Solid-state NMR of membrane peptides and proteins in the lipid cubic phase.

Solid-state nuclear magnetic resonance (ssNMR) is a powerful technique for studying membrane protein structure and dynamics. Ideally, measurements are performed with the protein in a lipid bilayer. However, homogenous reconstitution of functional protein into intact bilayers at sufficiently high concentrations is often difficult to achieve. In this work, we investigate the suitability of the lipid cubic phase (LCP), which incorporates a lipid bilayer, as an alternative medium for ssNMR of integral membrane peptides and proteins. The cubic mesophase has long been used to generate membrane protein crystals for use in X-ray crystallographic structure determination by the so-called in meso method and for protein functional and biophysical characterization. Preparing and handling protein-laden LCP is straightforward. LCP may therefore provide a valuable alternative to native membranes and other membrane mimetics for ssNMR. We tested this idea by conducting standard magic-angle spinning ssNMR experiments on LCP into which gramicidin, a ∼4-kDa transmembrane peptide, or bacterial lipoprotein signal peptidase II (LspA), a ∼20-kDa integral membrane enzyme, had been reconstituted. We report one- and two-dimensional ssNMR spectra for both gramicidin and LspA and the parameters for optimizing spectral quality. The high protein-carrying capacity of the cubic phase facilitated