A strip-shield improves the efficiency of a solenoid coil in probes for high-field solid-state NMR of lossy biological samples

A strip-shield inserted between a high inductance double-tuned solenoid coil and the glass tube containing the sample improves the efficiency of probes used for high-field solid-state NMR experiments on lossy aqueous samples of proteins and other biopolymers. A strip-shield is a coil liner consisting of thin copper strips layered on a PTFE (polytetrafluoroethylene) insulator. With lossy samples, the shift in tuning frequency is smaller, the reduction in Q, and RF-induced heating are all significantly reduced when the strip-shield is present. The performance of 800 MHz ¹H/¹⁵N and ¹H/¹³C double-resonance probes is demonstrated on aqueous samples of membrane proteins in phospholipid bilayers.     

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.     

An efficient (1)H/(31)P double-resonance solid-state NMR probe that utilizes a scroll coil

The construction and performance of a scroll coil double-resonance probe for solid-state NMR on stationary samples is described. The advantages of the scroll coil at the high resonance frequencies of (1)H and (31)P include: high efficiency, minimal perturbations of tuning by a wide range of samples, minimal RF sample heating of high dielectric samples of biopolymers in aqueous solution, and excellent RF homogeneity. The incorporation of a cable tie cinch for mechanical stability of the scroll coil is described. Experimental results obtained on a Hunter Killer Peptide 1 (HKP1) interacting with phospholipid bilayers of varying lipid composition demonstrate the capabilities of this probe on lossy aqueous samples.     

Reduction of RF-induced sample heating with a scroll coil resonator structure for solid-state NMR probes

Heating due to high power 1H decoupling limits the experimental lifetime of protein samples for solid-state NMR (SSNMR). Sample deterioration can be minimized by lowering the experimental salt concentration, temperature or decoupling fields; however, these approaches may compromise biological relevance and/or spectroscopic resolution and sensitivity. The desire to apply sophisticated multiple pulse experiments to proteins therefore motivates the development of probes that utilize the RF power more efficiently to generate a high ratio of magnetic to electric field in the sample. Here a novel scroll coil resonator structure is presented and compared to a traditional solenoid. The scroll coil is demonstrated to be more tolerant of high sample salt concentrations and cause less RF-induced sample heating. With it, the viable experimental lifetime of a microcrystalline ubiquitin sample has been extended by more than an order of magnitude. The higher B1 homogeneity and permissible decoupling fields enhance polarization transfer efficiency in 15N-13C correlation experiments employed for protein chemical shift assignments and structure determination.     

A low-E magic angle spinning probe for biological solid state NMR at 750 MHz

Crossed-coil NMR probes are a useful tool for reducing sample heating for biological solid state NMR. In a crossed-coil probe, the higher frequency ¹H field, which is the primary source of sample heating in conventional probes, is produced by a separate low-inductance resonator. Because a smaller driving voltage is required, the electric field across the sample and the resultant heating is reduced. In this work we describe the development of a magic angle spinning (MAS) solid state NMR probe utilizing a dual resonator. This dual resonator approach, referred to as “low-E,” was originally developed to reduce heating in samples of mechanically aligned membranes. The study of inherently dilute systems, such as proteins in lipid bilayers, via MAS techniques requires large sample volumes at high field to obtain spectra with adequate signal-to-noise ratio under physiologically relevant conditions. With the low-E approach, we are able to obtain homogeneous and sufficiently strong radiofrequency fields for both ¹H and ¹³C frequencies in a 4 mm probe with a ¹H frequency of 750 MHz. The performance of the probe using windowless dipolar recoupling sequences is demonstrated on model compounds as well as membrane-embedded peptides.     

Using a cross-coil to reduce RF heating by an order of magnitude in triple-resonance multinuclear MAS at high fields

Four different coil designs for use with MAS in triple-resonance multi-nuclear experiments at high fields are compared, using a combination of finite element analysis (FEA) software and NMR experiments, with respect to RF field strength per unit power and relative sample heating, as governed by mean E/B(1) within the sample region. A commercial FEA package, Microwave Studio 5.1 by Computer Simulation Technology (CST) is shown to obtain remarkably accurate agreement with the experiments in Q(L), L, B, E, and mode frequencies in all cases. A simplified treatment of RF heating in NMR MAS samples is derived and shown to agree with the NMR experimental results within about 10% for two representative stator designs. The coil types studied include: (1) a variable-pitch solenoid outside a ceramic coilform, (2) a conventional solenoid very closely spaced to the MAS rotor, (3) a scroll coil, and (4) a segmented saddle cross coil (XC) for (1)H with an additional solenoid over it for the two lower-frequency channels. The XC/solenoid is shown to offer substantial advantages in reduced decoupler heating, improved S/N, and improved compatibility with multinuclear tuning and high-power decoupling. This seems largely because the division of labor between two orthogonal coils allows them each, and their associated circuitry, to be separately optimized for their respective regimes.     

