Quadruple‐Resonance Magic‐Angle Spinning NMR Spectroscopy of Deuterated Solid Proteins

Abstract

(1)H-detected magic-angle spinning NMR experiments facilitate structural biology of solid proteins, which requires using deuterated proteins. However, often amide protons cannot be back-exchanged sufficiently, because of a possible lack of solvent exposure. For such systems, using (2)H excitation instead of (1)H excitation can be beneficial because of the larger abundance and shorter longitudinal relaxation time, T1, of deuterium. A new structure determination approach, "quadruple-resonance NMR spectroscopy", is presented which relies on an efficient (2)H-excitation and (2)H-(13)C cross-polarization (CP) step, combined with (1)H detection. We show that by using (2)H-excited experiments better sensitivity is possible on an SH3 sample recrystallized from 30 % H2O. For a membrane protein, the ABC transporter ArtMP in native lipid bilayers, different sets of signals can be observed from different initial polarization pathways, which can be evaluated further to extract structural properties.

The ABC exporter MsbA probed by solid state NMR – challenges and opportunities

“…Fast sample rotation about an axis inclined to the magnetic field at 54.7° (the 'magic angle'), averages all anisotropic interactions leading to well-resolved spectra (Figure 1). The first solid state NMR experiments on an ABC importer were described for ArtMP in 2D-crystals (Akbey et al, 2014;Lange et al, 2010). The studies were later complemented by EPR spectroscopy (Hellmich et al, 2012b) and by solution state NMR on the isolated NBDs (Hellmich et al, 2012a).…”

Advances in instrumentation and methodology for solid-state NMR of biological assemblies

“…This probe required high power for broadband excitation over the entire range of the wide-line deuterium spectrum, a necessity that can be greatly reduced with the implementation of MAS in conjunction with partial motional averaging [170,175]. An auxillary deuterium coil for decoupling in 2 Hlabeled proteins was developed to provide the necessary decoupling power with a simple modification to existing triple-resonance ( 1 H/ 13 C/ 15 N) biomolecular probes without the need for another instrument [176], while a quadruple-resonance probe operating at 600 MHz proton Larmor frequency was built for multidimensional experiments including 2 H excitation [177]. In multidimensional experiments, the signal-to-noise was demonstrated to be comparable or slightly better when initiating excitation through deuterium, and using Rotor Echo Short Pulse IRrAdiaTION (RESPIRATION) [172] and optimal control (OC) cross polarization pulse sequences allowed for lower rf power, better tolerance of rf inhomogeneity, and improved effciency of magnetization transfer [178].…”

“…In multidimensional experiments, the signal-to-noise was demonstrated to be comparable or slightly better when initiating excitation through deuterium, and using Rotor Echo Short Pulse IRrAdiaTION (RESPIRATION) [172] and optimal control (OC) cross polarization pulse sequences allowed for lower rf power, better tolerance of rf inhomogeneity, and improved effciency of magnetization transfer [178]. 2D experiments utilizing initial excitation on deuterium and hydrogen were compared, showing di erences in cross peaks that can be used to identify solvent-exposed structural components or membrane interiors due to their relative ability to back-exchange [177]. The Martin lab recently developed a quadruple-resonance ( 1 H/ 13 C/ 2 H/ 15 N) probe for use at 800 MHz 1 H Larmor frequency.…”

Organische Chemie 2014

“…In der hochauflösenden Festkörper‐MAS‐NMR‐Spektroskopie gab es im letzten Jahr erfreuliche Entwicklungen für Proteinstrukturbiologen, die mit kleineren Probenmengen auskommen. Die Gruppe um Oschkinat entwickelte eine Serie von Experimenten für deuterierte Proteine, bei der das Experiment nicht wie üblich mit Protonen‐ sondern mit Deuteriumanregung beginnt 151. Dies wird mit einem anschließenden Kreuzpolarisationsschritt auf 13C‐ und schlussendlicher 1H‐Detektion kombiniert.…”