a variety of transmit power levels. Experiments were conducted on a double-sided, counter wound spiral resonator designed to detect NMR signals from 13C nuclei at 14.1 T. Power-dependent nonlinearity was observed in the transmission coefficient and quality factor. The ability of the HTS resonators to accurately generate short pulses was studied in the time domain over the range power levels. The results of this study show that some form of Q switching is needed to get good transmit performance from HTS coils for 13C. For that purpose, the effect of adding a shorted transmission line stub to improve the pulse shapes and reduce phase transients was studied.

Inductively-coupled Frequency Tuning and Impedance Matching in HTS-based NMR Probes.

Nuclear Magnetic Resonance (NMR) probes based on High Temperature Superconducting (HTS) resonators have demonstrated significant gains in detection sensitivity. However, the widespread acceptance of this technology has been limited by some unresolved issues including the mechanical unreliability of the moveable inductive loops used to adjust tuning and matching. In order to improve reliability, we propose to implement frequency tuning and impedance matching of HTS resonators using fixed inductively coupled loops and variable capacitors. By analyzing the loss mechanisms associated with inductive loops, we predict that using a superconducting inductive loop for tuning and matching will not only improve the reliability of HTS probes, but also provide improvements in sensitivity.

HIV-1 Capsid Function Is Regulated by Dynamics: Quantitative Atomic-Resolution Insights by Integrating Magic-Angle-Spinning NMR, QM/MM, and MD.

HIV-1 CA capsid protein possesses intrinsic conformational flexibility, which is essential for its assembly into conical capsids and interactions with host factors. CA is dynamic in the assembled capsid, and residues in functionally important regions of the protein undergo motions spanning many decades of time scales. Chemical shift anisotropy (CSA) tensors, recorded in magic-angle-spinning NMR experiments, provide direct residue-specific probes of motions on nano- to microsecond time scales. We combined NMR, MD, and density-functional-theory calculations, to gain quantitative understanding of internal backbone dynamics in CA assemblies, and we found that the dynamically averaged 15N CSA tensors calculated by this joined protocol are in remarkable agreement with experiment. Thus, quantitative atomic-level understanding of the relationships between CSA tensors, local backbone structure, and motions in CA assemblies is achieved, demonstrating the power of integrating NMR experimental data and theory for characterizing atomic-resolution dynamics in biological systems.

(39) K and (23) Na relaxation times and MRI of rat head at 21.1 T.

At ultrahigh magnetic field strengths (B0 ≥ 7.0 T), potassium ((39) K) MRI might evolve into an interesting tool for biomedical research. However, (39) K MRI is still challenging because of the low NMR sensitivity and short relaxation times. In this work, we demonstrated the feasibility of (39) K MRI at 21.1 T, determined in vivo relaxation times of the rat head at 21.1 T, and compared (39) K and sodium ((23) Na) relaxation times of model solutions containing different agarose gel concentrations at 7.0 and 21.1 T. (39) K relaxation times were markedly shorter than those of (23) Na. Compared with the lower field strength, (39) K relaxation times were up to 1.9- (T1 ), 1.4- (T2S ) and 1.9-fold (T2L ) longer at 21.1 T. The increase in the (23) Na relaxation times was less pronounced (up to 1.2-fold). Mono-exponential fits of the (39) K longitudinal relaxation time at 21.1 T revealed T1 = 14.2 ± 0.1 ms for the healthy rat head. The (39) K transverse relaxation times were 1.8 ± 0.2 ms and 14.3 ± 0.3 ms for the short (T2S ) and long (T2L ) components, respectively. (23) Na relaxation times were markedly longer (T1 = 41.6 ± 0.4 ms; T2S = 4.9 ± 0.2 ms; T2L = 33.2 ± 0.2 ms). (39) K MRI of the healthy rat head could be performed with a nominal spatial resolution of 1 × 1 × 1 mm(3) within an acquisition time of 75 min. The increase in the relaxation times with magnetic field strength is beneficial for (23) Na and (39) K MRI at ultrahigh magnetic field strength. Our results demonstrate that (39) K MRI at 21.1 T enables acceptable image quality for preclinical research. Copyright © 2016 John Wiley & Sons, Ltd.

Effects of Dielectric Substrates and Ground Planes on Resonance Frequency of Archimedean Spirals.