Resonator with reduced sample heating and increased homogeneity for solid-state NMR

In the application of solid-state NMR to many systems, the presence of radiofrequency (rf) electric fields inside classical solenoidal coils causes heating of lossy samples. In particular, this is critical for proteins in ionic buffers. Rf sample heating increases proportional to frequency which may result in the need to reduce the rf pulse power to prevent partial or total sample deterioration. In the present paper, we propose a multifrequency-tunable NMR resonator where the sample is electrically shielded from the NMR coil by a conductive sheet that increases the magneto-electric ratio. Expressions for the B1 efficiency as function of magnetic and electric filling factors are derived that allow a direct comparison of different resonators. Rf efficiency, homogeneity, signal-to-noise, and rf sample heating are compared. NMR spectra at 700MHz on ethylene glycol, glycine, and a model protein were acquired to compare the resonators under realistic experimental conditions.     

Design of a high-power NMR probe for low-temperature studies

A low-temperature, high-power NMR probe head design is described which eliminates the problem of electric arc discharge commonly experienced during radiofrequency pulse cycling in a helium environment. A polychlorotrifluoroethylene (Kel-F) coil former, fitted with a solenoid coil, is heat-shrunk onto stainless-steel flanges and spot-welded inside a stainless-steel probe head assembly connected to a hollow coaxial transmission-line probe shaft. By this means, the sample coil and all high-voltage elements can effectively be isolated in a vacuum, while at the same time permitting good thermal contact between the sample and cryogenic gas. This design was used in NMR studies in the 4.6 K < or = T < or = 77 K temperature range for RF pulse durations < or = 50 ms (and longer for low RF amplitudes) and amplitudes up to approximately 60 G.     

Assigning solid-state NMR spectra of aligned proteins using isotropic chemical shifts

A method for assigning solid-state NMR spectra of membrane proteins aligned in phospholipid bicelles that makes use of isotropic chemical shift frequencies and assignments is demonstrated. The resonance assignments are based on comparisons of 15N chemical shift differences in spectra obtained from samples with their bilayer normals aligned perpendicular and parallel to the direction of the applied magnetic field.     

Estimation and measurement of flat or solenoidal coil inductance for radiofrequency NMR coil design

The inductance of a radiofrequency coil determines its compatibility with a given NMR probe circuit. However, calculation (or estimation) of inductance for radiofrequency coils of dimensions suitable for use in an NMR probe is not trivial, particularly for flat-coils. A comparison of a number of formulae for calculation of inductance is presented through the use of a straightforward inductance measurement circuit. This technique relies upon instrumentation available in many NMR laboratories rather than upon more expensive and specialized instrumentation often utilized in the literature. Inductance estimation methods are suggested and validated for both flat-coils and solenoids. These have proven very useful for fabrication of a number of new coils in our laboratory for use in static solid-state NMR probes operating at (1)H frequencies of 300 and 600MHz. Solenoidal coils with very similar measured and estimated inductances having inner diameters from 1 to 5mm are directly compared as an example of the practical application of inductance estimation for interchange of coils within an existing solid-state NMR probe.     

Solid-state NMR spectroscopy of a membrane protein in biphenyl phospholipid bicelles with the bilayer normal parallel to the magnetic field

Bicelles composed of the long-chain biphenyl phospholipid TBBPC (1-tetradecanoyl-2-(4-(4-biphenyl)butanoyl)-sn-glycero-3-PC) and the short-chain phospholipid DHPC align with their bilayer normals parallel to the direction of the magnetic field. In contrast, in typical bicelles the long-chain phospholipid is DMPC or DPPC, and the bilayers align with their normals perpendicular to the field. Samples of the membrane-bound form of the major coat protein of Pf1 bacteriophage in TBBPC bicelles are stable for several months, align magnetically over a wide range of temperatures, and yield well-resolved solid-state NMR spectra similar to those obtained from samples aligned mechanically on glass plates or in DMPC bicelle samples "flipped" with lanthanide ions so that their bilayer normals are parallel to the field. The order parameter of the TBBPC bicelle sample decreases from approximately 0.9 to 0.8 upon increasing the temperature from 20 degrees C to 60 degrees C. Since the frequency spans of the chemical shift and dipolar coupling interactions are twice as large as those obtained from proteins in DMPC bicelles without lanthanide ions, TBBPC bicelles provide an opportunity for structural studies with higher spectral resolution of the metal-binding membrane proteins without the risk of chemical or spectroscopic interference from the added lanthanide ions. In addition, the large temperature range of these samples is advantageous for the studies of membrane proteins that are unstable at elevated temperatures and for experiments requiring measurements as a function of temperature.     

