The radial mode matching (RMM) method has been used to calculate accurately the microwave field distribution of the TE0 1 1 mode in a spherical EPR cavity containing a linear aqueous sample, in order to understand in detail the factors affecting sensitivity in EPR measurements at X band. Specific details of the experiment were included in the calculations, such as the cavity geometry, the presence of a quartz dewar, the size of the aqueous sample, and the sample’s dielectric properties. From the field distribution, several key physical parameters were calculated, including cavity Q, filling factor, mean microwave magnetic field at the sample, and cavity efficiency parameter Λ. The dependence of EPR signal intensity on sample diameter for a cylindrical aqueous sample was calculated and measured experimentally for non-saturated and half-saturated samples. The optimal aqueous sample diameter was determined for both cases. The impact of sample temperature, conductivity, and cavity Q on sensitivity of EPR is discussed.
EPR; Water; Sensitivity; Cavity; Field distribution
To evaluate the efficiency and feasibility of intermolecular multiple quantum coherence (iMQC) magnetic resonance (MR) imaging for single cell detection, we obtained intermolecular double quantum coherence (iDQC) and conventional gradient echo (GE) images of macrophage cells labeled by contrast agents in gel. The iDQC images obtained with echo-planar readout visualized the labeled cells effectively and with a higher contrast than seen in conventional GE images, especially at low planar resolutions and with thick slices. This implies that iDQC imaging with contrast agents could be a good alternative to conventional MR imaging for detecting labeled single cells or cell tracking under favorable conditions.
We described a versatile spectrometer designed for the study of dynamic nuclear polarization (DNP) at low temperatures and high fields. The instrument functions both as an NMR spectrometer operating at 212 MHz (1H frequency) with DNP capabilities, and as a pulsed-EPR operating at 140 GHz. A coiled TE011 resonator acts as both an NMR coil and microwave resonator, and a double balanced (1H, 13C) radio frequency circuit greatly stabilizes the NMR performance. A new 140 GHz microwave bridge has also been developed, which utilizes a four-phase network and ELDOR channel at 8.75 GHz, that is then multiplied and mixed to obtain 140 GHz microwave pulses with an output power of 120 mW. Nutation frequencies obtained are as follows: 6 MHz on S = ½ electron spins, 100 kHz on 1H, and 50 kHz on 13C. We demonstrate basic EPR, ELDOR, ENDOR, and DNP experiments here. Our solid effect DNP results demonstrate an enhancement of 144 and sensitivity gain of 310 using OX063 trityl at 80 K and an enhancement of 157 and maximum sensitivity gain of 234 using Gd-DOTA at 20 K, which is significantly better performance than previously reported at high fields (>3 T).
Recent advances in electron paramagnetic resonance (EPR) include capabilities for applications to areas as diverse as archeology, beer shelf life, biological structure, dosimetry, in vivo imaging, molecular magnets, and quantum computing. Enabling technologies include multifrequency continuous wave, pulsed, and rapid scan EPR. Interpretation is enhanced by increasingly powerful computational models.
The rapidly-changing magnetic field of sinusoidal rapid scans creates background signals that are dominated by oscillations at the scan frequency. The background oscillations can be removed without acquiring off-resonance data. For data acquired in quadrature, up-field and down-field scan signals can be separated in the frequency domain. For each scan direction, the background oscillation can be calculated by fitting to the half cycle that does not contain the EPR signal. The extrapolated fit function is then subtracted from the half cycle that contains the EPR signal. By zeroing the array for the half cycles that do not contain the EPR signal, the signal-to-noise is improved and the data are corrected for non-orthogonality of the quadrature channels.
