High-Resolution NMR Spectroscopy of a GPCR in Aligned Bicelles

Abstract

Solid-state NMR spectra with single-site resolution of CXCR1, a G protein-coupled receptor (GPCR), were obtained in magnetically-aligned phospholipid bicelles. These results demonstrate that GPCRs in phospholipid bilayers are suitable samples for structure determination by solid-state NMR. The spectra also enable studies of drug-receptor interactions.G protein-coupled receptors (GPCRs) are prized targets for structure determination; however, with seven transmembrane helices and more than 300 residues, they are also among the most challenging. Strategically located in the membrane, these proteins regulate the physiological functions of cells in response to external chemical signals. The information is transmitted through the membrane by a change in conformation, and the resulting activation of a cognate G protein triggers myriad signaling pathways in the cytoplasm. About 1000 GPCRs have been identified in the human genome. Although most are for sensory functions, several hundred are potential drug receptors.The structure of only one GPCR has been determined, and that is of rhodopsin 1 , which responds to photons and not chemical ligands. Although efforts are being made to model GPCRs on the structure of rhodopsin and with other computational methods 2 , it is essential to determine their individual three-dimensional structures in order to understand their mechanisms of action and for structure-based drug design. A number of NMR studies of rhodopsin have been reported that illustrate the complexities encountered in applying both solution NMR to micelle 3 and magic angle spinning solid-state NMR to bilayer 3a, 4 samples of polytopic membrane proteins.

Human Cannabinoid 1 GPCR C-Terminal Domain Interacts with Bilayer Phospholipids to Modulate the Structure of its Membrane Environment

“…The results form the contextual foundation for the current study, which utilizes this hCB1(TMH7/H8) peptide as reconstituted and aligned in a defined dimyristoyl-sn-glycero-3-phosphocholine (DMPC) model-membrane system. Aligned bicelle samples afford high spectral resolution and are well-accepted for analyzing the structure of membrane proteins and investigating membrane properties using heteronuclear dipolar couplings (36)(37)(38)(39). With this experimental system, we now extend our previous findings by identifying and characterizing directly specific intermolecular interactions between hCB1(TMH7/H8) and its membrane phospholipid environment.…”

Interaction of Monomeric Interleukin-8 with CXCR1 Mapped by Proton-Detected Fast MAS Solid-State NMR

“…There are several examples where solid-state NMR methods have been used to characterize the structures of ligands bound to GPCRs (41)(42)(43)(44) as well as GPCRs themselves. Most prior studies of membrane proteins or their ligands have involved the direct detection of signals from labeled 13 C or 15 N sites; this continues to be a fruitful approach and we applied it in our earlier studies of CXCR1 (23,33,45). However, dramatic improvements in sensitivity have been obtained by the implementation of 1 H-detected magic angle spinning (MAS) solid-state NMR methods (46,47), especially when fast MAS is combined with perdeuteration of the samples (46,(48)(49)(50)(51).…”

Defining the sampling space in multidimensional NMR experiments: What should the maximum sampling time be?

“…9f) improves this number somewhat (0.487), but it is obvious that matched filtering is not the means to optimize the sensitivity. For large membrane protein samples [13], it will typically not be possible to record that many t 1 increments, and hence t s values on the order of a few ms are commonly applied [14][15][16][17]. An analysis of employing a sampling time of t s = 2 ms (procedure leading to box 1 in Fig.…”

“…Next, we introduce recipes for optimal sampling under various conditions. Finally, we demonstrate the power of efficient sampling as compared to conventional sampling in the context of 1 H-15 N separated-local-field (SLF) experiments for a 15 N labeled single crystal of N-acetyl-L-leucine (NAL) serving as a model system for uniaxially oriented membrane proteins.…”