Structure and Dynamics of Nanopore-Confined Membrane Proteins are Affected by Bilayer Lipid Composition

Abstract

Self-assembled nanotubular lipid bilayers confined within macroscopically aligned high-density homogeneous nanopore array structure of anodic aluminum oxide (AAO) membranes exhibit a high degree of macroscopic alignment that facilitates their studies by oriented sample solid state (OS SS) NMR and spin-labeling EPR methods. The principal advantages of the lipid nanotube arrays are in the applicability of this method to essentially any lipid composition and a tolerance of the macroscopic alignment to a wide range of environmental conditions such as, e.g., temperature, pH, and ionic strength. Here we describe 1) a recent progress in reconstructing small pore-forming and transmembrane peptides as well as large membrane protein complexes, such as photosynthetic reaction center (RC) protein in inorganic nanopores and 2) utilization of this technology to study lipid-induced changes in protein conformations by magnetic resonance methods. Specifically, by using OS SS NMR we have shown that the tilt and α-helix kink angles of Pf1 coat protein are affected by the bilayer composition on example of DOPC, POPC and DMPC bilayers. Effects of unsaturated lipids on Pf1 dynamics as evidenced by the changes in the linewidths in the Pf1 spectra are also reported. We relate these changes to the bilayer fluidity and its effects on the uniaxial rotational diffusion of the protein within the membrane. Further, we demonstrate the use of DEER spectroscopy to obtain long-range distance constraints for membrane protein systems incorporated into lipid nanotubes. The improved nanopore alignment technique described here provides a general method for studying lipid-induced structural conformations of membrane proteins under physiologically relevant conditions by magnetic resonance. Supported by U.S. DOE contract No. DE-FG02-02ER15354 to A.I.S. and NSF MRI 1229547 to A.A.N.