Improved understanding of the entrapment, transport, and release of drugs and small molecules within vesicles is important for drug delivery. Most methods rely on contrast agents or probe molecules; here, we propose a new MRI method to detect signal from water spins with restricted diffusion. This method is based on intermolecular double quantum coherences (iDQCs), which can probe the restricted diffusion characteristics at well-defined and tunable microscopic distance scales. By using an exceedingly short (and previously inaccessible) distance, the iDQC signal arises only from restricted diffusion spins and thereby provides a mechanism to directly image vesicle entrapment, transport, and release. Using uni- and multi-lamellar liposomes and polymersomes, we show how the composition, lamellar structure, vesicle size, and concentration affects the iDQC signal between coupled water spins at very short separation distances. The iDQC signal correlates well with conventional diffusion MRI and a proposed biexponential (multicompartmental) diffusion model. Finally, the iDQC signal was used to monitor dynamic changes in the lamellar structure as temperature-sensitive liposomes released their contents. These short distance iDQCs can probe the amount and diffusion of water entrapped in vesicles, which may be useful to further understand vesicle properties in materials science and drug delivery applications.
Intermolecular multiple quantum coherences; liposomes; restricted diffusion
We demonstrate 1H amide resonance line widths <300 Hz in 1H/15N heteronuclear correlation (HETCOR) spectra of membrane proteins in aligned phospholipid bilayers. This represents a substantial improvement over typically observed line widths of ~1 kHz. Furthermore, in a proton detected local field (PDLF) version of the experiment that measures heteronuclear dipolar couplings, line widths <130 Hz are observed. This dramatic line narrowing of 1H amide resonances enables many more individual signals to be resolved and assigned from uniformly 15N labeled membrane proteins in phospholipid bilayers under physiological conditions of temperature and pH. Finding that the decrease in line widths occurs only for membrane proteins that undergo fast rotational diffusion around the bilayer normal, but not immobile molecules, such as peptide single crystals, identifies a potential new direction for pulse sequence development that includes overall molecular dynamics in their design.
Solid-state NMR; Membrane proteins; Phospholipid bilayers; Line-narrowing
Initial steps in the development of a suite of triple-resonance 1H/13C/15N solid-state NMR experiments applicable to aligned samples of 13C and 15N labeled proteins are described. The experiments take advantage of the opportunities for 13C detection without the need for homonuclear 13C/13C decoupling presented by samples with two different patterns of isotopic labeling. In one type of sample, the proteins are ~20% randomly labeled with 13C in all backbone and side chain carbon sites and ~100% uniformly 15N labeled in all nitrogen sites; in the second type of sample, the peptides and proteins are 13C labeled at only the α-carbon and 15N labeled at the amide nitrogen of a few residues. The requirement for homonuclear 13C/13C decoupling while detecting 13C signals is avoided in the first case because of the low probability of any two 13C nuclei being bonded to each other; in the second case, the labeled 13Cα sites are separated by at least three bonds in the polypeptide chain. The experiments enable the measurement of the 13C chemical shift and 1H–13C and 15N–13C heteronuclear dipolar coupling frequencies associated with the 13Cα and 13C′ backbone sites, which provide orientation constraints complementary to those derived from the 15N labeled amide backbone sites. 13C/13C spin-exchange experiments identify proximate carbon sites. The ability to measure 13C–15N dipolar coupling frequencies and correlate 13C and 15N resonances provides a mechanism for making backbone resonance assignments. Three-dimensional combinations of these experiments ensure that the resolution, assignment, and measurement of orientationally dependent frequencies can be extended to larger proteins. Moreover, measurements of the 13C chemical shift and 1H–13C heteronuclear dipolar coupling frequencies for nearly all side chain sites enable the complete three-dimensional structures of proteins to be determined with this approach.
PISEMA; HETCOR; Isotopic labeling; Dipolar coupling; Oriented proteins
Statistical analysis reveals that, given a fixed acquisition time, linewidth (and thus pO2) can be more precisely determined from multiple scans with different modulation amplitudes and sweep widths than from a single scan. For a Lorentzian lineshape and an unknown but spatially uniform modulation amplitude, the analysis suggests the use of two scans, each occupying half of the total acquisition time. We term this mode of scanning as dual-scan acquisition. For unknown linewidths in a range [Γmin, Γmax], practical guidelines are provided for selecting the modulation amplitude and sweep width for each dual-scan component. Following these guidelines can allow for a 3–4 times reduction in spectroscopic acquisition time versus an optimized single-scan, without requiring hardware modifications. Findings are experimentally verified using L-band spectroscopy with an oxygen-sensitive particulate probe.
