Characterisation of styrene-maleic acid-solubilised G protein-coupled receptors

Detergents have historically been used to solubilise membrane proteins for structural studies and pharmacological research, however detergents can alter the lipid environment surrounding a membrane protein. The styrene-maleic acid (SMA) copolymer has been designed to solubilise membrane proteins from the cell membrane, with the lipid bilayer intact, thus forming styrene-maleic acid lipid particles (SMALP). This would retain the native conformation of the protein and therefore suitable for applications such as drug discovery. In this project, the adenosine 2A receptor (A2AR) and the calcitonin gene-related peptide receptor (CGRPR) were solubilised into SMALPs. Various techniques were used to characterise the SMA-solubilised receptors.Out of the SMA copolymers tested, SMA2000 was chosen as the copolymer to solubilise the GPCRs. The copolymer was also compared with the new diisobutylene-maleic acid (DIBMA) copolymer, which has better resistance to divalent cations than the SMA copolymer. Molecular techniques confirmed the expression of the GPCRs in membranes and after solubilisation into SMALPs. Radioligand binding assays demonstrated that the A2AR retained its binding capability when solubilised and purified. The binding assay showed that the A2AR was more stable in SMALPs than DIBMA lipid particles (DIBMALP). Various techniques were used to characterise the A2AR-SMALP, providing novel properties of GPCRs in SMALP. The x-ray radiolytic footprinting (XRF) was used to detect regions of the GPCR-SMALP which were exposed to hydroxyl modification. The transmembrane domain, and the intracellular surface of the SMA-solubilised A2AR were exposed to water, demonstrating the SMALPs can successfully be used in XRF. Fluorescence correlation spectroscopy (FCS) was implemented to characterise the pharmacology of a single ligand binding to a single receptor, where the pharmacological profile of the A2AR was successfully characterised when in a SMALP.SMALPs were also tested for their applicability in phage display in order to generate GPCR-specific nanobodies against receptors in their native conformation. The M13 phage used in this project were conjugated with a VHH nanobody. The A2AR-SMALP was immobilised onto ELISA plates for phage binding, where approximately 22% of the total phage were A2AR-SMALP specific, which was lower than the control Fab protein. Avi-tagged A2AR and CGRPR constructs were designed to improve the immobilisation of the SMALPs, to yield a higher enrichment of phage, specific to the GPCR of interest.Finally, the photoaffinity cross-linking assay was implemented in this project, which has potential implications in drug discovery as receptors can be locked into a particular conformation when cross-linked with a ligand. The technique can theoretically be applied to receptors in SMALPs. Residues of the extracellular loops 1 and 3 of the CGRPR were studied. The assay showed residues A199, N200 and N201 of the extracellular loop 1 forming crosslinks with the ligand, when substituted with azidophenylalanine. Overall, the project demonstrated techniques which are applicable to study SMA-solubilised receptors. Using the various techniques revealed novel properties of the GPCRs in SMALPs. SMALPs were also applied to the drug discovery technique, phage display, with limited success. Techniques were incorporated into this study to improve the applicability of SMALPs in phage display

Functional characterisation of G protein-coupled receptors

Characterisation of receptors can involve either assessment of their ability to bind ligands or measure receptor activation as a result of agonist or inverse agonist interactions. This review focuses on G protein-coupled receptors (GPCRs), examining techniques that can be applied to both receptors in membranes and after solubilisation. Radioligand binding remains a widely used technique, although there is increasing use of fluorescent ligands. These can be used in a variety of experimental designs, either directly monitoring ligand itself with techniques such as fluorescence polarisation or indirectly via resonance energy transfer (fluorescence/Forster resonance energy transfer, FRET and bioluminescence resonance energy transfer, BRET). Label free techniques such as isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR) are also increasingly being used. For GPCRs, the main measure of receptor activation is to investigate the association of the G protein with the receptor. The chief assay measures the receptor-stimulated binding of GTP or a suitable analogue to the receptor. The direct association of the G protein with the receptor has been investigated via resonance energy techniques. These have also been used to measure ligand-induced conformational changes within the receptor; a variety of experimental techniques are available to incorporate suitable donors and acceptors within the receptor

