A protocol for identifying the binding sites of small molecules on the cystic fibrosis transmembrane conductance regulator (CFTR) protein

Onofrio Laselva; Evgeniy V. Petrotchenko; C. Michael Hamilton; Zafar Qureshi; Christoph H. Borchers; Robert N. Young; Christine E. Bear

STAR Protocols (Jun 2022)

A protocol for identifying the binding sites of small molecules on the cystic fibrosis transmembrane conductance regulator (CFTR) protein

Onofrio Laselva,
Evgeniy V. Petrotchenko,
C. Michael Hamilton,
Zafar Qureshi,
Christoph H. Borchers,
Robert N. Young,
Christine E. Bear

Affiliations

Onofrio Laselva: Department of Medical and Surgical Sciences, University of Foggia, Foggia, Italy
Evgeniy V. Petrotchenko: Segal Cancer Proteomics Center, Lady Davis Institute, Jewish General Hospital, McGill University, Montreal, QC, Canada; Center for Computational and Data-Intensive Science and Engineering, Skolkovo Institute of Science and Technology, Moscow, Russia
C. Michael Hamilton: Department of Chemistry, Simon Fraser University, 8888 University Dr., Burnaby, BC V5A 1S6, Canada
Zafar Qureshi: Department of Chemistry, Simon Fraser University, 8888 University Dr., Burnaby, BC V5A 1S6, Canada
Christoph H. Borchers: Segal Cancer Proteomics Center, Lady Davis Institute, Jewish General Hospital, McGill University, Montreal, QC, Canada; Center for Computational and Data-Intensive Science and Engineering, Skolkovo Institute of Science and Technology, Moscow, Russia
Robert N. Young: Department of Chemistry, Simon Fraser University, 8888 University Dr., Burnaby, BC V5A 1S6, Canada
Christine E. Bear: Programme in Molecular Medicine, Hospital for Sick Children, 686 Bay Street, Toronto, ON M5G 0A4, Canada; Department of Physiology, University of Toronto, Toronto, ON, Canada; Department of Biochemistry, University of Toronto, Toronto, ON, Canada; Corresponding author

Journal volume & issue: Vol. 3, no. 2
p. 101258

Abstract

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Summary: We describe a protocol to identify the binding site(s) for a drug called ivacaftor that potentiates the CFTR chloride channel. We use photoaffinity probes—based on the structure of ivacaftor—to covalently modify the CFTR protein at the region that constitutes the drug binding site(s). We define the methods for photo-labeling CFTR, its membrane extraction, and enzymatic digestion using trypsin. We then describe the experimental methods to identify the modified peptides by using mass spectrometry.For complete details on the use and execution of this protocol, please refer to Laselva et al. (2021).

Published in STAR Protocols

ISSN: 2666-1667 (Online)
Publisher: Elsevier
Country of publisher: United States
LCC subjects: Science: Science (General)
Website: https://star-protocols.cell.com/

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