High and fast: NMR protein-proton side-chain assignments at 160 kHz and 1.2 GHz.

The NMR spectra of side-chain protons in proteins provide important information, not only about their structure and dynamics, but also about the mechanisms that regulate interactions between macromolecules. However, in the solid-state, these resonances are particularly difficult to resolve, even in relatively small proteins. We show that magic-angle-spinning (MAS) frequencies of 160 kHz, combined with a high magnetic field of 1200 MHz proton Larmor frequency, significantly improve their spectral resolution. We investigate in detail the gain for MAS frequencies between 110 and 160 kHz MAS for a model sample as well as for the hepatitis B viral capsid assembled from 120 core-protein (Cp) dimers. For both systems, we found a significantly improved spectral resolution of the side-chain region in the 1H-13C 2D spectra. The combination of 160 kHz MAS frequency with a magnetic field of 1200 MHz, allowed us to assign 61% of the aliphatic protons of Cp. The side-chain proton assignment opens up new possibilities for structural studies and further characterization of protein-protein or protein-nucleic acid interactions.

The effect of methyl group rotation on 1H-1H solid-state NMR spin-diffusion spectra.

Fast magic-angle spinning (MAS) NMR experiments open the way for proton-detected NMR studies and have been explored in the past years for a broad range of materials, comprising biomolecules and pharmaceuticals. Proton-spin diffusion (SD) is a versatile polarization-transfer mechanism and plays an important role in resonance assignment and structure determination. Recently, the occurrence of negative cross peaks in 2D 1H-1H SD-based spectra has been reported and explained with higher-order SD effects, in which the chemical shifts of the involved quadruple of nuclei need to compensate each other. We herein report negative cross peaks in SD-based spectra observed for a variety of small organic molecules involving methyl groups. We combine experimental observations with numerical and analytical simulations to demonstrate that the methyl groups can give rise to coherent (SD) as well as incoherent (Nuclear Overhauser Enhancement, NOE) effects, both in principle manifesting themselves as negative cross peaks in the 2D spectra. Analytical calculations and simulations however show that higher-order coherent contributions dominate the experimentally observed negative peaks in our systems. Methyl groups are prone to the observation of such higher order coherent effects. Due to their low-frequency shifted 1H resonances, the chemical-shift separation relative to for instance aromatic protons in spatial proximity is substantial (>4.7 ppm in the studied examples) preventing any sizeable second-order spin-diffusion processes, which would mask the negative contribution to the peaks.

Structural conservation of HBV-like capsid proteins over hundreds of millions of years despite the shift from non-enveloped to enveloped life-style.

The discovery of nackednaviruses provided new insight into the evolutionary history of the hepatitis B virus (HBV): The common ancestor of HBV and nackednaviruses was non-enveloped and while HBV acquired an envelope during evolution, nackednaviruses remained non-enveloped. We report the capsid structure of the African cichlid nackednavirus (ACNDV), determined by cryo-EM at 3.7 Å resolution. This enables direct comparison with the known capsid structures of HBV and duck HBV, prototypic representatives of the mammalian and avian lineages of the enveloped Hepadnaviridae, respectively. The sequence identity with HBV is 24% and both the ACNDV capsid protein fold and the capsid architecture are very similar to those of the Hepadnaviridae and HBV in particular. Acquisition of the hepadnaviral envelope was thus not accompanied by a major change in capsid structure. Dynamic residues at the spike tip are tentatively assigned by solid-state NMR, while the C-terminal domain is invisible due to dynamics. Solid-state NMR characterization of the capsid structure reveals few conformational differences between the quasi-equivalent subunits of the ACNDV capsid and an overall higher capsid structural disorder compared to HBV. Despite these differences, the capsids of ACNDV and HBV are structurally highly similar despite the 400 million years since their separation.

Probing a Hydrogen-π Interaction Involving a Trapped Water Molecule in the Solid State.

The detection and characterization of trapped water molecules in chemical entities and biomacromolecules remains a challenging task for solid materials. We herein present proton-detected solid-state Nuclear Magnetic Resonance (NMR) experiments at 100 kHz magic-angle spinning and at high static magnetic-field strengths (28.2 T) enabling the detection of a single water molecule fixed in the calix[4]arene cavity of a lanthanide complex by a combination of three types of non-covalent interactions. The water proton resonances are detected at a chemical-shift value close to zero ppm, which we further confirm by quantum-chemical calculations. Density Functional Theory calculations pinpoint to the sensitivity of the proton chemical-shift value for hydrogen-π interactions. Our study highlights how proton-detected solid-state NMR is turning into the method-of-choice in probing weak non-covalent interactions driving a whole branch of molecular-recognition events in chemistry and biology.

