High-resolution solid-state MAS NMR of proteins—Crh as an example

Abstract

Solid-state NMR spectroscopy provides unique possibilities for the structural investigation of insoluble molecules at the atomic level. Recent efforts aim at solving the complete structures of biological macromolecules using high-resolution magic angle spinning NMR. Structurally homogenous samples of [(13)C,(15)N]-labeled proteins have to be used in this type of studies. Microcrystalline model proteins present valuable tools for the developments of methods towards this goal. This review discusses recent progress in the field, using the Crh protein as an illustrative example. We discuss strategies for resonance assignments and for the determination of structure and dynamics, as well as techniques for the detection of protein interaction partners and folding mechanisms by solid-state NMR methods.

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

“…The 1 H spectra (recorded with the EASY scheme to suppress 1 H rotor and probe backgrounds [29] ) are shown in Figure 1a for compound 1. The strong contribution of the remaining 1 H-1 H homonuclear dipolar couplings to the NMR line width [1] leads, as expected, even in the MAS spectra recorded at a spinning frequency of 110 kHz to 1 H-lines which are still one to two orders of magnitude broader than those in solution-state NMR. As can be seen in particular for the 1 H-resonances of the methyl groups appearing at lowest ppm-values (0 to 0.4 ppm) or the HP-region (5.5 to 6.5 ppm) mentioned above and highlighted in magenta in Figure 1c, the resolution of the spectra improves significantly by going from 30 to 110 kHz.…”

“…The residual 1 H line width is determined by higher-order terms in the NMR average Hamiltonian, which are not averaged sufficiently at MAS frequencies typically available in NMR laboratories. [1,2] Recent progress in the development of solidstate NMR probes operating at MAS frequencies > 100 kHz, in particular the nowadays commercially-available 0.7 mm probes, has led to significant resolution enhancement in 1 H-detected MAS experiments, mainly in the field of biomolecules where the 1 H lines are becoming narrow enough to use protons for protein resonance assignment. [3][4][5] In combination with high magnetic field strengths, even the structure calculation for proteins became accessible.…”

“…Spatial connectivities extracted from 2D 1 H-1 H correlation experiments at a rotation frequency of 110 kHz: 2D 1 H-1 H SD spectra of compound 1 (a) and 2 (b) as well as back-to-back (BaBa-xy16) single-quantum double-quantum correlation spectra (c) and (d). Some representative assignments are indicated in the spectra.…”

Prions

“…NMR of fibrils that exhibit significant disorder, as judged from the heterogeneous line broadening in NMR spectra, often only allow the measurement of a limited number of distances between selective labels, which then leads to the establishment of structural models satisfying these restraints. In cases where the local order is high enough such that they yield narrow lines under magic-angle spinning conditions 6,24,31,34,39 (e.g., HET-s(218-289), 31 Ure2p, 6 or the Y145Stop variant or PrP 39 ), structural studies of fibrils have many common elements with solid-state NMR high-resolution 3D structure determination of crystalline proteins [40][41][42][43][44][45] and basically the same strategies can be applied. 40,[43][44][45][46][47][48] They include, as in solution, sequential resonance assignments, followed by the collection of a large set of distance restraints used in structure calculations.…”

Towards further understanding of protein crystallization : phase diagram, protein interactions, nucleation kinetics and growth kinetics

“…Another recent advance for the structure determination of protein molecules is the solid-state magic-angle-spinning NMR spectroscopy. Although protein structure determined by the solid-state NMR has not been reported, the solid-state NMR spectroscopy provides a possibility to determine the structures of the insoluble proteins at an atomic level [91]. The advances in both instrumentation and software provide a promising possibility to improve the low throughput of the structure determination of protein structures.…”