Biochemical measurements of ligand binding to eukaryotic membrane proteins are challenging because they can require large amounts of purified protein. For this reason, ligand binding is preferentially evaluated on soluble domains rather than on the full length proteins. In this chapter, we describe the use of fluorescence size exclusion chromatography-based thermostability (FSEC-TS) as an assay to monitor ligand binding to the full length mammalian ion channel HCN4. FSEC-TS monitors the effect of the ligand on the thermal denaturation curve of the protein by following the fluorescence of a fused GFP protein. Changes in the melting temperature (Tm) provide a quantitative value for measuring ligand-protein interaction. As a proof of concept, we describe here the protocol for monitoring the binding of the second messenger cAMP and of the known HCN drug Ivabradine to the purified GFP-HCN4 channel. cTMP, a known non-binder of HCN channels, is used as a control. Due to the small amount of protein required, the assay represents a high-throughput screening system for evaluating binding of small molecules to full length proteins.

Detection of ligand binding to purified HCN channels using fluorescence-based size exclusion chromatography / A. Saponaro, A.S. Sharifzadeh, A. Moroni (METHODS IN ENZYMOLOGY). - In: Methods in Enzymology[s.l] : Academic Press, 2021 Feb. - ISBN 9780323853743. - pp. 105-123 [10.1016/bs.mie.2021.01.043]

Detection of ligand binding to purified HCN channels using fluorescence-based size exclusion chromatography

A. Saponaro
Primo
;
A. Moroni
Ultimo
2021-02

Abstract

Biochemical measurements of ligand binding to eukaryotic membrane proteins are challenging because they can require large amounts of purified protein. For this reason, ligand binding is preferentially evaluated on soluble domains rather than on the full length proteins. In this chapter, we describe the use of fluorescence size exclusion chromatography-based thermostability (FSEC-TS) as an assay to monitor ligand binding to the full length mammalian ion channel HCN4. FSEC-TS monitors the effect of the ligand on the thermal denaturation curve of the protein by following the fluorescence of a fused GFP protein. Changes in the melting temperature (Tm) provide a quantitative value for measuring ligand-protein interaction. As a proof of concept, we describe here the protocol for monitoring the binding of the second messenger cAMP and of the known HCN drug Ivabradine to the purified GFP-HCN4 channel. cTMP, a known non-binder of HCN channels, is used as a control. Due to the small amount of protein required, the assay represents a high-throughput screening system for evaluating binding of small molecules to full length proteins.
cAMP; Eukaryotic membrane proteins; Fluorescence size exclusion chromatography-based thermostability assay; HCN4 channels; Ivabradine; Ligand-protein interaction; Animals; Chromatography, Gel; Ligands; Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels
Settore BIO/09 - Fisiologia
Settore BIO/10 - Biochimica
Settore BIO/11 - Biologia Molecolare
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/2434/854993
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