Biomolecular solid-state NMR spectroscopy at 1200 MHz: the gain in resolution.

Progress in NMR in general and in biomolecular applications in particular is driven by increasing magnetic-field strengths leading to improved resolution and sensitivity of the NMR spectra. Recently, persistent superconducting magnets at a magnetic field strength (magnetic induction) of 28.2 T corresponding to 1200 MHz proton resonance frequency became commercially available. We present here a collection of high-field NMR spectra of a variety of proteins, including molecular machines, membrane proteins, viral capsids, fibrils and large molecular assemblies. We show this large panel in order to provide an overview over a range of representative systems under study, rather than a single best performing model system. We discuss both carbon-13 and proton-detected experiments, and show that in 13C spectra substantially higher numbers of peaks can be resolved compared to 850 MHz while for 1H spectra the most impressive increase in resolution is observed for aliphatic side-chain resonances.

A spin-thermodynamic approach to characterize spin dynamics in TEMPO-based samples for dissolution DNP at 7 T field.

The spin dynamics of dissolution DNP samples consisting of 4.5 M [13C]urea in a mixture of (1/1)Vol glycerol/water using 4-Oxo-TEMPO as a radical was investigated. We analyzed the DNP dynamics as function of radical concentration at 7 T and 3.4 T static magnetic field as well as function of deuteration of the solvent matrix at the high field. The spin dynamics could be reproduced in all cases, at least qualitatively, by a thermodynamic model based on spin temperatures of the nuclear Zeeman baths and an electron non-Zeeman (dipolar) bath. We find, however, that at high field (7 T) and low radical concentrations (25 mM) the nuclear spins do not reach the same spin temperature indicating a weak coupling of the two baths. At higher radical concentrations, as well as for all radical concentrations at low field (3.4 T), the two nuclear Zeeman baths reach the same spin temperature within experimental errors. Additionally, the spin system was prepared with different initial conditions. For these cases, the thermodynamic model was able to predict the time evolution of the system well. While the DNP profiles do not give clear indications to a specific polarization transfer mechanism, at high field (7 T) increased coupling is seen. The EPR line shapes cannot clarify this in absence of ELDOR type experiments, nevertheless DNP profiles and dynamics under frequency-modulated microwave irradiation illustrate the expected increase in coupling between electrons with increasing radical concentration.

Microscale 3D imaging by magnetic resonance force microscopy using full-volume Fourier- and Hadamard-encoding.

Three-dimensional spatially resolved full-volume imaging by magnetic resonance force microscopy at room temperature is described. Spatial resolution in z-dimension is achieved by using the magnetic-field gradient of a ferromagnetic particle that is also used for the force detection of the magnetic resonance. The gradient of the radiofrequency pulses generated by two separate wire-bonded microcoils is used for spatial resolution in x- and y-dimension. To enhance the sensitivity of our measurement Hadamard- and Fourier-encoding schemes are applied due to their multiplex effect. Measurements were taken on a patterned (NH4)2SO4 crystal sample. From the calculated magnetic field distributions, a 3D image was reconstructed with a voxel volume of about 5 µm3 (1.2 µm × 3.0 µm × 1.4 µm in x-, y- and z-dimension).

Detection of liquids by magnetic resonance force microscopy in the gradient-on-cantilever geometry.

We demonstrate the detection of picoliter amounts of water and triethylenetetramine by a magnetic-resonance-force-microscopy (MRFM) setup operated in the gradient-on-cantilever geometry at room temperature. A magnetic field gradient is produced by a ferromagnetic SmCo particle glued to the tip of a micromechanical resonator (cantilever). The liquids are enclosed in a micro-capillary to protect them from the high vacuum environment needed for sensitive detection. We describe simple spectroscopic experiments as proton T1 - relaxation, Rabi nutation curves and Hahn-echo measurements.

CONFINE-MAS: a magic-angle spinning NMR probe that confines the sample in case of a rotor explosion.

Magic-angle spinning (MAS) is mandatory in solid-state NMR experiments to achieve resolved spectra. In rare cases, instabilities in the rotation or damage of either the rotor or the rotor cap can lead to a so called "rotor crash" involving a disintegration of the sample container and possibly the release of an aerosol or of dust. We present a modified design of a 3.2 mm probe with a confining chamber which in case of a rotor crash prevents the release of aerosols and possibly hazardous materials. 1D and 2D NMR experiments show that such a hazardous material-confining MAS probe ("CONFINE-MAS" probe) has a similar sensitivity compared to a standard probe and performs equally well in terms of spinning stability. We illustrate the CONFINE-MAS probe properties and performance by application to a fungal amyloid.

