Lipid bilayer membranes are essential for conformational stability and function of membrane proteins. Currently, lipid nanodiscs, liposomes, bicelles, and micelles of various compositions are utilized to solubilize a broad range of membrane proteins while substrate-supported lipid bilayers are employed to place and pattern membrane proteins on surfaces. The latter method is also suitable for macroscopic alignment of membrane proteins – an essential prerequisite of high-resolution magnetic resonance and other spectroscopic studies. However, maintaining macroscopic alignment of such samples or those in the form of magnetically aligned bicelles or nanodiscs over a broad range of experimental conditions such pH, ionic strength, and temperature is proven to be a difficult if not impossible task. Here we describe methods for forming self-assembled lipid nanotubular bilayers inside cylindrical nanopores composed of anodic aluminum oxide (AAO). Such hybrid nanostructures, named lipid nanotube arrays, represent a new type of substrate-supported and macroscopically aligned lipid bilayers of defined curvature that have many attractive features for biophysical studies of membranes and membrane proteins. Optical properties of AAO allow for assessing the integrity of membrane protein complexes by UV-vis while high density of the deposited lipids and proteins enable examination by other biophysical methods, including differential scanning calorimetry (DSC), quartz crystal microbalance (QCM), and magnetic resonance. The latter studies have shown that the individual lipids in such nanopore-confined structures maintain fast uniaxial diffusion and a high degree of macroscopic alignment. The macroscopic alignment enables detailed studies of the effects of lipid composition on the structure of integral membrane proteins by solid state NMR, EPR, DEER, and HYSCORE. Accessibility of either both or mainly inner leaflets of the nanotubular bilayers to water-soluble species provides for studies of protein, peptides, and drug binding. Finally, we demonstrate the use of such hybrid nanostructures in QCM biosensor capable of quantifying changes in specific volume upon thermal phase transition of sub-microliter volumes of lipids bilayers and membrane proteins.