Fast Magic-Angle-Spinning NMR Reveals the Evasive Hepatitis†B Virus Capsid C-Terminal Domain.

Experimentally determined protein structures often feature missing domains. One example is the C-terminal domain (CTD) of the hepatitis B virus capsid protein, a functionally central part of this assembly, crucial in regulating nucleic-acid interactions, cellular trafficking, nuclear import, particle assembly and maturation. However, its structure remained elusive to all current techniques, including NMR. Here we show that the recently developed proton-detected fast magic-angle-spinning solid-state NMR at >100 kHz MAS allows one to detect this domain and unveil its structural and dynamic behavior. We describe the experimental framework used and compare the domain's behavior in different capsid states. The developed approaches extend solid-state NMR observations to residues characterized by large-amplitude motion on the microsecond timescale, and shall allow one to shed light on other flexible protein domains still lacking their structural and dynamic characterization.

Proton-phosphorous connectivities revealed by high-resolution proton-detected solid-state NMR.

Proton-detected solid-state NMR enables atomic-level insight in solid-state reactions, for instance in heterogeneous catalysis, which is fundamental for deciphering chemical reaction mechanisms. We herein introduce a phosphorus-31 radiofrequency channel in proton-detected solid-state NMR at fast magic-angle spinning. We demonstrate our approach using solid-state 1H/31P and 1H/13C correlation experiments at high magnetic fields (850 and 1200 MHz) and high spinning frequencies (100 kHz) to characterize four selected PH-containing compounds from the chemistry of phosphane-borane frustrated Lewis pairs. Frustrated Lewis pairs have gained high interest in the past years, particularly due to their capabilities of activating and binding small molecules, such as di-hydrogen, however, their analytical characterization especially in the solid state is still limited. Our approach reveals proton-phosphorus connectivities providing important information on spatial proximity and chemical bonding within such compounds. We also identify protons that show strongly different chemical-shift values compared to the solution state, which we attribute to intermolecular ring-current effects. The most challenging example presented herein is a cyclotrimeric frustrate Lewis pair-associate comprising three crystallographically distinct phosphonium entities that are unambiguously distinguished by our approach. Such 31P spin-filtered proton-detected NMR can be easily extended to other material classes and can strongly impact the structural characterization of reaction products of hydrogen-activated phosphane/borane FLPs, heterogeneous catalysts and solid-state reactions in general.

Correction of field instabilities in biomolecular solid-state NMR by simultaneous acquisition of a frequency reference.

With the advent of faster magic-angle spinning (MAS) and higher magnetic fields, the resolution of biomolecular solid-state nuclear magnetic resonance (NMR) spectra has been continuously increasing. As a direct consequence, the always narrower spectral lines, especially in proton-detected spectroscopy, are also becoming more sensitive to temporal instabilities of the magnetic field in the sample volume. Field drifts in the order of tenths of parts per million occur after probe insertion or temperature change, during cryogen refill, or are intrinsic to the superconducting high-field magnets, particularly in the months after charging. As an alternative to a field-frequency lock based on deuterium solvent resonance rarely available for solid-state NMR, we present a strategy to compensate non-linear field drifts using simultaneous acquisition of a frequency reference (SAFR). It is based on the acquisition of an auxiliary 1D spectrum in each scan of the experiment. Typically, a small-flip-angle pulse is added at the beginning of the pulse sequence. Based on the frequency of the maximum of the solvent signal, the field evolution in time is reconstructed and used to correct the raw data after acquisition, thereby acting in its principle as a digital lock system. The general applicability of our approach is demonstrated on 2D and 3D protein spectra during various situations with a non-linear field drift. SAFR with small-flip-angle pulses causes no significant loss in sensitivity or increase in experimental time in protein spectroscopy. The correction leads to the possibility of recording high-quality spectra in a typical biomolecular experiment even during non-linear field changes in the order of 0.1 ppm h-1 without the need for hardware solutions, such as stabilizing the temperature of the magnet bore. The improvement of linewidths and peak shapes turns out to be especially important for 1H-detected spectra under fast MAS, but the method is suitable for the detection of carbon or other nuclei as well.

