Approximately 30% of the proteins expressed from the human genome correspond to membrane proteins. They perform a wide variety of cellular functions and are very attractive drug targets, with almost half of all pharmaceutics targeting these proteins. G protein coupled receptors (GPCRs) are the largest class of integral membrane proteins comprised of seven transmembrane-spanning helices, an extracellular N-terminus, and an intracellular C-terminus. CXCR1 is a class A, rhodopsin-like GPCR which couples to the Gi G-proteins, and has one high-affinity ligand, interleukin-8 (IL8). Many methods used to study membrane protein structure, function, and dynamics require significant modifications to be made to the protein of interest, resulting in an environment that is far from the native phospholipid bilayer. However, NMR spectroscopy is a powerful technique which allows the study of unmodified membrane proteins in near-native lipid environments and at physiological temperatures and pH.
IL8 interacts with CXCR1 at two sites - the N-terminus (Site I) and the extracellular loops (Site II). To probe these interactions, 1H fast MAS solid-state NMR was used to study IL8 bound to CXCR1 in pnhospholipid bilayers. A majority of the IL8 residues become immobilized upon receptor binding and their chemical shifts significantly perturbed, indicating that the environment and dynamics of the ligand are affected by interactions with CXCR1. Additional long-range distance restraints for the IL8-CXCR1 interaction were obtained via paramagnetic relaxation enhancement (PRE) experiments utilizing incorporation of a metal-chelating unnatural amino acid, 2-amino-3-(8-hydroxyquinolin-3-yl) propanoic acid (HQA), into various CXCR1 constructs positioned at residues near the ligand binding sites.
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. The use of detergents can be especially detrimental to membrane proteins. We have developed styrene-maleic acid (SMA) macrodiscs which provide a detergent-free phospholipid bilayer environment for biophysical and functional studies of membrane proteins under physiological conditions. They are particularly well suited for structure determination by oriented-sample solid-state NMR and high-resolution solid-state NMR spectra of membrane proteins have been obtained in these discs.