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

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

Authors

Aaron J Horsey

D Holliday

Deborah A Briggs

Aaron J Horsey

Nicholas D Holliday

Stephen J Briddon

Ian D Kerr

; Ian

Derek Kerr



Abstract

ABCG2 is one of a triumvirate 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 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 a fluorescence 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. Response to Reviewers: Dear Michael We were delighted to read the reviewer comments for our paper and their acknowledgement that the work represents an interesting new method for understanding ABCG2 pharmacology. We have incorporated the vast majority of their suggestions in the revised manuscript and would welcome this now being acceptable for publication. Yours faithfully Ian Kerr, on behalf of all authors

Journal Article Type Article
Journal BBA - Biomembranes
Print ISSN 0005-2736
Publisher Elsevier
Peer Reviewed Peer Reviewed
APA6 Citation Horsey, A. J., Briggs, D. A., Holliday, N. D., Briddon, S. J., & Kerr, I. D. (in press). Application of fluorescence correlation spectroscopy to study substrate binding in styrene maleic acid lipid copolymer encapsulated ABCG2. BBA - Biomembranes,
Keywords ABC transporter; multidrug resistance; SMALP; pharmacology; fluorescence; photon counting histogram