Calculating order parameter profiles utilizing magnetically aligned phospholipid bilayers for 2H solid-state NMR studies

Solid-state deuterium NMR spectroscopy was used to study the structural and dynamic properties of stearic acid-d(35) in magnetically aligned phospholipid bilayers as a function of temperature. Magnetically aligned phospholipid bilayers or bicelles are model systems, which mimic biological membranes for magnetic resonance studies. Paramagnetic lanthanide ions (Yb(3+)) were added to align the bicelles such that the bilayer normal is colinear with the direction of the static magnetic field. The corresponding order parameters of the stearic acid-d(35) probe were calculated and compared with values obtained from unoriented samples in the literature. The addition of cholesterol to the bicelle system decreases the fluidity of the phospholipid bilayers and increases the ordering of the acyl chains of stearic acid-d(35). This study demonstrates the feasibility of utilizing magnetically aligned bicelles for calculating 2H order parameter profiles for non-biological systems such as polymer-grafted membranes and Schiff's base complexes.     

Design of small volume HX and triple-resonance probes for improved limits of detection in protein NMR experiments

Three- and four-frequency nuclear magnetic-resonance probes have been designed for the study of small amounts of protein. Both "HX" (1H, X, and 2H channels) and "triple-resonance" (1H, 15N, 13C, and 2H) probes were implemented using a single transmit/receive coil and multiple-frequency impedance matching circuits. The coil used was a six-turn solenoid with an observe volume of 15 microl. A variable pitch design was used to improve the B1 homogeneity of the coil. Two-dimensional HSQC spectra of approximately 1mM single labeled 15N- and double labeled 15N/13C-proteins were acquired in experimental times of approximately 2h. Triple-resonance capability of the small-volume triple-resonance probe was demonstrated by acquiring three-dimensional HNCO spectra from the same protein samples. In addition to enabling very small quantities of protein to be used, the extremely short pulse widths (1H = 4, 15N = 4, and 13C = 2 micros) of this particular design result in low power decoupling and wide-bandwidth coverage, an important factor for the ever-higher operating frequencies used for protein NMR studies.     

Sensitivity and resolution enhancement in solid-state NMR spectroscopy of bicelles

Magnetically aligned bicelles are becoming attractive model membranes to investigate the structure, dynamics, geometry, and interaction of membrane-associated peptides and proteins using solution- and solid-state NMR experiments. Recent studies have shown that bicelles are more suitable than mechanically aligned bilayers for multidimensional solid-state NMR experiments. In this work, we describe experimental aspects of the natural abundance (13)C and (14)N NMR spectroscopy of DMPC/DHPC bicelles. In particular, approaches to enhance the sensitivity and resolution and to quantify radio-frequency heating effects are presented. Sensitivity of (13)C detection using single pulse excitation, conventional cross-polarization (CP), ramp-CP, and NOE techniques are compared. Our results suggest that the proton decoupling efficiency of the FLOPSY pulse sequence is better than that of continuous wave decoupling, TPPM, SPINAL, and WALTZ sequences. A simple method of monitoring the water proton chemical shift is demonstrated for the measurement of sample temperature and calibration of the radio-frequency-induced heating in the sample. The possibility of using (14)N experiments on bicelles is also discussed.     

水化磷脂层中蛋白质和多肽的高分辨固体核磁共振波谱学

有序样品的固体核磁共振(NMR)已快速发展成测定蛋白质和多肽在“仿真”水化磷脂层中高分辨结构的重要谱学方法. 由于与膜相连的蛋白质和多肽的结构、动力学和功能往往都和其周边自然环境密切相关,因此人们把蛋白质和多肽有序排列于水化磷脂层中进行固体NMR测量, 从而获得与取向相关的各向异性自旋相互作用. 这些取向约束可作为结构参数重构蛋白质在水化磷脂层中的高分辨三维结构. 近十年来在样品制备,NMR探头和实验方法方面的显著发展,极大地促进了有序样品的固体NMR的发展,并使之成为测定与膜相连的蛋白质和多肽结构的有效方法. 该综述介绍有序样品的固体NMR谱学方法,并总结此领域里的最新研究进展.     

Radiation damping in microcoil NMR probes

Radiation damping arises from the field induced in the receiver coil by large bulk magnetization and tends to selectively drive this magnetization back to equilibrium much faster than relaxation processes. The demand for increased sensitivity in mass-limited samples has led to the development of microcoil NMR probes that are capable of obtaining high quality NMR spectra with small sample volumes (nL-microL). Microcoil probes are optimized to increase sensitivity by increasing either the sample-to-coil ratio (filling factor) of the probe or quality factor of the detection coil. Though radiation damping effects have been studied in standard NMR probes, these effects have not been measured in the microcoil probes. Here a systematic evaluation of radiation damping effects in a microcoil NMR probe is presented and the results are compared with similar measurements in conventional large volume samples. These results show that radiation-damping effects in microcoil probe is much more pronounced than in 5 mm probes, and that it is critically important to optimize NMR experiments to minimize these effects. As microcoil probes provide better control of the bulk magnetization, with good RF and B0 inhomogeneity, in addition to negligible dipolar field effects due to nearly spherical sample volumes, these probes can be used exclusively to study the complex behavior of radiation damping.     