Spectroscopic glimpses of the transition state of ATP hydrolysis trapped in a bacterial DnaB helicase.

The ATP hydrolysis transition state of motor proteins is a weakly populated protein state that can be stabilized and investigated by replacing ATP with chemical mimics. We present atomic-level structural and dynamic insights on a state created by ADP aluminum fluoride binding to the bacterial DnaB helicase from Helicobacter pylori. We determined the positioning of the metal ion cofactor within the active site using electron paramagnetic resonance, and identified the protein protons coordinating to the phosphate groups of ADP and DNA using proton-detected 31P,1H solid-state nuclear magnetic resonance spectroscopy at fast magic-angle spinning > 100 kHz, as well as temperature-dependent proton chemical-shift values to prove their engagements in hydrogen bonds. 19F and 27Al MAS NMR spectra reveal a highly mobile, fast-rotating aluminum fluoride unit pointing to the capture of a late ATP hydrolysis transition state in which the phosphoryl unit is already detached from the arginine and lysine fingers.

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.

Temperature-Dependent Solid-State NMR Proton Chemical-Shift Values and Hydrogen Bonding.

Temperature-dependent NMR experiments are often complicated by rather long magnetic-field equilibration times, for example, occurring upon a change of sample temperature. We demonstrate that the fast temporal stabilization of a magnetic field can be achieved by actively stabilizing the temperature of the magnet bore, which allows quantification of the weak temperature dependence of a proton chemical shift, which can be diagnostic for the presence of hydrogen bonds. Hydrogen bonding plays a central role in molecular recognition events from both fields, chemistry and biology. Their direct detection by standard structure-determination techniques, such as X-ray crystallography or cryo-electron microscopy, remains challenging due to the difficulties of approaching the required resolution, on the order of 1 Å. We, herein, explore a spectroscopic approach using solid-state NMR to identify protons engaged in hydrogen bonds and explore the measurement of proton chemical-shift temperature coefficients. Using the examples of a phosphorylated amino acid and the protein ubiquitin, we show that fast magic-angle spinning (MAS) experiments at 100 kHz yield sufficient resolution in proton-detected spectra to quantify the rather small chemical-shift changes upon temperature variations.

Dimer Organization of Membrane-Associated NS5A of Hepatitis†C Virus as Determined by Highly Sensitive 1 H-Detected Solid-State NMR.

The Hepatitis C virus nonstructural protein 5A (NS5A) is a membrane-associated protein involved in multiple steps of the viral life cycle. Direct-acting antivirals (DAAs) targeting NS5A are a cornerstone of antiviral therapy, but the mode-of-action of these drugs is poorly understood. This is due to the lack of information on the membrane-bound NS5A structure. Herein, we present the structural model of an NS5A AH-linker-D1 protein reconstituted as proteoliposomes. We use highly sensitive proton-detected solid-state NMR methods suitable to study samples generated through synthetic biology approaches. Spectra analyses disclose that both the AH membrane anchor and the linker are highly flexible. Paramagnetic relaxation enhancements (PRE) reveal that the dimer organization in lipids requires a new type of NS5A self-interaction not reflected in previous crystal structures. In conclusion, we provide the first characterization of NS5A AH-linker-D1 in a lipidic environment shedding light onto the mode-of-action of clinically used NS5A inhibitors.

Residual dipolar line width in magic-angle spinning proton solid-state NMR.

Magic-angle spinning is routinely used to average anisotropic interactions in solid-state nuclear magnetic resonance (NMR). Due to the fact that the homonuclear dipolar Hamiltonian of a strongly coupled spin system does not commute with itself at different time points during the rotation, second-order and higher-order terms lead to a residual dipolar line broadening in the observed resonances. Additional truncation of the residual broadening due to isotropic chemical-shift differences can be observed. We analyze the residual line broadening in coupled proton spin systems based on theoretical calculations of effective Hamiltonians up to third order using Floquet theory and compare these results to numerically obtained effective Hamiltonians in small spin systems. We show that at spinning frequencies beyond 75 kHz, second-order terms dominate the residual line width, leading to a 1/ωr dependence of the second moment which we use to characterize the line width. However, chemical-shift truncation leads to a partial ωr-2 dependence of the line width which looks as if third-order effective Hamiltonian terms are contributing significantly. At slower spinning frequencies, cross terms between the chemical shift and the dipolar coupling can contribute in third-order effective Hamiltonians. We show that second-order contributions not only broaden the line, but also lead to a shift of the center of gravity of the line. Experimental data reveal such spinning-frequency-dependent line shifts in proton spectra in model substances that can be explained by line shifts induced by the second-order dipolar Hamiltonian.

