Solution NMR: A powerful tool for structural and functional studies of membrane proteins in reconstituted environments

Abstract

Edited by Karen G. Fleming A third of the genes in prokaryotic and eukaryotic genomes encode membrane proteins that are either essential for signal transduction and solute transport or function as scaffold structures. Unlike many of their soluble counterparts, the overall structural and functional organization of membrane proteins is sparingly understood. Recent advances in X-ray crystallography, cryo-EM, and nuclear magnetic resonance (NMR) are closing this gap by enabling an in-depth view of these ever-elusive proteins at atomic resolution. Despite substantial technological advancements, however, the overall proportion of membrane protein entries in the Protein Data Bank (PDB) remains <4%. This paucity is mainly attributed to difficulties associated with their expression and purification, propensity to form large multisubunit complexes, and challenges pertinent to identification of an ideal detergent, lipid, or detergent/lipid mixture that closely mimic their native environment. NMR is a powerful technique to obtain atomic-resolution and dynamic details of a protein in solution. This is accomplished through an assortment of isotopic labeling schemes designed to acquire multiple spectra that facilitate deduction of the final protein structure. In this review, we discuss current approaches and technological developments in the determination of membrane protein structures by solution NMR and highlight recent structural and mechanistic insights gained with this technique. We also discuss strategies for overcoming size limitations in NMR applications, and we explore a plethora of membrane mimetics available for the structural and mechanistic understanding of these essential cellular proteins.

The structural basis of T-cell receptor (TCR) activation: An enduring enigma

“…In that study, the authors determined an ensemble of cryoEM structures of yeast TRiC/ CCT at various nucleotide concentrations that included both open and closed states, revealing an unforeseen allosteric network at atomic resolution. Visualizing the cytoplasmic domains of CD3 subunits may require cryoEM analysis of the TCR-CD3 complex in nondetergent systems, such as nanodiscs or amphipols, that may more faithfully reproduce a natural lipid environment (67,71). Finally, none of the conventional structural methods used to study TCR triggering (X-ray crystallography, NMR, and cryoEM) take into account mechanical force, as posited in the mechanosensor model.…”

Structure and Dynamics of GPCRs in Lipid Membranes: Physical Principles and Experimental Approaches

“…This means that by altering q , the suitability of this system can be adjusted for a range of biophysical techniques. For example, DMPC-DHPC bicelles prepared with q values > 2.3 will align with a magnetic field and can be used in oriented-sample solid-state NMR in order to establish 15 N-labelled amide bond orientations and the TM helix angle of the incorporated proteins relative to the bilayer normal [ 237 , 238 ], whilst q values ≤ 0.7 allow bicelles to be sufficiently small for solution-state NMR to be carried out [ 239 ].…”

“…Unfortunately the large size of liposomes even when using SUVs generally precludes their use for solution NMR studies ( Figure 4 ) aiming at a characterisation directly on the intact receptor [ 237 ]. However, waterLOGSY-based NMR spectroscopy has been successfully applied to study the membrane association of the truncated C terminus of the B-type CGRP receptor [ 265 ].…”

The Potential of 19F NMR Application in GPCR Biased Drug Discovery

“…Most recently, its applications have expanded into resolving the cryo-EM structures of alternative complex III in a supercomplex with cytochrome oxidase [79]. In comparison to nanodiscs and detergent systems, an overall smaller molecular weight assembly might offer favorable relaxation advantages for NMR application [80], though no NMR studies of SMALP-reconstituted GPCRs have been reported. An adaptable phospholipid membrane mimetic system was also developed for solution state NMR studies and it has been tested in several proteins, including GPCR [81].…”