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

Abstract

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 A_2A receptor (A_2AR) 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, τ_D = 68 ± 2 μs; diffusion coefficient, D = 287 ± 15 μm² s⁻¹). In the presence of A_2AR-SMALP, bound ligand was also evident (τ_D = 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 A_2AR. 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.