NMR structural studies of membrane proteins in bilayer environments

Abstract

Membrane protein research focused on studying the structure, function, and interactions of proteins in membrane environments by NMR spectroscopy will be presented. Sample preparation is challenging because membrane proteins are generally low expressing in heterologous systems, hydrophobic, and difficult to re‐fold into their active conformations – any variation from the native structure results in inactive and/or mis‐folded protein. Typically, the purification or extraction of these proteins requires the use of detergents, which can be detrimental to their structure and function. Therefore, an important goal is the development and implementation of detergent‐free environments for NMR studies of membrane proteins.The main membrane environments used for NMR studies include micelles, bicelles, liposomes, and nano‐ and macro‐discs. Studying the same protein in different environments can reveal significant differences in the structure, conformation, and orientation of the protein. For example, the HIV‐1 protein Vpu, studied using static oriented sample solid state NMR, in different lipid bicelle environments resulted in different tilt angles of the protein in the model membrane as well as the absence (or presence) of a kink in the transmembrane helix, depending on the bilayer‐forming phospholipids. Another example is the structure obtained for the hepatitis C protein p7. In DPC micelles, the protein is a hexamer whereas in DHPC micelles the protein is a monomer, having the typical two‐transmembrane spanning protein topology.A macrodisc is a detergent‐free model membrane system composed of phospholipid bilayers surrounded by a peptide, protein, or styrene‐maleic acid (SMA) polymer belt, with a diameter of > 30nm. They serve to immobilize and magnetically align embedded membrane proteins. Examples of proteins having between one and seven trans‐membrane helices will be described, including G‐protein coupled receptors (GPCRs).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

NMR-Based Methods for Protein Analysis

“…The most essential prerequisite for membrane protein structural analysis is the use of a proper membrane environment to ensure that both structures and functions of membrane proteins are retained. Several technologies have been developed for in vitro reconstitution of proteins in the membranous environment, such as proteoliposomes, 133 bicelles, 134,135 styreme maleic acid lipid particles (SMALPs), 136 and nanodiscs 137 to study the structure of membrane proteins by using solution NMR methods. Recently, vesicles such as phospholipid vesicles 138 and outer membrane vesicles (OMVs) 139 have been prepared as cell membrane models for membrane protein structural analysis in their native membrane environments.…”