Superconducting self-resonant spiral structures are of current interest for applications both in metamaterials and as probe coils for nuclear magnetic resonance (NMR) spectroscopy for high-sensitivity chemical analysis. Accurate spiral models are available in the literature for behavior of a spiral below and up to self-resonance. However, knowledge of the higher modes is also important. We present the relationships between the spiral parameters and the multiple mode frequencies of single sided spirals on dielectric substrates as modeled by method of moments simulation. In the absence of a ground plane, we find that the mode frequency has a linear though not necessarily harmonic dependence on the mode number. The effect of a thick substrate can be approximated by an effective dielectric constant. But when the thickness is less than 20% of the spiral trace width (router - rinner) this approximation is no longer accurate. We have developed a simple empirical formula to predict the higher modes.

An Empirical Expression to Predict the Resonant Frequencies of Archimedean Spirals.

This work presents an empirical formula to accurately determine the frequencies of the fundamental and higher order resonances of an Archimedean spiral in a uniform dielectric medium in the absence of a ground plane. The formula is based on method-of-moments simulations which have been experimentally validated. This empirical formula is widely applicable to a broad range of spirals from thin-ring to disk-shaped (ratio of inner to outer radii 0 to 1), with 10 or more turns.

In vivo chlorine and sodium MRI of rat brain at 21.1 T.

OBJECT: MR imaging of low-gamma nuclei at the ultrahigh magnetic field of 21.1 T provides a new opportunity for understanding a variety of biological processes. Among these, chlorine and sodium are attracting attention for their involvement in brain function and cancer development. MATERIALS AND METHODS: MRI of (35)Cl and (23)Na were performed and relaxation times were measured in vivo in normal rat (n = 3) and in rat with glioma (n = 3) at 21.1 T. The concentrations of both nuclei were evaluated using the center-out back-projection method. RESULTS: T 1 relaxation curve of chlorine in normal rat head was fitted by bi-exponential function (T 1a = 4.8 ms (0.7) T 1b = 24.4 ± 7 ms (0.3) and compared with sodium (T 1 = 41.4 ms). Free induction decays (FID) of chlorine and sodium in vivo were bi-exponential with similar rapidly decaying components of [Formula: see text] ms and [Formula: see text] ms, respectively. Effects of small acquisition matrix and bi-exponential FIDs were assessed for quantification of chlorine (33.2 mM) and sodium (44.4 mM) in rat brain. CONCLUSION: The study modeled a dramatic effect of the bi-exponential decay on MRI results. The revealed increased chlorine concentration in glioma (~1.5 times) relative to a normal brain correlates with the hypothesis asserting the importance of chlorine for tumor progression.

Magic angle spinning and oriented sample solid-state NMR structural restraints combine for influenza a M2 protein functional insights.

As a small tetrameric helical membrane protein, the M2 proton channel structure is highly sensitive to its environment. As a result, structural data from a lipid bilayer environment have proven to be essential for describing the conductance mechanism. While oriented sample solid-state NMR has provided a high-resolution backbone structure in lipid bilayers, quaternary packing of the helices and many of the side-chain conformations have been poorly restrained. Furthermore, the quaternary structural stability has remained a mystery. Here, the isotropic chemical shift data and interhelical cross peaks from magic angle spinning solid-state NMR of a liposomal preparation strongly support the quaternary structure of the transmembrane helical bundle as a dimer-of-dimers structure. The data also explain how the tetrameric stability is enhanced once two charges are absorbed by the His37 tetrad prior to activation of this proton channel. The combination of these two solid-state NMR techniques appears to be a powerful approach for characterizing helical membrane protein structure.

Multidimensional oriented solid-state NMR experiments enable the sequential assignment of uniformly 15N labeled integral membrane proteins in magnetically aligned lipid bilayers.

Oriented solid-state NMR is the most direct methodology to obtain the orientation of membrane proteins with respect to the lipid bilayer. The method consists of measuring (1)H-(15)N dipolar couplings (DC) and (15)N anisotropic chemical shifts (CSA) for membrane proteins that are uniformly aligned with respect to the membrane bilayer. A significant advantage of this approach is that tilt and azimuthal (rotational) angles of the protein domains can be directly derived from analytical expression of DC and CSA values, or, alternatively, obtained by refining protein structures using these values as harmonic restraints in simulated annealing calculations. The Achilles' heel of this approach is the lack of suitable experiments for sequential assignment of the amide resonances. In this Article, we present a new pulse sequence that integrates proton driven spin diffusion (PDSD) with sensitivity-enhanced PISEMA in a 3D experiment ([(1)H,(15)N]-SE-PISEMA-PDSD). The incorporation of 2D (15)N/(15)N spin diffusion experiments into this new 3D experiment leads to the complete and unambiguous assignment of the (15)N resonances. The feasibility of this approach is demonstrated for the membrane protein sarcolipin reconstituted in magnetically aligned lipid bicelles. Taken with low electric field probe technology, this approach will propel the determination of sequential assignment as well as structure and topology of larger integral membrane proteins in aligned lipid bilayers.

