It is well established that lipid bilayer membranes provide an optimal native environment for membrane proteins to fold and function but currently, only a handful of spectroscopic methods are suitable for structural studies of such membrane-embedded assemblies. Even then, detailed data on the orientation of protein domains and sidechains with respect to the lipid bilayer normal and lipid-water interface are hard to obtain. However, such essential structural data and, particularly, angular restraints with respect to the bilayer normal, could be obtained by employing macroscopically aligned lipid systems. One of the best developed such system is the bicelles, formed by self-assembly from a mixture of long- and short-chain phospholipids that align macroscopically in sufficiently high static magnetic field of modern NMR spectrometers and a narrow temperature range. Interestingly, a previous small angle X-ray scattering study demonstrated that the alignment persisted even without external magnetic field (Langmuir, 2011, 27, 9122–9130). Here, we employ a more accessible method of spin-labeling EPR to study several-day-long remnant macroscopic alignment of bicelles. Unexpectedly, lipid macrodiscs formed by peptoids also demonstrated similarly remnant macroscopic alignment if the sample temperature is maintained >30 o C. However, the order was lost immediately once the sample temperature dropped below the threshold value, regardless of whether the external magnetic field was maintained. At lower temperatures, the use of nanotemplates, such as those provided by well-organized nanoporous anodic aluminum oxide (AAO), appears to be the only suitable approach to maintain macroscopic alignment. Advantages of such an alignment for structural studies are demonstrated by double electron-electron resonance (DEER) of transmembrane peptides oriented perpendicular to the external magnetic field.
Cao et al. (2026) studied this question.