A Time-Saving Strategy for MAS NMR Spectroscopy by Combining Nonuniform Sampling and Paramagnetic Relaxation Assisted Condensed Data Collection

Abstract

We present a timesaving strategy for acquiring 3D magic angle spinning NMR spectra for chemical shift assignments in proteins and protein assemblies in the solid state. By simultaneous application of non-uniform sampling (NUS) and paramagnetic-relaxation-assisted condensed data collection (PACC), we can attain 16-fold time reduction in the 3D experiments without sacrificing the signal-to-noise ratio or the resolution. We demonstrate that with appropriate concentration of paramagnetic dopant introduced into the sample the overwhelming majority of chemical shifts are not perturbed, with the exception of a limited number of shifts corresponding to residues located at the surface of the protein, which exhibit small perturbations. This approach enables multi-dimensional MAS spectroscopy in samples of intrinsically low sensitivity and/or high spectral congestion where traditional experiments fail, and is especially beneficial for structural and dynamics studies of large proteins and protein assemblies.

Mapping protein–protein interactions by double-REDOR-filtered magic angle spinning NMR spectroscopy

“…The U- 13 C, 15 N-labeled LC8 sample containing 5 mM Cu(II)-EDTA was prepared as described previously. (Sun et al 2012) The LC8 protein precipitated from PEG was packed into a 1.9 mm Bruker MAS rotor. The U- 13 C, 15 N CAP-Gly sample was prepared by controlled precipitation from polyethylene glycol (PEG) as described previously (Marulanda et al 2004; Sun et al 2009), and 24.4 mg of the precipitate were packed into a 3.2 mm Bruker rotor.…”

Combined zero-quantum and spin-diffusion mixing for efficient homonuclear correlation spectroscopy under fast MAS: broadband recoupling and detection of long-range correlations

“…MLF powder sample was packed into a 1.6 mm Agilent MAS rotor for subsequent NMR experiments at 14.1 T. U- 13 C, 15 N-dynein light chain LC8 protein was expressed in Escherichia coli , purified, doped with Cu(II)-EDTA to the final concentration of Cu 2+ of 5 mM, and precipitated by controlled precipitation from PEG-3350, as described previously (Marulanda et al 2004; Sun et al 2012b). 9.6 mg of LC8 precipitate were packed into a 1.9 mm Bruker MAS rotor for subsequent NMR experiments at 20.0 T. Expression and purification of HIV-1 CA (NL4–3 A92E construct) were performed as reported previously (Han et al 2013).…”

NMR of sedimented, fibrillized, silica-entrapped and microcrystalline (metallo)proteins

“…This is made easier by fast spinning that mitigates the need for heteronuclear decoupling, thus lowering the power deposition on the sample and on the coil [106]. Such approach has been successfully used to different kinds of samples, including microcrystalline proteins [107], membrane proteins [108,109] and protein aggregates [110].…”