Experimental Characterization of the Hepatitis B Virus Capsid Dynamics by Solid-State NMR.

Protein plasticity and dynamics are important aspects of their function. Here we use solid-state NMR to experimentally characterize the dynamics of the 3.5 MDa hepatitis B virus (HBV) capsid, assembled from 240 copies of the Cp149 core protein. We measure both T 1 and T 1ρ relaxation times, which we use to establish detectors on the nanosecond and microsecond timescale. We compare our results to those from a 1 microsecond all-atom Molecular Dynamics (MD) simulation trajectory for the capsid. We show that, for the constituent residues, nanosecond dynamics are faithfully captured by the MD simulation. The calculated values can be used in good approximation for the NMR-non-detected residues, as well as to extrapolate into the range between the nanosecond and microsecond dynamics, where NMR has a blind spot at the current state of technology. Slower motions on the microsecond timescale are difficult to characterize by all-atom MD simulations owing to computational expense, but are readily accessed by NMR. The two methods are, thus, complementary, and a combination thereof can reliably characterize motions covering correlation times up to a few microseconds.

Asparagine and Glutamine Side-Chains and Ladders in HET-s(218-289) Amyloid Fibrils Studied by Fast Magic-Angle Spinning NMR.

Asparagine and glutamine side-chains can form hydrogen-bonded ladders which contribute significantly to the stability of amyloid fibrils. We show, using the example of HET-s(218-289) fibrils, that the primary amide side-chain proton resonances can be detected in cross-polarization based solid-state NMR spectra at fast magic-angle spinning (MAS). J-coupling based experiments offer the possibility to distinguish them from backbone amide groups if the spin-echo lifetimes are long enough, which turned out to be the case for the glutamine side-chains, but not for the asparagine side-chains forming asparagine ladders. We explore the sensitivity of NMR observables to asparagine ladder formation. One of the two possible asparagine ladders in HET-s(218-289), the one comprising N226 and N262, is assigned by proton-detected 3D experiments at fast MAS and significant de-shielding of one of the NH2 proton resonances indicative of hydrogen-bond formation is observed. Small rotating-frame 15N relaxation-rate constants point to rigidified asparagine side-chains in this ladder. The proton resonances are homogeneously broadened which could indicate chemical exchange, but is presently not fully understood. The second asparagine ladder (N243 and N279) in contrast remains more flexible.

Protein Side-Chain-DNA Contacts Probed by Fast Magic-Angle Spinning NMR.

Protein-nucleic acid interactions are essential in a variety of biological events ranging from the replication of genomic DNA to the synthesis of proteins. Noncovalent interactions guide such molecular recognition events, and protons are often at the center of them, particularly due to their capability of forming hydrogen bonds to the nucleic acid phosphate groups. Fast magic-angle spinning experiments (100 kHz) reduce the proton NMR line width in solid-state NMR of fully protonated protein-DNA complexes to such an extent that resolved proton signals from side-chains coordinating the DNA can be detected. We describe a set of NMR experiments focusing on the detection of protein side-chains from lysine, arginine, and aromatic amino acids and discuss the conclusions that can be obtained on their role in DNA coordination. We studied the 39 kDa enzyme of the archaeal pRN1 primase complexed with DNA and characterize protein-DNA contacts in the presence and absence of bound ATP molecules.

Protein NMR Spectroscopy at 150†kHz Magic-Angle Spinning Continues To Improve Resolution and Mass Sensitivity.

Spectral resolution is the key to unleashing the structural and dynamic information contained in NMR spectra. Fast magic-angle spinning (MAS) has recently revolutionized the spectroscopy of biomolecular solids. Herein, we report a further remarkable improvement in the resolution of the spectra of four fully protonated proteins and a small drug molecule by pushing the MAS rotation frequency higher (150 kHz) than the more routinely used 100 kHz. We observed a reduction in the average homogeneous linewidth by a factor of 1.5 and a decrease in the observed linewidth by a factor 1.25. We conclude that even faster MAS is highly attractive and increases mass sensitivity at a moderate price in overall sensitivity.