Dissolution DNP using trityl radicals at 7 T field.

Dissolution DNP has become an important method to generate highly polarized substrates such as pyruvic acid for in vivo imaging and localized spectroscopy. In a quest to further increase the polarization levels, which is important for in vivo MRI employing 13C detection, we describe the design and implementation of a new DNP polarizer that is suitable for dissolution operation at 7 T static magnetic field and a temperature of 1.4 K. We describe all important sample preparation steps and experimental details necessary to optimize trityl based samples for use in our polarizer at this higher field. In [1-13C]-pyruvic acid polarization levels of about 56% are achieved, compared to typical polarization levels of about 35-45% at a standard field of 3.4 T. At the same time, the polarization build-up time increases significantly from about 670 s at 3.4 T to around 1300-1900 s at 7 T, depending on the trityl derivate used. We also investigate the effect of adding trace amounts of Gd3+ to the samples. While one trityl compound does not exhibit any benefit, the other profits significantly, boosting achievable polarization by 6%.

Line-Broadening in Low-Temperature Solid-State NMR Spectra of Fibrils.

The temperature-dependent resonance-line broadening of HET-s(218-289) in its amyloid form is investigated in the range between 110 K and 280 K. Significant differences are observed between residues in the structured hydrophobic triangular core, which are broadened the least and can be detected down to 100 K, and in the solvent-exposed parts, which are broadened the most and often disappear from the observed spectrum around 200 K. Below the freezing of the bulk water, around 273 K, the protein fibrils are still surrounded by a layer of mobile water whose thickness decreases with temperature, leading to drying out of the fibrils.

Hexagonal ice in pure water and biological NMR samples.

Ice, in addition to "liquid" water and protein, is an important component of protein samples for NMR spectroscopy at subfreezing temperatures but it has rarely been observed spectroscopically in this context. We characterize its spectroscopic behavior in the temperature range from 100 to 273 K, and find that it behaves like pure water ice. The interference of magic-angle spinning (MAS) as well as rf multiple-pulse sequences with Bjerrum-defect motion greatly influences the ice spectra.

Protein resonance assignment at MAS frequencies approaching 100 kHz: a quantitative comparison of J-coupling and dipolar-coupling-based transfer methods.

We discuss the optimum experimental conditions to obtain assignment spectra for solid proteins at magic-angle spinning (MAS) frequencies around 100 kHz. We present a systematic examination of the MAS dependence of the amide proton T 2' times and a site-specific comparison of T 2' at 93 kHz versus 60 kHz MAS frequency. A quantitative analysis of transfer efficiencies of building blocks, as they are used for typical 3D experiments, was performed. To do this, we compared dipolar-coupling and J-coupling based transfer steps. The building blocks were then combined into 3D experiments for sequential resonance assignment, where we evaluated signal-to-noise ratio and information content of the different 3D spectra in order to identify the best assignment strategy. Based on this comparison, six experiments were selected to optimally assign the model protein ubiquitin, solely using spectra acquired at 93 kHz MAS. Within 3 days of instrument time, the required spectra were recorded from which the backbone resonances have been assigned to over 96%.

Cross-polarization for dissolution dynamic nuclear polarization.

Dynamic nuclear polarization (DNP) in combination with subsequent dissolution of the sample allows the detection of low-γ nuclei in the solution state with a signal gain of up to tens of thousand times compared to experiments starting from Boltzmann conditions. The long polarization build-up times of typically more than one hour are a drawback of this technique. The combination of dissolution DNP with cross-polarization (CP) in the solid state was shown to have the potential to overcome this disadvantage. In this article we discuss the cross-polarization step under dissolution DNP conditions in more detail. We show that adiabatic half-passage pulses allow us to enhance the CP efficiency in power-limited DNP probes. As a low-power alternative to Hartmann-Hahn CP we also demonstrate the applicability of frequency-swept de- and re-magnetization pulses for polarization transfer via dipolar order. We investigate the implications and restrictions of the common solid-state DNP mechanisms to the DNP-CP technique and apply a spin-thermodynamic model based on the thermal-mixing mechanism. The model allows us to investigate the dynamics of the polarization levels in a system with two nuclear Zeeman reservoirs and explains the enhanced DNP efficiency upon solvent deuteration within a spin-thermodynamic picture.

