Frank Engelke

Dynamic nuclear polarization at high magnetic fields in liquids.

MPI for Biophysical Chemistry Gottingen, Am Fassberg 11, 37077 Gottingen, Germany b Free University Berlin, Inst. of Experimental Physics, Arnimallee 14, 14195 Berlin, Germany Magnetic Resonance Center (CERM) and Department of Chemistry, University of Florence, Via Luigi Sacconi 6, 50019 Sesto Fiorentino, Italy Bruker Biospin GmbH, Silberstreifen 4, 76287 Rheinstetten, Germany e Technische Universitat Munchen, Department of Chemistry, Lichtenbergstr. 4, 85747 Garching, Germany Goethe University Frankfurt, Max von Laue Strasse 7, 60438 Frankfurt, Germany

Facing and Overcoming Sensitivity Challenges in Biomolecular NMR Spectroscopy

In the Spring of 2013, NMR spectroscopists convened at the Weizmann Institute in Israel to brainstorm on approaches to improve the sensitivity of NMR experiments, particularly when applied in biomolecular settings. This multi-author interdisciplinary Review presents a state-of-the-art description of the primary approaches that were considered. Topics discussed included the future of ultrahigh-field NMR systems, emerging NMR detection technologies, new approaches to nuclear hyperpolarization, and progress in sample preparation. All of these are orthogonal efforts, whose gains could multiply and thereby enhance the sensitivity of solid- and liquid-state experiments. While substantial advances have been made in all these areas, numerous challenges remain in the quest of endowing NMR spectroscopy with the sensitivity that has characterized forms of spectroscopies based on electrical or optical measurements. These challenges, and the ways by which scientists and engineers are striving to solve them, are also addressed.

Structure of fully protonated proteins by proton-detected magic-angle spinning NMR.

Significance Protein structure determination is key to the detailed description of many biological processes. The critical factor that would allow general application of magic-angle spinning (MAS) solid-state NMR to this end is improvement in sensitivity and resolution for as many nuclear spins as possible. This is achieved here with detection of resolved 1H resonances in protonated proteins by increasing MAS rates to frequencies of 100 kHz and above. For large proteins and assemblies, ultrafast spinning narrows spectral resonances better than Brownian motion on which solution NMR relies, removing a fundamental barrier to the NMR study of large systems. This is exploited here to determine the de novo structure of a 28-kDa protein dimer in a 2.5-MDa viral capsid assembly. Protein structure determination by proton-detected magic-angle spinning (MAS) NMR has focused on highly deuterated samples, in which only a small number of protons are introduced and observation of signals from side chains is extremely limited. Here, we show in two fully protonated proteins that, at 100-kHz MAS and above, spectral resolution is high enough to detect resolved correlations from amide and side-chain protons of all residue types, and to reliably measure a dense network of 1H-1H proximities that define a protein structure. The high data quality allowed the correct identification of internuclear distance restraints encoded in 3D spectra with automated data analysis, resulting in accurate, unbiased, and fast structure determination. Additionally, we find that narrower proton resonance lines, longer coherence lifetimes, and improved magnetization transfer offset the reduced sample size at 100-kHz spinning and above. Less than 2 weeks of experiment time and a single 0.5-mg sample was sufficient for the acquisition of all data necessary for backbone and side-chain resonance assignment and unsupervised structure determination. We expect the technique to pave the way for atomic-resolution structure analysis applicable to a wide range of proteins.

Amplifying Dynamic Nuclear Polarization of Frozen Solutions by Incorporating Dielectric Particles

There is currently great interest in understanding the limits on NMR signal enhancements provided by dynamic nuclear polarization (DNP), and in particular if the theoretical maximum enhancements can be achieved. We show that over a 2-fold improvement in cross-effect DNP enhancements can be achieved in MAS experiments on frozen solutions by simply incorporating solid particles into the sample. At 9.4 T and ∼105 K, enhancements up to εH = 515 are obtained in this way, corresponding to 78% of the theoretical maximum. We also underline that degassing of the sample is important to achieve highest enhancements. We link the amplification effect to the dielectric properties of the solid material, which probably gives rise to scattering, diffraction, and amplification of the microwave field in the sample. This is substantiated by simulations of microwave propagation. A reduction in sample heating at a given microwave power also likely occurs due to reduced dielectric loss. Simulations indicate that the microwave field (and thus the DNP enhancement) is inhomogeneous in the sample, and we deduce that in these experiments between 5 and 10% of the solution actually yields the theoretical maximum signal enhancement of 658. The effect is demonstrated for a variety of particles added to both aqueous and organic biradical solutions.