EPR; Spectroscopy; Oximetry; Overmodulation; Cramér-Rao lower bound; LiNc-BuO
Modern solid-state NMR methods can acquire high-resolution protein spectra for structure determination. However, these methods use rapid sample spinning and intense decoupling fields that can heat and denature the protein being studied. Here we present a strategy to avoid destroying valuable samples. We advocate first creating a sacrificial sample, which contains unlabeled protein (or no protein) in buffer conditions similar to the intended sample. This sample is then doped with the chemical shift thermometer Sm2Sn2O7. We introduce a pulse scheme called TCUP (for Temperature Calibration Under Pulseload) that can characterize the heating of this sacrificial sample rapidly, under a variety of experimental conditions, and with high temporal resolution. Sample heating is discussed with respect to different instrumental variables such as spinning speed, decoupling strength and duration, and cooling gas flow rate. The effects of different sample preparation variables are also discussed, including ionic strength, the inclusion of cryoprotectants, and the physical state of the sample (i.e. liquid, solid, or slurry). Lastly, we discuss probe detuning as a measure of sample thawing that does not require retuning the probe or using chemical shift thermometer compounds. Use of detuning tests and chemical shift thermometers with representative sample conditions makes it possible to maximize the efficiency of the NMR experiment while retaining a functional sample.
Temperature calibration; TCUP; sample preservation; ionic strength; cryoprotectant; sacrificial sample; chemical shift thermometer
We demonstrate the determination of quantitative rates of molecular reorientation in the solid state with rotating frame (R1ρ) relaxation measurements. Reorientation of the carbon chemical shift anisotropy (CSA) tensor was used to probe site-specific conformational exchange in a model system, d6-dimethyl sulfone (d6-DMS). The CSA as a probe of exchange has the advantage that it can still be utilized when there is no dipolar mechanism (i.e. no protons attached to the site of interest). Other works have presented R1ρ measurements as a general indicator of dynamics, but this study extracts quantitative rates of molecular reorientation from the R1ρ values. Some challenges of this technique include precise knowledge of sample temperature and determining the R20 contribution to the observed relaxation rate from interactions other than molecular reorientation, such as residual dipolar couplings or fast timescale dynamics; determination of this term is necessary in order to quantify the exchange rate due to covariance between the 2 terms. Low-temperature experiments measured an R20 value of 1.8 ± 0.2 s−1 Allowing for an additional relaxation term (R20), which was modeled as both temperature-dependent and temperature-independent, rates of molecular reorientation were extracted from field strength-dependent R1ρ measurements at 4 different temperatures and the activation energy was determined from these exchange rates. The activation energies determined were 74.7 ± 4.3 kJ/mol and 71.7 ± 2.9 kJ/mol for the temperature-independent and temperature-dependent R20 models respectively, in excellent agreement with literature values. The results of this study suggest important methodological considerations for the application of the method to more complicated systems such as proteins, such as the importance of deuterating samples and the need to make assumptions regarding the R20 contribution to relaxation.
Rotating frame relaxation; chemical shift anisotropy; dynamics; molecular reorientation; dimethyl sulfone
A solid state NMR experiment is introduced for probing motions on the millisecond time scale, based on dephasing and refocusing 1H-13C or 1H-15N dipolar couplings. The method is related to the previously described Centerband-Only Detection of Exchange or CODEX experiment. The use of an R-type dipolar recoupling sequence takes advantage of the strong 1H-13C or 1H-15N dipolar coupling, while suppressing the effect of 1H-1H homonuclear coupling. This approach paves the way to detect both the correlation time and reorientational angle of the dynamics in fully protonated samples. The performance of this pulse sequence is demonstrated using imidazole methyl sulfonate.