A sedimented sample of a 59†kDa dodecameric helicase yields high-resolution solid-state NMR spectra.

Crystal clear: Preparing solid-state NMR samples that yield high-resolution spectra displaying high sensitivity is time-consuming and complicated. A sample of the 59 kDa protein DnaB, prepared simply by preparative centrifugation, provides spectra that are as good as the ones from carefully grown microcrystals.

A multi-sample 94 GHz dissolution dynamic-nuclear-polarization system.

We describe the design and initial performance results of a multi-sample dissolution dynamic-nuclear-polarization (DNP) polarizer based on a Helium-temperature NMR cryostat for use in a wide-bore NMR magnet with a room-temperature bore. The system is designed to accommodate up to six samples in a revolver-style sample changer that allows changing samples at liquid-Helium temperature and at pressures ranging from ambient pressure down to 1 mbar. The multi-sample setup is motivated by the desire to do repetitive in vivo measurements and to characterize the DNP process by investigating samples of different chemical composition. The system can be loaded with up to six samples simultaneously to reduce sample loading and unloading. Therefore, series of experiments can be carried out faster and more reliably. The DNP probe contains an oversized microwave cavity and includes EPR and NMR capabilities for monitoring the DNP process. In the solid state, DNP enhancements corresponding to ∼45% polarization for [1-(13)C]pyruvic acid with a trityl radical have been measured. In the initial liquid-state acquisition experiments described here, the polarization was found to be ∼13%, corresponding to an enhancement factor exceeding 16,000 relative to thermal polarization at 9.4 T and ambient temperature.

Characterization of different water pools in solid-state NMR protein samples.

We observed and characterized two distinct signals originating from different pools of water protons in solid-state NMR protein samples, namely from crystal water which exchanges polarization with the protein (on the NMR timescale) and is located in the protein-rich fraction at the periphery of the magic-angle spinning (MAS) sample container, and supernatant water located close to the axis of the sample container. The polarization transfer between the water and the protein can be probed by two-dimensional exchange spectroscopy, and we show that the supernatant water does not interact with protein on the timescale of the experiments. The two water pools have different spectroscopic properties, including resonance frequency, longitudinal, transverse and rotating frame relaxation times. The supernatant water can be removed almost completely physically or can be frozen selectively. Both measures lead to an enhancement of the quality factor of the probe circuit, accompanied by an improvement of the experimental signal/noise, and greatly simplify solvent-suppression by substantially reducing the water signal. We also present a tool, which allows filling solid-state NMR sample containers in a more efficient manner, greatly reducing the amount of supernatant water and maximizing signal/noise.

Magnetic double resonance in force microscopy.

Magnetic-resonance force microscopy is combined with cross-polarization and spin-decoupling NMR techniques to obtain double-resonance NMR signals of micrometer-scaled objects. The effective one-dimensional spatial resolution obtained in our experiments performed on a KPF6 single crystal sample is approximately 0.5 microm. The spectral linewidth of 900 Hz is sample limited. The described double-resonance techniques can introduce new chemical specificity to the magnetic-force sensor.

Switched-angle spinning applied to bicelles containing phospholipid-associated peptides.

In a model study, the proton NMR spectrum of the opioid pentapeptide leucine-enkephalin associated with bicelles is investigated. The spectral resolution for a static sample is limited due to the large number of anisotropic interactions, in particular strong proton-proton couplings, but resolution is greatly improved by magic-angle sample spinning. Here we present two-dimensional switched-angle spinning NMR experiments, which correlate the high-resolution spectrum of the membrane-bound peptide under magic-angle spinning with its anisotropic spectrum, leading to well-resolved spectra. The two-dimensional spectrum allows the exploitation of the high resolution of the isotropic spectrum, while retaining the structural information imparted by the anisotropic interactions in the static spectrum. Furthermore, switched-angle spinning techniques are demonstrated that allow one to record the proton spectrum of ordered bicellar phases as a function of the angle between the rotor axis and the magnetic field direction, thereby scaling the dipolar interactions by a predefined factor.