Application of fluorescence correlation spectroscopy to study substrate binding in styrene maleic acid lipid copolymer encapsulated ABCG2

Horsey, Aaron J; Briggs, Deborah A; Holliday, Nicholas D.; Briddon, Stephen J.; Kerr, Ian D.

Abstract

© 2020 The Authors ABCG2 is one of a trio of human ATP binding cassette transporters that have the ability to bind and transport a diverse array of chemical substrates out of cells. This so-called “multidrug” transport has numerous physiological consequences including effects on how drugs are absorbed into and eliminated from the body. Understanding how ABCG2 is able to interact with multiple drug substrates remains an important goal in transporter biology. Most drugs are believed to interact with ABCG2 through the hydrophobic lipid bilayer and experimental systems for ABCG2 study need to incorporate this. We have exploited styrene maleic acid to solubilise ABCG2 from HEK293T cells overexpressing the transporter, and confirmed by dynamic light scattering and fluorescence correlation spectroscopy (FCS) that this results in the extraction of SMA lipid copolymer (SMALP) particles that are uniform in size and contain a dimer of ABCG2, which is the predominant physiological state. FCS was further employed to measure the diffusion of a fluorescent ABCG2 substrate (BODIPY-prazosin) in the presence and absence of SMALP particles of purified ABCG2. Autocorrelation analysis of FCS traces enabled the mathematical separation of free BODIPY-prazosin from drug bound to ABCG2 and allowed us to show that combining SMALP extraction with FCS can be used to study specific drug: transporter interactions.

Citation

Horsey, A. J., Briggs, D. A., Holliday, N. D., Briddon, S. J., & Kerr, I. D. (2020). Application of fluorescence correlation spectroscopy to study substrate binding in styrene maleic acid lipid copolymer encapsulated ABCG2. BBA - Biomembranes, 1862(6), https://doi.org/10.1016/j.bbamem.2020.183218

• Journal Article Type: Article
• Acceptance Date: Jan 30, 2020
• Online Publication Date: Feb 11, 2020
• Publication Date: Jun 1, 2020
• Deposit Date: Feb 13, 2020
• Publicly Available Date: Feb 13, 2020
• Journal: Biochimica et Biophysica Acta (BBA) - Biomembranes
• Print ISSN: 0005-2736
• Electronic ISSN: 1879-2642
• Publisher: Elsevier
• Peer Reviewed: Peer Reviewed
• Volume: 1862
• Issue: 6
• Article Number: 183218
• DOI: https://doi.org/10.1016/j.bbamem.2020.183218
• Keywords: Biophysics; Cell Biology; Biochemistry
• Public URL: https://nottingham-repository.worktribe.com/output/3958245
• Publisher URL: https://www.sciencedirect.com/science/article/pii/S0005273620300444
• Additional Information: This article is maintained by: Elsevier; Article Title: Application of fluorescence correlation spectroscopy to study substrate binding in styrene maleic acid lipid copolymer encapsulated ABCG2; Journal Title: Biochimica et Biophysica Acta (BBA) - Biomembranes; CrossRef DOI link to publisher maintained version: https://doi.org/10.1016/j.bbamem.2020.183218; Content Type: article; Copyright: © 2020 The Authors. Published by Elsevier B.V.