Accommodation of Pulsed Field Gradients with Cascade Field Regulation in Powered Magnets.

High magnetic fields significantly improve the resolution and sensitivity of nuclear magnetic resonance (NMR) spectroscopy measurements, which presents exciting research opportunities in areas of chemistry, biology, and material science. Powered magnets can provide much higher magnetic fields than persistent mode superconducting magnets but suffer from temporal magnetic field fluctuations due to power supply ripple and variations in cooling water temperature and flow rate which make powered magnets non-viable for high resolution NMR experiments. Previous work has demonstrated that a multi-rate sampled data cascade control system may be used to improve the resolution of NMR experiments in powered magnets. Despite these advances in reducing temporal magnetic field fluctuations, the field regulation design does not accommodate the use of pulsed field gradients, which are necessary in many NMR experiments. This work presents a control topology which accommodates the use of pulsed field gradient signals with the field regulation system. This control approach is verified using NMR measurements.

High-field QCPMG NMR of large quadrupolar patterns using resistive magnets.

Spectroscopy in a high magnetic field reduces second-order quadrupolar shift while increasing chemical shift. It changes the scale between quadrupolar and chemical shift of half-integer quadrupolar spins. The application of QCPMG multiple echo for acquiring large quadrupolar pattern under the high magnetic field of a 25 T resistive magnet is presented for acquiring large quadrupolar patterns. It shows that temporal field fluctuations and spatial homogeneity of the Keck magnet at the NHMFL contribute about +/- 20 ppm in line broadening. NMR patterns which have breadths of hundreds to thousands of kilohertz can be efficiently recorded using a combination of QCPMG and magnetic field stepping with negligible hindrance from the inhomogeneity and field fluctuations of powered magnets.

Achieving 1 ppm field homogeneity above 24 T: Application of differential mapping for shimming Keck and the Series Connected Hybrid magnets at the NHMFL.

Powered resistive and resistive-superconductive hybrid magnets can reach fields higher than superconducting NMR magnets but lack the field homogeneity and temporal stability needed for high resolution NMR. Due to field fluctuations in powered magnets, commercially available mapping systems fail to produce maps of these magnets with sufficient reproducibility, thus hampering attempts to improve homogeneity of the field they generate. Starting with a commercial mapper, we built a mapping system which uses a two-channel (measurement + reference) mapper probe. We used this system to map and then to shim two magnets of Florida Bitter type at the National High Magnetic Field Laboratory in Tallahassee, FL. With a combination of passive (ferromagnetic) and active shims we achieved 2.3 ppm homogeneity in 1 cm diameter spherical volume (dsv) at 25.0 T in the Keck resistive magnet, and 0.9 ppm homogeneity in 1 cm dsv at 23.5, 28.2, and 35.2 T in the series-connected resistive-superconductive hybrid (SCH) magnet.

Investigating molecular recognition and biological function at interfaces using piscidins, antimicrobial peptides from fish.

We studied amidated and non-amidated piscidins 1 and 3, amphipathic cationic antimicrobial peptides from fish, to characterize functional and structural similarities and differences between these peptides and better understand the structural motifs involved in biological activity and functional diversity among amidated and non-amidated isoforms. Antimicrobial and hemolytic assays were carried out to assess their potency and toxicity, respectively. Site-specific high-resolution solid-state NMR orientational restraints were obtained from (15)N-labeled amidated and non-amidated piscidins 1 and 3 in the presence of hydrated oriented lipid bilayers. Solid-state NMR and circular dichroism results indicate that the peptides are alpha-helical and oriented parallel to the membrane surface. This orientation was expected since peptide-lipid interactions are enhanced at the water-bilayer interface for amphipathic cationic antimicrobial peptides. (15)N solid-state NMR performed on oriented samples demonstrate that piscidin experiences fast, large amplitude backbone motions around an axis parallel to the bilayer normal. Under the conditions tested here, piscidin 1 was confirmed to be more antimicrobially potent than piscidin 3 and antimicrobial activity was not affected by amidation. In light of functional and structural similarities between piscidins 1 and 3, we propose that their topology and fast dynamics are related to their mechanism of action.

