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

Rachael L. Grime, Joelle Goulding, Romez Uddin, Leigh A. Stoddart, Stephen J. Hill, David Poyner, S. Briddon, Mark Wheatley

Research output: Contribution to journalArticle

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 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.

Original languageEnglish
Pages (from-to)11518-11525
Number of pages8
JournalNanoscale
Volume12
Issue number21
Early online date19 May 2020
DOIs
Publication statusPublished - 4 Jun 2020

Bibliographical note

Open Access Article. Published on 19 May 2020. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.
This work was supported by funding from the Biotechnology and Biological Sciences Research Council (BBSRC: BB/R016615/1 and BB/R016755/1) to MW and DRP and the Medical Research Council (MRC: MR/N020081/1) to SH and SB. RLG was supported by a BBSRC-MIBTP award to RLG and MW.

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