Untersuchung einer Wasserstoff‐π Wechselwirkung in einem eingeschlossenen Wassermolekül im Festkörper

Der Nachweis und die Charakterisierung von eingeschlossenen Wassermolekülen in chemischen Gebilden und Biomakromolekülen ist weiterhin eine Herausforderung für feste Materialien. Wir präsentieren hier Protonen-detektierte Festkörper-Kernspinresonanzspektroskopie (NMR) Experimente bei Rotationsfrequenzen von 100 kHz um den magischen Winkel und bei hohen statischen Magnetfeldstärken (28.2 T), die den Nachweis eines einzelnen Wassermoleküls ermöglichen, das im Calix[4]aren-Hohlraum eines Lanthan-Komplexes durch eine Kombination von drei Arten nicht-kovalenter Wechselwirkungen fixiert ist. Die Protonenresonanzen des Wassers werden bei einer chemischen Verschiebung nahe Null ppm nachgewiesen, was wir durch quantenchemische Berechnungen bestätigen. Berechnungen mit der Dichtefunktionaltheorie zeigen, wie empfindlich der Wert der chemischen Verschiebung der Protonen auf Wasserstoff-π-Wechselwirkungen reagiert. Unsere Studie unterstreicht, wie sich die Protonen-detektierte Festkörper NMR zur Methode der Wahl für die Untersuchung schwacher nicht-kovalenter Wechselwirkungen entwickelt, die einen ganzen Zweig molekularer Erkennungsvorgänge in der Chemie und Biologie bestimmen

Solid-State NMR with ever faster Magic-Angle Spinning: Proton Linewidth, Protein Dynamics and Biomolecular Applications