Single molecule binding of a ligand to a G-protein-coupled receptor in real time using fluorescence correlation spectroscopy, rendered possible by nano-encapsulation in styrene maleic acid lipid particles

The fundamental importance of membrane proteins in cellular processes has driven a marked increase in the use of membrane mimetic approaches for studying and exploiting these proteins. Nano-encapsulation strategies which preserve the native lipid bilayer environment are particularly attractive. Consequently, the use of poly(styrene co-maleic acid) (SMA) has been widely adopted to solubilise proteins directly from cell membranes by spontaneously forming "SMA Lipid Particles" (SMALPs). G-protein-coupled receptors (GPCRs) are ubiquitous "chemical switches", are central to cell signalling throughout the evolutionary tree, form the largest family of membrane proteins in humans and are a major drug discovery target. GPCR-SMALPs that retain binding capability would be a versatile platform for a wide range of down-stream applications. Here, using the adenosine A2A receptor (A2AR) as an archetypical GPCR, we show for the first time the utility of fluorescence correlation spectroscopy (FCS) to characterise the binding capability of GPCRs following nano-encapsulation. Unbound fluorescent ligand CA200645 exhibited a monophasic autocorrelation curve (dwell time, τD = 68 ± 2 μs; diffusion coefficient, D = 287 ± 15 μm2 s-1). In the presence of A2AR-SMALP, bound ligand was also evident (τD = 625 ± 23 μs; D = 30 ± 4 μm2 s-1). Using a non-receptor control (ZipA-SMALP) plus competition binding confirmed that this slower component represented binding to the encapsulated A2AR. Consequently, the combination of GPCR-SMALP and FCS is an effective platform for the quantitative real-time characterisation of nano-encapsulated receptors, with single molecule sensitivity, that will have widespread utility for future exploitation of GPCR-SMALPs in general

Photoaffinity cross-linking and unnatural amino acid mutagenesis reveal insights into calcitonin gene-related peptide binding to the calcitonin receptor-like receptor/receptor activity-modifying protein 1 (CLR/RAMP1) complex

Calcitonin gene-related peptide (CGRP) binds to the complex of the calcitonin receptor-like receptor (CLR) with receptor activity-modifying protein 1 (RAMP1). How CGRP interacts with the transmembrane domain (including the extracellular loops) of this family B receptor remains unclear. In this study, a photoaffinity cross-linker, p-azido l-phenylalanine (azF), was incorporated into CLR, chiefly in the second extracellular loop (ECL2) using genetic code expansion and unnatural amino acid mutagenesis. The method was optimized to ensure efficient photolysis of azF residues near the transmembrane bundle of the receptor. A CGRP analogue modified with fluorescein at position 15 was used for detection of ultraviolet-induced cross-linking. The methodology was verified by confirming the known contacts of CGRP to the extracellular domain of CLR. Within ECL2, the chief contacts were I284 on the loop itself and L291, at the top of the fifth transmembrane helix (TM5). Minor contacts were noted along the lip of ECL2 between S286 and L290 and also with M223 in TM3 and F349 in TM6. Full length molecular models of the bound receptor complex suggest that CGRP sits at the top of the TM bundle, with Thr6 of the peptide making contacts with L291 and H295. I284 is likely to contact Leu12 and Ala13 of CGRP, and Leu16 of CGRP is at the ECL/extracellular domain boundary of CLR. The reduced potency, Emax, and affinity of [Leu16Ala]-human α CGRP are consistent with this model. Contacts between Thr6 of CGRP and H295 may be particularly important for receptor activation

Expression and purification of recombinant G protein-coupled receptors: A review

Given their extensive role in cell signalling, GPCRs are significant drug targets; despite this, many of these receptors have limited or no available prophylaxis. Novel drug design and discovery significantly rely on structure determination, of which GPCRs are typically elusive. Progress has been made thus far to produce sufficient quantity and quality of protein for downstream analysis. As such, this review highlights the systems available for recombinant GPCR expression, with consideration of their advantages and disadvantages, as well as examples of receptors successfully expressed in these systems. Additionally, an overview is given on the use of detergents and the styrene maleic acid (SMA) co-polymer for membrane solubilisation, as well as purification techniques