Millisecond Dynamics; Solid state NMR; CODEX; R-CODEX; Reorientation angle; R1871; 1H homonuclear coupling; imidazole
This study aims to (1) undertake and analyse 1D and 2D MR correlation spectroscopy from human soleus muscle in vivo at 7T, and (2) determine T1 and T2 relaxation time constants at 7T field strength due to their importance in sequence design and spectral quantitation.
Six healthy, male volunteers were consented and scanned on a 7T whole-body scanner (Siemens AG, Erlangen, Germany). Experiments were undertaken using a 28 cm diameter detunable birdcage coil for signal excitation and an 8.5 cm diameter surface coil for signal reception. The relaxation time constants, T1 and T2 were recorded using a STEAM sequence, using the ‘progressive saturation’ method for the T1 and multiple echo times for T2. The 2D L-Correlated SpectroscopY (L-COSY) method was employed with 64 increments (0.4 ms increment size) and eight averages per scan, with a total time of 17 min.
T1 and T2 values for the metabolites of interest were determined. The L-COSY spectra obtained from the soleus muscle provided information on lipid content and chemical structure not available, in vivo, at lower field strengths. All molecular fragments within multiple lipid compartments were chemically shifted by 0.20–0.26 ppm at this field strength. 1D and 2D L-COSY spectra were assigned and proton connectivities were confirmed with the 2D method.
In vivo 1D and 2D spectroscopic examination of muscle can be successfully recorded at 7T and is now available to assess lipid alterations as well as other metabolites present with disease. T1 and T2 values were also determined in soleus muscle of male healthy volunteers.
MR spectroscopy; L-COSY; 7 Tesla; Soleus muscle; In vivo; Human; Intra-myocellular and extramyocellular; lipids
Hyperpolarized 13C offers high signal-to-noise ratios for imaging metabolic activity in vivo, but care must be taken when designing pulse sequences because the magnetization cannot be recovered once it has decayed. It has a short lifetime, on the order of minutes, and gets used up by each RF excitation. In this paper, we present a new dynamic chemical-shift imaging method that uses specialized RF pulses designed to maintain most of the hyperpolarized substrate while providing adequate SNR for the metabolic products. These are multiband, variable flip angle, spectral-spatial RF pulses that use spectral selectivity to minimally excite the injected prepolarized 13C-pyruvate substrate. The metabolic products of lactate and alanine are excited with a larger flip angle to increase SNR. This excitation was followed by an RF amplitude insensitive double spin-echo and an echo-planar flyback spectral-spatial readout gradient. In vivo results in rats and mice are presented showing improvements over constant flip angle RF pulses. The metabolic products are observable for a longer window because the low pyruvate flip angle preserves magnetization, allowing for improved observation of spatially varying metabolic reactions.
Spectral-spatial RF pulses; multiband RF pulses; hyperpolarization; dynamic MRSI; metabolic imaging
NMR structure determination is frequently hindered by an insufficient amount of distance information for determining the correct fold of the protein in its early stages. In response we introduce a simple and general structure-based metric that can be used to incorporate NMR-based restraints on protein surface accessibility. This metric is inversely proportional to the sum of the inverse square distances to neighboring heavy atoms. We demonstrate the use of this restraint using a dataset from the water to protein magnetization transfer experiment on the protein Bax and the solvent paramagnetic relaxation enhancement experiment on the protein ubiquitin and Qua1 homodimer. The calculated solvent accessibility values using the new empirical function are well correlated with the experimental data. By incorporating an associated energy term into Xplor-NIH, we show that structure calculation with a limited number of additional experimental restraints, improves both the precision and accuracy of the resulting structures. This new empirical energy term will have general applicability to other types of solvent accessibility data.
