Trehalose-protected lipid membranes for determining membrane protein structure and insertion

Abstract

Trehalose preserves lipid bilayers during dehydration and rehydration by replacing water to form hydrogen bonds between its own OH groups and lipid headgroups. We compare the lipid conformation and dynamics between trehalose-protected lyophilized membranes and hydrated membranes, to assess the suitability of the trehalose-containing membrane as a matrix for membrane protein structure determination. 31 P spectra indicate that the lipid headgroup of trehalose-protected dry POPC membrane (TRE-POPC) have an effective phase transition temperature that is ~50 K higher than that of the hydrated POPC membrane. In contrast, the acyl chains have similar transition temperatures in the two membranes. Intramolecular lipid 13 C'-31 P distances are the same in TRE-POPC and crystalline POPC, indicating that the lipid headgroup and glycerol backbone conformation is unaffected by trehalose incorporation. Intermolecular 13 C-31 P distances between a membrane peptide and the lipid headgroups are 10% longer in the hydrated membrane at 226 K than in the trehalose-protected dry membrane at 253 K. This is attributed to residual motions in the hydrated membrane, manifested by the reduced 31 P chemical shift anisotropy, even at the low temperature of 226 K. Thus, trehalose lyoprotection facilitates the study of membrane protein structure by allowing experiments to be conducted at higher temperatures than possible with the hydrated membranes.

Investigation of the Curvature Induction and Membrane Localization of the Influenza Virus M2 Protein Using Static and Off-Magic-Angle Spinning Solid-State Nuclear Magnetic Resonance of Oriented Bicelles

“…In general, for non-spinning membrane samples, the 31 P CSA is determined by both the chemical structure of the headgroup and lipid reorientational motions. Immobilized lipid headgroups at low temperature give a rigid-limit 31 P chemical shift span of ~190 ppm 40 . Fast uniaxial rotational diffusion and wobble of the molecular axis in liquid-crystalline lamellar bilayers average this CSA to ~45 ppm, and the maximum intensity occurs at the upfield edge of the power pattern.…”

Matrix‐Free DNP‐Enhanced NMR Spectroscopy of Liposomes Using a Lipid‐Anchored Biradical

“…For cases in which the sample of interest can afford a dried state and cryogenic temperatures, it should then be possible to directly perform DNP on a powder with optimal filling factor of the rotor, just as it is usually the case for standard solid‐state NMR. Protecting liposomes in the dried state using trehalose has been demonstrated by Tang et al 27. However, the DNP experiment performed on lyophilized liposomes with N ‐propyl‐PALMIPOL embedded, gave almost no signal enhancement ( ε DNP =1.5).…”

“…Indeed, a wide range of organisms are using the non‐reductive disaccharide trehalose to survive in adverse conditions such as sub‐zero temperatures25 or in the absence of water 26. In the context of solid‐state NMR spectroscopy, trehalose has been successfully used in the past to protect lipid membranes in near‐dry conditions27 and for the application of matrix‐free DNP on proteins 17. In addition to its biocompatibility, the amount of trehalose needed to cryo‐ or dry‐protect lipids is small, limiting thus the occupied volume in the NMR rotor and the intensity of unwanted additive peaks in the spectra.…”

Cryoprotection of lipid membranes for high-resolution solid-state NMR studies of membrane peptides and proteins at low temperature

“…Three resolved carbonyl peaks and doublets for G2 and C are observed, indicating that glycerol creates two discrete conformations in the DMPC headgroup, backbone and the beginning of the acyl chains. These two conformations are likely related to the two isomers in the crystal structures of DMPC (Nomura, Lintuluoto and Morigaki, 2011) and other phosphatidylcholine lipids (Tang, Waring and Hong, 2007). Each isomer can have two different carbonyl chemical shifts due to the inequivalence of sn -1 and sn -2 chains, thus a maximum of four C′ peaks is possible.…”