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

doi:10.1016/j.xpro.2022.101258

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

STAR Protoc. 2022 Apr 7;3(2):101258. doi: 10.1016/j.xpro.2022.101258. eCollection 2022 Jun 17.

Authors

Onofrio Laselva¹, Evgeniy V Petrotchenko^{2

3}, C Michael Hamilton⁴, Zafar Qureshi⁴, Christoph H Borchers^{2

3}, Robert N Young⁴, Christine E Bear^{5

6

7}

Affiliations

¹ Department of Medical and Surgical Sciences, University of Foggia, Foggia, Italy.
² Segal Cancer Proteomics Center, Lady Davis Institute, Jewish General Hospital, McGill University, Montreal, QC, Canada.
³ Center for Computational and Data-Intensive Science and Engineering, Skolkovo Institute of Science and Technology, Moscow, Russia.
⁴ Department of Chemistry, Simon Fraser University, 8888 University Dr., Burnaby, BC V5A 1S6, Canada.
⁵ Programme in Molecular Medicine, Hospital for Sick Children, 686 Bay Street, Toronto, ON M5G 0A4, Canada.
⁶ Department of Physiology, University of Toronto, Toronto, ON, Canada.
⁷ Department of Biochemistry, University of Toronto, Toronto, ON, Canada.

Abstract

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).

Keywords: Cell Biology; Cell Membrane; Cell-based Assays; Mass Spectrometry; Molecular/Chemical Probes; Protein Biochemistry.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Binding Sites
Cystic Fibrosis Transmembrane Conductance Regulator* / genetics
Cystic Fibrosis* / drug therapy
Humans
Ion Transport
Mutation

Substances

CFTR protein, human
Cystic Fibrosis Transmembrane Conductance Regulator

Grants and funding

CIHR/Canada