Macrodiscs, which are magnetically alignable lipid bilayer discs with diameters of >30 nm, were obtained by solubilizing protein-containing liposomes with styrene-maleic acid copolymers. Macrodiscs provide a detergent-free phospholipid bilayer environment for biophysical and functional studies of membrane proteins under physiological conditions. The narrow resonance linewidths observed from membrane proteins in styrene-maleic acid macrodiscs advance structure determination by oriented sample solid-state NMR spectroscopy.

Membrane proteins have always presented technical challenges for structural studies because of their requirement for a lipid environment. Multiple approaches exist including X-ray crystallography and electron microscopy that can give significant insights into their structure and function. However, nuclear magnetic resonance (NMR) is unique in that it offers the possibility of determining the structures of unmodified membrane proteins in their native environment of phospholipid bilayers under physiological conditions. Furthermore, NMR enables the characterization of the structure and dynamics of backbone and side chain sites of the proteins alone and in complexes with both small molecules and other biopolymers. The learning curve has been steep for the field as most initial studies were performed under non-native environments using modified proteins until ultimately progress in both techniques and instrumentation led to the possibility of examining unmodified membrane proteins in phospholipid bilayers under physiological conditions. This review aims to provide an overview of the development and application of NMR to membrane proteins. It highlights some of the most significant structural milestones that have been reached by NMR spectroscopy of membrane proteins; especially those accomplished with the proteins in phospholipid bilayer environments where they function.

The use of paramagnetic constraints in protein NMR is an active area of research because of the benefits of long-range distance measurements (>10 Å). One of the main issues in successful execution is the incorporation of a paramagnetic metal ion into diamagnetic proteins. The most common metal ion tags are relatively long aliphatic chains attached to the side chain of a selected cysteine residue with a chelating group at the end where it can undergo substantial internal motions, decreasing the accuracy of the method. An attractive alternative approach is to incorporate an unnatural amino acid (UAA) that binds metal ions at a specific site on the protein using the methods of molecular biology. Here we describe the successful incorporation of the unnatural amino acid 2-amino-3-(8-hydroxyquinolin-3-yl) propanoic acid (HQA) into two different membrane proteins by heterologous expression in E. coli. Fluorescence and NMR experiments demonstrate complete replacement of the natural amino acid with HQA and stable metal chelation by the mutated proteins. Evidence of site-specific intra- and inter-molecular PREs by NMR in micelle solutions sets the stage for the use of HQA incorporation in solid-state NMR structure determinations of membrane proteins in phospholipid bilayers.

The human chemokine interleukin-8 (IL-8; CXCL8) is a key mediator of innate immune and inflammatory responses. This small, soluble protein triggers a host of biological effects upon binding and activating CXCR1, a G proteincoupled receptor, located in the cell membrane of neutrophils. Here, we describe 1 H-detected magic angle spinning solid-state NMR studies of monomeric IL-8 (1-66) bound to full-length and truncated constructs of CXCR1 in phospholipid bilayers under physiological conditions. Cross-polarization experiments demonstrate that most backbone amide sites of IL-8 (1-66) are immobilized and that their chemical shifts are perturbed upon binding to CXCR1, demonstrating that the dynamics and environments of chemokine residues are affected by interactions with the chemokine receptor. Comparisons of spectra of IL-8 (1-66) bound to full-length CXCR1 (1-350) and to N-terminal truncated construct NT-CXCR1 (39-350) identify specific chemokine residues involved in interactions with binding sites associated with N-terminal residues (binding site-I) and extracellular loop and helical residues (binding site-II) of the receptor. Intermolecular paramagnetic relaxation enhancement broadening of IL-8 (1-66) signals results from interactions of the chemokine with CXCR1 (1-350) containing Mn 2þ chelated to an unnatural amino acid assists in the characterization of the receptor-bound form of the chemokine.

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.

Compelling evidence indicates that repair of damage to the central nervous system (CNS) is inhibited by the presence of protein factors within myelin that prevent axonal regrowth. Myelin growth inhibitors and their common receptor have been identified as targets in the treatment of spinal cord injury, MS, ALS and stroke. We have determined the NMR structure of one of the myelin growth inhibitors, the neurite outgrowth inhibitor (Nogo). We studied the structure of this protein alone and in the presence of dodecylphosphocholine micelles to mimic the natural cell membrane environment. To understand interactions between Nogo and lipid, we hypothesize that aromatic groups and a negative charge hyperconserved among this family of proteins drive the remarkably strong association of Nogo-66 with a phosphocholine surface. We modeled the docking of dodecylphosphocholine (DPC) with Nogo-66 and found that a lipid choline group could form a stable salt bridge with Glu26 and serve as a membrane anchor point.To test the role of the Glu26 anion in binding choline, we mutated this residue to alanine and assessed the structural consequences, the association with lipid and the affinity for the Nogo receptor. Paramagnetic probes allowed us to define portions of the growth inhibitor that are accessible to solvent (and consequently the Nogo receptor). Using computational docking methods, NMR data and mutagenesis results, we calculated the optimal protein-protein interface between our structure of Nogo and the Nogo receptor. This model has inspired the development of compounds that can bind and block Nogo.

a finding consistent with the results of experimental mutagenesis studies. It was found that lysine snorkeling allows for critical interactions which control tilt of the domain in the membrane. This study has allowed for detailed characterization of the protein-lipid interactions involved in modulating b3 tilt to the membrane and, ultimately, aIIbb3 activation.