Nanopore-Confined Bilayers: A Model of Biomembranes with Defined Curvature and a Tool for Oriented Sample Magnetic Resonance

Abstract

It is well established that biological function of cellular membranes is determined by both membrane proteins and the membrane biochemical composition. Moreover, the membrane shape/local curvature may also play a role in modulating membrane properties and the proteins’ function. While specific biomembrane compositions are more readily replicated in biophysical bench experiments, formation of bilayers of specific shapes and curvature represents a more challenging task. Macroscopic alignment of such membranes – an important prerequisite of high resolution magnetic resonance and other studies – is even more difficult especially over a broad range of experimental conditions such pH, ionic strength, and temperature. 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 both membrane biophysics and structure-function protein studies by spectroscopic techniques. 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 DSC, 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 effects of lipid composition on structure of integral membrane proteins by solid state oriented sample NMR, EPR and DEER. Accessibility of either both or mainly inner leaflet of the nanotubular bilayers to water-soluble species provides for studies of protein, peptides, and drug binding. Supported by DE-FG02-02ER15354 to AIS.