protein structure; structure refinement; solvent accessibility; solvent PRE
We describe the design and implementation of a novel tunable 250 GHz gyrotron oscillator with >10 W output power over most of a 3 GHz band and >35 W peak power. The tuning bandwidth and power are sufficient to generate a >1 MHz nutation frequency across the entire nitroxide EPR lineshape for cross effect DNP, as well as to excite solid effect transitions utilizing other radicals, without the need for sweeping the NMR magnetic field. Substantially improved tunability is achieved by implementing a long (23 mm) interaction cavity that can excite higher order axial modes by changing either the magnetic field of the gyrotron or the cathode potential. This interaction cavity excites the rotating TE5,2,q mode, and an internal mode converter outputs a high-quality microwave beam with >94% Gaussian content. The gyrotron was integrated into a DNP spectrometer, resulting in a measured DNP enhancement of 54 on the membrane protein bacteriorhodopsin.
Dynamic Nuclear Polarization; Instrumentation; Gyrotron
The influence of g tensor anisotropy on spin dynamics of paramagnetic centers having real or effective spin of 1/2 is studied. The g anisotropy affects both the excitation and the detection of EPR signals, producing noticeable differences between conventional continuous-wave (cw) EPR and pulsed EPR spectra. The magnitudes and directions of the spin and magnetic moment vectors are generally not proportional to each other, but are related to each other through the g tensor. The equilibrium magnetic moment direction is generally parallel to neither the magnetic field nor the spin quantization axis due to the g anisotropy. After excitation with short microwave pulses, the spin vector precesses around its quantization axis, in a plane that is generally not perpendicular to the applied magnetic field. Paradoxically, the magnetic moment vector precesses around its equilibrium direction in a plane exactly perpendicular to the external magnetic field. In the general case, the oscillating part of the magnetic moment is elliptically polarized and the direction of precession is determined by the sign of the g tensor determinant (g tensor signature). Conventional pulsed and cw EPR spectrometers do not allow determination of the g tensor signature or the ellipticity of the magnetic moment trajectory. It is generally impossible to set a uniform spin turning angle for simple pulses in an unoriented or ‘powder’ sample when g tensor anisotropy is significant.
Phase-contrast (PC) magnetic resonance imaging (MRI) with hyperpolarized 3He is potentially useful for developing and testing patient-specific models of pulmonary airflow. One challenge, however, is that PC-MRI provides apparent values of local 3He velocity that not only depend on actual airflow but also on gas diffusion. This not only blurs laminar flow patterns in narrow airways but also introduces anomalous airflow structure that reflects gas-wall interactions. Here, both effects are predicted in a live rat using computational fluid dynamics (CFD), and for the first time, simulated patterns of apparent 3He gas velocity are compared with in-vivo PC-MRI. Results show 1) that correlations (R2) between measured and simulated airflow patterns increase from 0.23 to 0.79 simply by accounting for apparent 3He transport, and 2) that remaining differences are mainly due to uncertain airway segmentation and partial volume effects stemming from relatively coarse MRI resolution. Higher-fidelity testing of pulmonary airflow predictions should therefore be possible with future imaging improvements.
3He MRI; CFD; Pulmonary Airflow; Convection-Diffusion
We demonstrate the feasibility of one-dimensional and two-dimensional 1H–13C double resonance NMR experiments with dynamic nuclear polarization (DNP) at 9.4 T and temperatures below 20 K, including both 1H–13C cross-polarization and 1H decoupling, and discuss the effects of polarizing agent type, polarizing agent concentration, temperature, and solvent deuteration. We describe a two-channel low-temperature DNP/NMR probe, capable of carrying the radio-frequency power load required for 1H–13C cross-polarization and high-power proton decoupling. Experiments at 8 K and 16 K reveal a significant T2 relaxation of 13C, induced by electron spin flips. Carr–Purcell experiments and numerical simulations of Carr–Purcell dephasing curves allow us to determine the effective correlation time of electron flips under our experimental conditions. The dependence of the DNP signal enhancement on electron spin concentration shows a maximum near 80 mM. Although no significant difference in the absolute DNP enhancements for triradical (DOTOPA-TEMPO) and biradical (TOTAPOL) dopants was found, the triradical produced greater DNP build-up rates, which are advantageous for DNP experiments. Additionally the feasibility of structural measurements on 13C-labeled biomolecules was demonstrated with a two-dimensional 13C–13C exchange spectrum of selectively 13C-labeled β-amyloid fibrils.
