Solid-State NMR with ever faster Magic-Angle Spinning: Proton Linewidth, Protein Dynamics and Biomolecular Applications
Embargoed until 2023-03-22
- Doctoral Thesis
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. Show more
External linksSearch print copy at ETH Library
SubjectSolid-state NMR; fast MAS; proton detection; Linewidths; Protein dynamics; Resolution; hydrogen bonds; magnetic fields; Polarization transfer
Organisational unit03496 - Meier, Beat H. / Meier, Beat H.
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