Nucleotide Binding Modes in a Motor Protein Revealed by 31 P- and 1 H-Detected MAS Solid-State NMR Spectroscopy.

Protein-nucleic acid interactions play important roles not only in energy-providing reactions, such as ATP hydrolysis, but also in reading, extending, packaging, or repairing genomes. Although they can often be analyzed in detail with X-ray crystallography, complementary methods are needed to visualize them in complexes, which are not crystalline. Here, we show how solid-state NMR spectroscopy can detect and classify protein-nucleic interactions through site-specific 1 H- and 31 P-detected spectroscopic methods. The sensitivity of 1 H chemical-shift values on noncovalent interactions involved in these molecular recognition processes is exploited allowing us to probe directly the chemical bonding state, an information, which is not directly accessible from an X-ray structure. We show that these methods can characterize interactions in easy-to-prepare sediments of the 708 kDa dodecameric DnaB helicase in complex with ADP:AlF4- :DNA, and this despite the very challenging size of the complex.

100 kHz MAS Proton-Detected NMR Spectroscopy of Hepatitis B Virus Capsids.

We sequentially assigned the fully-protonated capsids made from core proteins of the Hepatitis B virus using proton detection at 100 kHz magic-angle spinning (MAS) in 0.7 mm rotors and compare sensitivity and assignment completeness to previously obtained assignments using carbon-detection techniques in 3.2 mm rotors and 17.5 kHz MAS. We show that proton detection shows a global gain of a factor ~50 in mass sensitivity, but that signal-to-noise ratios and completeness of the assignment was somewhat higher for carbon-detected experiments for comparable experimental times. We also show that deuteration and HN back protonation improves the proton linewidth at 100 kHz MAS by a factor of 1.5, from an average of 170-110 Hz, and by a factor of 1.3 compared to deuterated capsids at 60 kHz MAS in a 1.3 mm rotor. Yet, several HN protons cannot be back-exchanged due to solvent inaccessibility, which results in a total of 15% of the amides missing in the spectra.

Quantifying proton NMR coherent linewidth in proteins under fast MAS conditions: a second moment approach.

Proton detected solid-state NMR under fast magic-angle-spinning (MAS) conditions is currently redefining the applications of solid-state NMR, in particular in structural biology. Understanding the contributions to the spectral linewidth is thereby of paramount importance. When disregarding the sample-dependent inhomogeneous contributions, the NMR proton linewidth is defined by homogeneous broadening, which has incoherent and coherent contributions. Understanding and disentangling these different contributions in multi-spin systems like proteins is still an open issue. The coherent contribution is mainly caused by the dipolar interaction under MAS and is determined by the molecular structure and the proton chemical shifts. Numerical simulation approaches based on numerically exact direct integration of the Liouville-von Neumann equation can give valuable information about the lineshape, but are limited to small spin systems (<12 spins). We present an alternative simulation method for the coherent contributions based on the rapid and partially analytic calculation of the second moments of large spin systems. We first validate the method on a simple system by predicting the 19F linewidth in CaF2 under MAS. We compare simulation results to experimental data for microcrystalline ubiquitin (deuterated 100% back-exchanged at 110 kHz and fully-protonated at 125 kHz). Our results quantitatively explain the observed linewidth per-residue basis for the vast majority of residues.

Characterization of H2 -Splitting Products of Frustrated Lewis Pairs: Benefit of Fast Magic-Angle Spinning.

Proton spectroscopy in solid-state NMR on catalytic materials offers new opportunities in structural characterization, in particular of reaction products of catalytic reactions such as hydrogenation reactions. Unfortunately, the 1 H NMR line widths in magic-angle spinning solid-state spectra are often broadened by an incomplete averaging of 1 H-1 H dipolar couplings. We herein discuss two model compounds, namely the H2 -splitting products of two phosphane-borane Frustrated Lewis Pairs (FLPs), to study potentials and limitations of proton solid-state NMR experiments employing magic-angle spinning frequencies larger than 100 kHz at a static magnetic field strength of 20.0 T. The 1 H lines are homogeneously broadened as illustrated by spin-echo decay experiments. We study two structurally similar materials which however show significant differences in 1 H line widths which we explain by differences in their 1 H-1 H dipolar networks. We discuss the benefit of fast MAS experiments up to 110 kHz to detect the resonances of the H+ /H- pair in the hydrogenation products of FLPs.