1H and 13C dynamic nuclear polarization in aqueous solution with a two-field (0.35 T/14 T) shuttle DNP spectrometer.

Dynamic nuclear polarization (DNP) permits increasing the NMR signal of nuclei by pumping the electronic spin transitions of paramagnetic centers nearby. This method is emerging as a powerful tool to increase the inherent sensitivity of NMR in structural biology aiming at detection of macromolecules. In aqueous solution, additional technical issues associated with the penetration of microwaves in water and heating effects aggravate the performance of the experiment. To examine the feasibility of low-field (9.7 GHz/0.35 T) DNP in high resolution NMR, we have constructed the prototype of a two-field shuttle DNP spectrometer that polarizes nuclei at 9.7 GHz/0.35 T and detects the NMR spectrum at 14 T. We report our first (1)H and (13)C DNP enhancements with this spectrometer. Effective enhancements up to 15 were observed for small molecules at (1)H 600 MHz/14 T as compared to the Boltzmann signal. The results provide a proof of principle for the feasibility of a shuttle DNP experiment and open up perspectives for the application potential of this method in solution NMR.

Nanosecond Time Scale Motions in Proteins Revealed by High-Resolution NMR Relaxometry

Understanding the molecular determinants underlying protein function requires the characterization of both structure and dynamics at atomic resolution. Nuclear relaxation rates allow a precise characterization of protein dynamics at the Larmor frequencies of spins. This usually limits the sampling of motions to a narrow range of frequencies corresponding to high magnetic fields. At lower fields one cannot achieve sufficient sensitivity and resolution in NMR. Here, we use a fast shuttle device where the polarization builds up and the signals are detected at high field, while longitudinal relaxation takes place at low fields 0.5 < B0 < 14.1 T. The sample is propelled over a distance up to 50 cm by a blowgun-like system in about 50 ms. The analysis of nitrogen-15 relaxation in the protein ubiquitin over such a wide range of magnetic fields offers unprecedented insights into molecular dynamics. Some key regions of the protein feature structural fluctuations on nanosecond time scales, which have so far been overlooked in high-field relaxation studies. Nanosecond motions in proteins may have been underestimated by traditional high-field approaches, and slower supra-τc motions that have no effect on relaxation may have been overestimated. High-resolution relaxometry thus opens the way to a quantitative characterization of nanosecond motions in proteins.

Continuous flow hyperpolarized 129Xe-MAS NMR studies of microporous materials

A magic angle spinning (MAS) NMR probe has been developed to allow in situ measurements of NMR spectra. Two applications are targeted with this device: i) in situ and operando MAS NMR spectroscopy of working catalysts and ii) the hyperpolarized (HP) 129Xe spectroscopy of porous materials under MAS and continuous flow conditions. The construction of the MAS probe is described and the usefulness of this system is demonstrated by studying the adsorption of hyperpolarized xenon on AlPO-41 and ITQ-6 zeolites. The high stability of the HP xenon flow allowed us to perform two-dimensional exchange experiments under MAS conditions, in a short time and with very good resolution.

Instrumentation for solid-state dynamic nuclear polarization with magic angle spinning NMR

Advances in dynamic nuclear polarization (DNP) instrumentation and methodology have been key factors in the recent growth of solid-state DNP NMR applications. We review the current state of the art of solid-state DNP NMR instrumentation primarily based on available commercial platforms. We start with a general system overview, including options for microwave sources and DNP NMR probes, and then focus on specific developments for DNP at 100K with magic angle spinning (MAS). Gyrotron microwave sources, passive components to transmit microwaves, the DNP MAS probe, a cooling device for low-temperature MAS, and sample preparation procedures including radicals for DNP are considered.

A theoretical approach for the calculation of the Hartmann-Hahn matching under cross polarization and magic-angle spinning

Abstract A method for the calculation of the Hartmann-Hahn matching curve in 1 H 13 C cross polarization and magic-angle spinning NMR experiments is proposed. The theoretical results are compared to experimental data. The basis of the theoretical approach is a two-step autocorrelation function calculation using Moris integrodifferential equation as a starting point for the introduction of suitable approximations (Anderson-Weiss-type approximation and short correlation limit) to find a description of the spin dynamics of the coupled 1 H and 13 C spin system under magic-angle-spinning conditions.

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.