Dynamic nuclear polarization; Double resonance probe; Polarizing agent; Transverse relaxation
Several techniques currently exist for measuring tissue oxygen; however technical difficulties have limited their usefulness and general application. We report a recently developed electron paramagnetic resonance (EPR) oximetry approach with multiple probe implantable resonators (IRs) that allow repeated measurements of oxygen in tissue at depths of greater than 10 mm.
The EPR signal to noise (S/N) ratio of two probe IRs was compared with that of LiPc deposits. The feasibility of intracranial tissue pO2 measurements by EPR oximetry using IRs was tested in normal rats and rats bearing intracerebral F98 tumors. The dynamic changes in the tissue pO2 were assessed during repeated hyperoxia with carbogen breathing.
A 6–10 times increase in the S/N ratio was observed with IRs as compared to LiPc deposits. The mean brain pO2 of normal rats was stable and increased significantly during carbogen inhalation in experiments repeated for 3 months. The pO2 of F98 glioma declined gradually, while the pO2 of contralateral brain essentially remained the same. Although a significant increase in the glioma pO2 was observed during carbogen inhalation, this effect declined in experiments repeated over days.
EPR oximetry with IRs provides a significant increase in S/N ratio. The ability to repeatedly assess orthotopic glioma pO2 is likely to play a vital role in understanding the dynamics of tissue pO2 during tumor growth and therapies designed to modulate tumor hypoxia. This information could then be used to optimize chemoradiation by scheduling treatments at times of increased glioma oxygenation.
Carbogen; EPR oximetry; F98 glioma; Implantable resonator; Intracranial tumor; pO2
This work describes our first efforts to implement SWIFT (SWeep Imaging with Fourier Transformation) in continuous mode for imaging and spectroscopy. We connected a standard quadrature hybrid with a quad coil and acquired NMR signal during continuous radiofrequency excitation. We utilized a chirped radiofrequency pulse to minimize the instantaneous radiofrequency field during excitation of the spin system for the target flip angle and bandwidth. Due to the complete absence of “dead time”, continuous SWIFT has the potential to extend applications of MRI and spectroscopy in studies of spin systems having extremely fast relaxation or broad chemical shift distributions beyond the range of existing MRI sequences.
MRI; spectroscopy; continuous wave; sweep imaging; SWIFT
Using the DUMAS (Dual acquisition Magic Angle Spinning) solid-state NMR approach, we created new pulse schemes that enable the simultaneous acquisition of three dimensional (3D) experiments on uniformly 13C, 15N labeled proteins. These new experiments exploit the simultaneous cross-polarization (SIM-CP) from 1H to 13C and 15N to acquire two 3D experiments simultaneously. This is made possible by bidirectional polarization transfer between 13C and 15N and the long living 15N z-polarization in solid state NMR. To demonstrate the power of this approach, four 3D pulse sequences (NCACX, CANCO, NCOCX, CON(CA)CX,) are combined into two pulse sequences (3D DUMAS-NCACX-CANCO, 3D DUMAS-NCOCX-CON(CA)CX) that allow simultaneous acquisition of these experiments, reducing the experimental time by approximately half. Importantly, the 3D DUMAS-NCACX-CANCO experiment alone makes it possible to obtain the majority of the backbone sequential resonance assignments for microcrystalline U-13C,15N ubiquitin. The DUMAS approach is general and applicable to many 3D experiments, nearly doubling the performance of NMR spectrometers.
