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

Research output: Contribution to journalArticle

Authors

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

External organisations

  • University of Birmingham

Abstract

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.

Details

Original languageEnglish
Article number183218
Pages (from-to)1-11
Number of pages11
JournalBiochimica et Biophysica Acta (BBA) - Biomembranes
Volume1862
Issue number6
Early online date11 Feb 2020
Publication statusPublished - 1 Jun 2020
Externally publishedYes

Keywords

  • ABC transporter, pharmacology, multidrug resistance, membrane protein, SMALP, fluorescence, fluorescence correlation spectroscopy, photon counting histogram