Over the last decades solid-state nuclear magnetic resonance (NMR) has emerged as a powerful tool for structural, functional and dynamic studies of biomolecules and materials. In this context protons may provide particularly valuable information due to their potential of forming hydrogen bonds, contributing to thermodynamic stability, supramolecular assembling or molecular recognition (e.g. protein-DNA/RNA interactions). The incomplete averaging of large 1H-1H dipolar couplings under magic-angle spinning (MAS) typically leads to broad spectral lines in proton-detected spectra. However, significant advances in spinning technology and access to spinning frequencies exceeding 100 kHz provide nowadays sufficient spectral resolution even in molecular systems with dense proton dipolar networks like fully protonated proteins, enabling practical applications. In this thesis we investigate both methodological and application aspects of proton-detected solid-state NMR in a spinning regime ranging from 60-170 kHz. Thereby, we will discuss the challenges and possibilities of this technique, whose constant evolution is driven by the access to ever faster spinning frequencies. In the first part of this work we focus on the factors influencing resolution in proton-detected spectra and how it can be improved by going to faster MAS frequencies and higher magnetic field strengths. We report remarkable proton linewidth narrowing in the spectra of a series of fully protonated proteins when pushing with an 0.5 mm probe-head prototype the sample rotation frequency to 150 kHz compared to the more routinely used 100 kHz. Furthermore, we find an improvement in mass sensitivity by comparison with spectra recorded in an 0.7 mm rotor operating at 100 kHz, despite the reduction in sample volume by a factor of 0.52 (from 0.59μL to 0.31μL). We attribute this observation to higher coil efficiency, linewidth narrowing and improvements in polarization-transfer efficiency. Systematic experimental studies monitoring the homogeneous linewidths in fully protonated molecules over the MAS range 60-170kHz reveal that the CH2 resonances in the small drug molecule Meldonium and in two phosphorylated amino acids profit the most from an increase in spinning frequency. To rationalize these findings we developed an alternative simulation method based on second-moment calculations to estimate the portion of the experimental linewidth that originates from the homonuclear dipolar network. Compared to conventional Liouville- von Neumann-based simulation programs, this second-moment approach is much less expensive and allows for performing calculations until convergence is reached also in large multi-spin systems. We established it on the model protein Ubiquitin and used it to prove that the Meldonium linewidth is indeed dominated by coherent dipolar effects and can thereby be improved significantly by faster spinning. Using this simulation approach we also illustrate how the proximity to CH2 groups can be particularly detrimental, due to the typically small spatial and spectral distances between the ethyl protons generating large residual linewidth contributions. In light of these results we also understand the experimental linewidth differences observed for two H2-splitting products of Frustrated Lewis Pairs that we present as an example to illustrate the great potential of proton-detected solid-state NMR for structural studies of functionalized catalytic materials. Our linewidth simulations predict an additional coherent linewidth narrowing effect, when increasing the external magnetic field strength. We verify this prediction experimentally by comparing homogeneous proton linewidths in spectra of proteins and phosphorylated amino acids recorded at 20.0T (850MHz) and 28.2T (1.2GHz). Finally, we show how it is possible to obtain unprecedented resolution and almost baseline separation for the CH2 resonances in o-phospho-L-serine, using a combination of 160 kHz MAS and 28.2 T. In the second part of this thesis we use the strength of solid-state NMR to experimen- tally investigate protein dynamics. Measuring longitudinal and rotating-frame relaxation rate constants and using a detectors approach for data analysis, we obtain information on residue-specific amplitudes of motion over the nanosecond, hundreds of nanoseconds and microsecond timescales. In a first study we characterize the dynamics of the hepatitis B virus capsid. In particular we find that it shows larger amplitudes of microsecond motion within the capsid spike. Thanks to the detectors approach we were able to extract similar information from a recently published 1 μs molecular dynamics (MD) trajectory. A comparison between NMR and MD data reveals overall good agreement for the faster nanosecond motions thereby experimentally validating the MD results, but larger discrepancies for the slower motions on the timescales approaching the end of the trajectory. For these timescales it is therefore advisable to mostly rely on the NMR results. We also investigated the temperature dependence of the dynamics of the archaeal RNA polymerase subunit complex Rpo4-7 over the temperature range from 17 °C to 60°. In addition to proving the stability of the molecule under these conditions we observed in particular rather small differences for the nanosecond motions but a large thermal activation of the slower microsecond motions with increasing temperature. In a third part we explore different possibilities of using proton-detected solid-state NMR to study hydrogen bonding. We characterize asparagine ladder formation in HET-s(218-289) fibrils by using different spectral and relaxation properties of the NH2 proton sidechain resonances. We use a 1H-31P correlation experiment to generate protein-nucleotide correlation signals, proving spatial proximity and describing on a local level the nucleotide binding modes of the ATP-hydrolysis transition state in the bacterial DnaB helicase in complex with DNA. Finally, we use the temperature dependence of the proton chemical shifts as a direct proof for hydrogen bond formation. We extend this approach known from solution-state NMR to the solid-state using o-phospho-L-serine and ubiquitin as model compounds and apply it to further elucidate the protein-nucleotide interactions of the DnaB helicase. In the remaining chapters we characterize the performance of basic polarization transfer steps like spin-diffusion, INEPT and cross polarization and comment on the feasibility and potential of five-dimensional SO-APSY in the fast MAS regime. We show how a specific arginine labeling scheme can be used in combination with spin diffusion and non-refocused INEPT transfers to investigate the highly flexible C-terminal domain of the full-length HBV capsid, which has so far always escaped detection in conventional carbon and proton- detected NMR. Finally, we show some additional biomolecular applications of state-of the art proton-detected NMR. In particular we show techniques that can be used to probe protein side-chain-DNA contacts in the archaeal primase pRN1 and discuss preliminary fingerprint spectra of the prion sup35 and the hepatitis D virus antigen proteins

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 ω −2 r 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.ISSN:2699-001

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.ISSN:1463-9084ISSN:1463-907

Characterization of H-2-Splitting Products of Frustrated Lewis Pairs: Benefit of Fast Magic-Angle Spinning

ISSN:1439-4235ISSN:1439-764

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.ISSN:2296-889

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 associated 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. The simulations however reveal that higher order coherent contributions dominate the experimentally observed negative peaks. 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 superimpose the negative contribution to the peaks

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 2D¹H–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.ISSN:1463-9084ISSN:1463-907

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 the magnetic field can be achieved by actively stabilizing the temperature which allows to quantify the weak temperature dependence of the 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.<br /

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

[Image: see text] 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