Complete resolution of the solid-state NMR spectrum of a uniformly ¹⁵ N-labeled membrane protein in phospholipid bilayers

Abstract

ABSTRACT

Hydration-optimized oriented phospholipid bilayer samples for solid-state NMR structural studies of membrane proteins

“…Finally, while the samples described in this report were prepared by organic solvent deposition of mixed lipids and protein on glass slides, oriented lipid bilayer samples for solid-state NMR structural studies can also be prepared by depositing aqueous suspensions of protein/lipid vesicles, obtained by detergent dialysis [9,27]. The method of choice for reconstitution is protein-dependent, and both methods yield highly oriented lipid bilayer samples that are ready for hydration optimization in reduced humidity atmosphere.…”

“…When highly oriented samples are prepared, multi-dimensional NMR experiments, that are based on the 1 H/ 15 N PI-SEMA pulse sequence [6,7], yield high-resolution spectra [8,9] where the frequencies measured for each resonance depend on the orientation of the corresponding molecular site with respect to the direction of the applied magnetic field, and thus, serve as orientational restraints for three-dimensional structure determination. For membrane proteins in oriented bilayers, the sample orientation is fixed, and each frequency reflects the orientation of a specific site in the protein with respect to the membrane.…”

NMR of membrane proteins in micelles and bilayers: The FXYD family proteins

“…They require the use of solid-state NMR methods since the associated polypeptides are immobile on the 10 4 Hz timescale of the dipolar and chemical shift interactions. Bilayers can be completely aligned between glass plates, and the resulting spectra from the incorporated proteins have line widths that are similar to those of single crystals [70]. Bicelles, prepared by mixing long chain and short chain lipids, can be aligned magnetically perpendicular to the direction to the magnetic field, or flipped to the parallel orientation by the addition of lanthanide ions [71], and have been used recently to obtain high-resolution spectra of membrane proteins [72].…”

“…In all cases the thinnest available glass slides are utilized to obtain the best filling factor in the coil of the probe. With carefully prepared samples it is possible to obtain 15 N resonance line widths of less than 3 ppm [70].…”

Nuclear Magnetic Resonance Structural Studies of Membrane Proteins in Micelles and Bilayers

“…The polarization inversion with exchange at the magic angle (PISEMA) experiment (51) gives high-resolution, two-dimensional (2D), 1 H-15 N dipolar coupling and 15 N chemical shift correlation spectra of oriented membrane proteins where the individual resonances contain orientation restraints for structure determination (4,52). The spectra were obtained with a cross polarization contact time of 1 ms, a 1 H 90° pulse width of 5 µs, and continuous 1 H decoupling of 63 kHz rf field strength.…”