Construction of Protein-Based Biosensors Using Ligand-Directed Chemistry for Detecting Analyte Binding.
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 GABAAreceptor which permits quantitative analysis of the ligand affinity.
Graduate School of Engineering, Kyoto University, Katsura, Kyoto, Japan.
Kei
K
Yamaura
Graduate School of Engineering, Kyoto University, Katsura, Kyoto, Japan.
Shigeki
S
Kiyonaka
Graduate School of Engineering, Kyoto University, Katsura, Kyoto, Japan; Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency, Kawaguchi, Saitama, Japan. Electronic address: ihamachi@sbchem.kyoto-u.ac.jp.
Itaru
I
Hamachi
D000818:Animals; D015374:Biosensing Techniques / Q000379:methods*; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D005456:Fluorescent Dyes / Q000032:analysis / Q000378:metabolism; D058785:GABA-A Receptor Agonists / Q000494:pharmacology; D058787:GABA-A Receptor Antagonists / Q000494:pharmacology; D057809:HEK293 Cells; D006801:Humans; D008024:Ligands; D008856:Microscopy, Fluorescence / Q000379:methods; D008958:Models, Molecular; D061848:Optical Imaging / Q000379:methods; D011963:Receptors, GABA-A / Q000032:analysis / Q000378:metabolism*; D013050:Spectrometry, Fluorescence / Q000379:methods
D005456:Fluorescent Dyes; D058785:GABA-A Receptor Agonists; D058787:GABA-A Receptor Antagonists; D008024:Ligands; D011963:Receptors, GABA-A
Chemical labeling; Drug screening; Fluorescent biosensor; GABA(A) receptor; Ligand binding; Membrane protein