High Q calcium titanate cylindrical dielectric resonators for magnetic resonance microimaging

At high magnetic fields radiation losses, wavelength effects, self-resonance, and the high resistance of typical components all contribute to increased losses in conventional RF coil designs. High permittivity ceramic dielectric resonators create strong uniform magnetic fields in a compact structure at high frequencies and can potentially solve some of the challenges of high field coil design. In this study an NMR probe was constructed for operation at 600 MHz (14.1 T) using an inductively fed CaTiO₃ (relative permittivity of 156) cylindrical hollow bore dielectric resonator. The design has an unmatched Q value greater than 2000, and the electric field is largely confined to the dielectric itself, with near zero values in the hollow bore which accommodates the sample. Experimental and simulation mapping of the RF field show good agreement, with the ceramic resonator giving a pulse width approximately 25% less than a loop gap resonator of similar inner dimensions. High resolution images, with voxel dimensions less than 50 μm³, have been acquired from fixed zebrafish samples, showing excellent delineation of several fine structures.     

Magnetically aligned phospholipid bilayers in weak magnetic fields: optimization,mechanism, and advantages for X-band EPR studies

Our lab is developing a spin-labeled EPR spectroscopic technique complementary to solid-state NMR studies to study the structure, orientation, and dynamics of uniaxially aligned integral membrane proteins inserted into magnetically aligned discotic phospholipid bilayers, or bicelles. The focus of this study is to optimize and understand the mechanisms involved in the magnetic alignment process of bicelle disks in weak magnetic fields. Developing experimental conditions for optimized magnetic alignment of bicelles in low magnetic fields may prove useful to study the dynamics of membrane proteins and its interactions with lipids, drugs, steroids, signaling events, other proteins, etc. In weak magnetic fields, the magnetic alignment of Tm(3+)-doped bicelle disks was thermodynamically and kinetically very sensitive to experimental conditions. Tm(3+)-doped bicelles were magnetically aligned using the following optimized procedure: the temperature was slowly raised at a rate of 1.9K/min from an initial temperature being between 298 and 307K to a final temperature of 318K in the presence of a static magnetic field of 6300G. The spin probe 3beta-doxyl-5alpha-cholestane (cholestane) was inserted into the bicelle disks and utilized to monitor bicelle alignment by analyzing the anisotropic hyperfine splitting for the corresponding EPR spectra. The phases of the bicelles were determined using solid-state 2H NMR spectroscopy and compared with the corresponding EPR spectra. Macroscopic alignment commenced in the liquid crystalline nematic phase (307K), continued to increase upon slowly raising the temperature, and was well-aligned in the liquid crystalline lamellar smectic phase (318K).     

The effects of cholesterol on magnetically aligned phospholipid bilayers: a solid-state NMR and EPR spectroscopy study

This paper presents the first time that both solid-state NMR spectroscopy and EPR spectroscopy are used to study the effects of cholesterol on magnetically aligned phospholipid bilayers (bicelles). Solid-state deuterium NMR spectroscopy was carried out using both chain perdeuterated 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC-d(54)) and a partially deuterated beta-[2,2,3,4,4,6-(2)H(6)]cholesterol (cholesterol-d(6)). Also, EPR spectroscopy was carried out utilizing a 3 beta-doxyl-5 alpha-cholestane (cholestane) spin probe incorporated into magnetically aligned bilayers to provide a more complete picture about the ordering and dynamics of the phospholipid and cholesterol molecules in the bicelle membrane system. The results demonstrate that cholesterol was successfully incorporated into the phospholipid bilayers. The molecular order parameters extracted directly from the (2)H NMR spectra of both DMPC-d(54) and cholesterol-d(6) were compared to that from the EPR study of cholestane. The order parameters indicate that the sterol was motionally restricted, and that the DMPC had high order and low motion for the hydrocarbon segments close to the head groups of the phospholipids and less order and more rapid motion toward the terminal methyl groups. Both methods clearly indicate an overall increase in the degree of ordering of the molecules in the presence of cholesterol and a decrease in the degree of ordering at higher temperatures. However, EPR spectroscopy and (2)H NMR spectroscopy exhibit different degrees of sensitivity in detecting the phospholipid molecular motions in the membrane. Finally, cholesterol increases the minimum alignment temperature necessary to magnetically align the phospholipid bilayers.