Construction of Protein-Based Biosensors Using Ligand-Directed Chemistry for Detecting Analyte Binding

Methods Enzymol. 2017:589:253-280. doi: 10.1016/bs.mie.2017.02.006. Epub 2017 Mar 7.

Abstract

Protein-based fluorescent biosensors are powerful tools for quantitative detection of biomolecules or drugs with high sensitivity under physiological conditions. However, conventional methods for construction of biosensors require structural data with high resolution or amino acid sequence information in most cases, which hampers applicability of this method to structurally complicated receptor proteins. To sidestep such limitations, we recently developed a new method that employs ligand-directed chemistry coupled with a bimolecular fluorescence quenching and recovery system, which enabled the conversion of various kinds of membrane-bound receptors to "turn-on" type fluorescent sensors. Here, we describe a protocol for construction of "turn-on" type fluorescent biosensors based on the GABAA receptor which permits quantitative analysis of the ligand affinity.

Keywords: Chemical labeling; Drug screening; Fluorescent biosensor; GABA(A) receptor; Ligand binding; Membrane protein.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Biosensing Techniques / methods*
  • Drug Evaluation, Preclinical / methods*
  • Fluorescent Dyes / analysis
  • Fluorescent Dyes / metabolism
  • GABA-A Receptor Agonists / pharmacology
  • GABA-A Receptor Antagonists / pharmacology
  • HEK293 Cells
  • Humans
  • Ligands
  • Microscopy, Fluorescence / methods
  • Models, Molecular
  • Optical Imaging / methods
  • Receptors, GABA-A / analysis
  • Receptors, GABA-A / metabolism*
  • Spectrometry, Fluorescence / methods

Substances

  • Fluorescent Dyes
  • GABA-A Receptor Agonists
  • GABA-A Receptor Antagonists
  • Ligands
  • Receptors, GABA-A