Dual Acquisition Magic Angle Spinning; SIM-CP; DUMAS; Solid-state NMR; proteins. 3D DUMAS-NCACX-CANCO; 3D DUMAS-NCOCX-CON(CA)CX; microcrystalline ubiquitin
In this work we describe a large volume 340 mL 1H-X magnetic resonance (MR) probe for studies of hyperpolarized compounds at 0.0475 T. 1H/13C and 1H/15N probe configurations are demonstrated with the potential for extension to 1H/129Xe. The primary applications of this probe are preparation and quality assurance of 13C and 15N hyperpolarized contrast agents using PASADENA (parahydrogen and synthesis allow dramatically enhanced nuclear alignment) and other parahydrogen-based methods of hyperpolarization. The probe is efficient and permits 62 μs 13C excitation pulses at 5.3 Watts, making it suitable for portable operation. The sensitivity and detection limits of this probe, tuned to 13C, are compared with a commercial radio frequency (RF) coil operating at 4.7 T. We demonstrate that low field MR of hyperpolarized contrast agents could be as sensitive as conventional high field detection and outline potential improvements and optimization of the probe design for preclinical in vivo MRI. PASADENA application of this low-power probe is exemplified with 13C hyperpolarized 2-hydroxyethyl propionate-1-13C,2,3,3-d3.
hyperpolarization; multi-nuclear; magnetic resonance probe; parahydrogen; 13C; 15N; low field
NMR relaxation methods probe biomolecular motions over a wide range of timescales. In particular, the rotating frame spin-lock R1ρ and Carr–Purcell–Meiboom–Gill (CPMG) R2 experiments are commonly used to characterize μs to ms dynamics, which play a critical role in enzyme folding and catalysis. In an effort to complement these approaches, we introduced the Heteronuclear Adiabatic Relaxation Dispersion (HARD) method, where dispersion in rotating frame relaxation rate constants (longitudinal R1ρ and transverse R2ρ) is created by modulating the shape and duration of adiabatic full passage (AFP) pulses. Previously, we showed the ability of the HARD method to detect chemical exchange dynamics in the fast exchange regime (kex ~ 104–105 s−1). In this article, we show the sensitivity of the HARD method to slower exchange processes by measuring R1ρ and R2ρ relaxation rates for two soluble proteins (ubiquitin and 10C RNA ligase). One advantage of the HARD method is its nominal dependence on the applied radio frequency field, which can be leveraged to modulate the dispersion in the relaxation rate constants. In addition, we also include product operator simulations to define the dynamic range of adiabatic R1ρ and R2ρ that is valid under all exchange regimes. We conclude from both experimental observations and simulations that this method is complementary to CPMG-based and rotating frame spin-lock R1ρ experiments to probe conformational exchange dynamics for biomolecules. Finally, this approach is germane to several NMR-active nuclei, where relaxation rates are frequency-offset independent.
Adiabatic relaxation dispersion; Rotating frame relaxation; NMR; Proteins
The use of susceptibility matching to minimize spectral distortion of biphasic samples layered in a standard 5 mm NMR tube is described. The approach uses magic angle spinning (MAS) to first extract chemical shift differences by suppressing bulk magnetization. Then, using biphasic coaxial samples, magnetic susceptibilities are matched by titration with a paramagnetic salt. The matched phases are then layered in a standard NMR tube where they can be shimmed and examined. Line widths of two distinct spectral lines, selected to characterize homogeneity in each phase, are simultaneously optimized. Two-dimensional distortion-free, slice-resolved spectra of an octanol/water system illustrate the method. These data are obtained using a 2D stepped-gradient pulse sequence devised for this application. Advantages of this sequence over slice-selective methods are that acquisition efficiency is increased and processing requires only conventional software.
NMR; MAS; magic angle spinning; susceptibility; biphasic; shimming; demagnetization field; octanol; bulk magnetism; chemical shift