Low-E probe for (19)F-(1)H NMR of dilute biological solids.

Sample heating induced by radio frequency (RF) irradiation presents a significant challenge to solid state NMR experiments in proteins and other biological systems, causing the sample to dehydrate which may result in distorted spectra and a damaged sample. In this work we describe a large volume, low-E (19)F-(1)H solid state NMR probe, which we developed for the 2D (19)F CPMG studies of dilute membrane proteins in a static and electrically lossy environment at 600MHz field. In (19)FCPMG and related multi-pulse (19)F-(1)H experiments the sample is heated by the conservative electric fields E produced in the sample coil at both (19)F and (1)H frequencies. Instead of using a traditional sample solenoid, our low-E (19)F-(1)H probe utilizes two orthogonal loop-gap resonators in order to minimize the conservative electric fields responsible for sample heating. Absence of the wavelength effects in loop-gap resonators results in homogeneous RF fields and enables the study of large sample volumes, an important feature for the dilute protein preparations. The orthogonal resonators also provide intrinsic isolation between the (19)F and (1)H channels, which is another major challenge for the (19)F-(1)H circuits where Larmor frequencies are only 6% apart. We detail steps to reduce (19)F background signals from the probe, which included careful choice of capacitor lubricants and manufacture of custom non-fluorinated coaxial cables. Application of the probe for two-dimensional (19)F CPMG spectroscopy in oriented lipid membranes is demonstrated with Flufenamic acid (FFA), a non-steroidal anti-inflammatory drug.

Using low-E resonators to reduce RF heating in biological samples for static solid-state NMR up to 900 MHz.

RF heating of solid-state biological samples is known to be a destabilizing factor in high-field NMR experiments that shortens the sample lifetime by continuous dehydration during the high-power cross-polarization and decoupling pulses. In this work, we describe specially designed, large volume, low-E 15N-1H solid-state NMR probes developed for 600 and 900 MHz PISEMA studies of dilute membrane proteins oriented in hydrated and dielectrically lossy lipid bilayers. The probes use an orthogonal coil design in which separate resonators pursue their own aims at the respective frequencies, resulting in a simplified and more efficient matching network. Sample heating at the 1H frequency is minimized by a loop-gap resonator which produces a homogeneous magnetic field B1 with low electric field E. Within the loop-gap resonator, a multi-turn solenoid closely matching the shape of the sample serves as an efficient observe coil. We compare power dissipation in a typical lossy bilayer sample in the new low-E probe and in a previously reported 15N-1H probe which uses a double-tuned 4-turn solenoid. RF loss in the sample is measured in each probe by observing changes in the 1H 360 degrees pulse lengths. For the same values of 1H B1 field, sample heating in the new probe was found to be smaller by an order of magnitude. Applications of the low-E design to the PISEMA study of membrane proteins in their native hydrated bilayer environment are demonstrated at 600 and 900 MHz.

Single Scan 2D NMR Spectroscopy on a 25 T Bitter Magnet.

2D NMR relies on monitoring systematic changes in the phases incurred by spin coherences as a function of an encoding time t(1), whose value changes over the course of independent experiments. The intrinsic multiscan nature of such protocols implies that resistive and/or hybrid magnets, capable of delivering the highest magnetic field strengths but possessing poor temporal stabilities, become unsuitable for 2D NMR acquisitions. It is here shown with a series of homo- and hetero-nuclear examples that such limitations can be bypassed using recently proposed 2D "ultrafast" acquisition schemes, which correlate interactions along all spectral dimensions within a single scan.

Optimizing ssNMR experiments for dilute proteins in heterogeneous mixtures at high magnetic fields.

Solid-state NMR spectroscopy at high magnetic fields is proving to be an effective technique in structural biology, particularly for proteins which are not amenable to traditional X-ray and solution NMR approaches. Several parameters can be selected to provide optimal sensitivity, improve sample stability, and ensure biological relevance for ssNMR measurements on protein samples. These include selection of sample conditions, NMR probe design, and design of pulse experiments. Here, we demonstrate and evaluate several engineering and experimental approaches for pursuing measurements on dilute proteins in heterogeneous mixtures.