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Methods Mol Biol. 2016;1408:93-105. doi: 10.1007/978-1-4939-3512-3_7.

A Fluorometric Activity Assay for Light-Regulated Cyclic-Nucleotide-Monophosphate Actuators.

Author information

1
Institut für Biologie, Biophysikalische Chemie, Humboldt-Universität zu Berlin, Berlin, Germany.
2
Department of Molecular Sensory Systems, Research Center Caesar, Bonn, Germany.
3
Institut für Biologie, Neurobiologie, Freie Universität Berlin, Berlin, Germany.
4
Institut für Biologie, Biophysikalische Chemie, Humboldt-Universität zu Berlin, Berlin, Germany. andreas.moeglich@uni-bayreuth.de.
5
Faculty of Biology, Chemistry and Earth Sciences, Lehrstuhl für Biochemie, Universität Bayreuth, Building NW III, Universitätsstraße 30, Bayreuth, 95440, Germany. andreas.moeglich@uni-bayreuth.de.

Abstract

As a transformative approach in neuroscience and cell biology, optogenetics grants control over manifold cellular events with unprecedented spatiotemporal definition, reversibility, and noninvasiveness. Sensory photoreceptors serve as genetically encoded, light-regulated actuators and hence embody the cornerstone of optogenetics. To expand the scope of optogenetics, ever more naturally occurring photoreceptors are being characterized, and synthetic photoreceptors with customized, light-regulated function are being engineered. Perturbational control over intracellular cyclic-nucleotide-monophosphate (cNMP) levels is achieved via sensory photoreceptors that catalyze the making and breaking of these second messengers in response to light. To facilitate discovery, engineering and quantitative characterization of such light-regulated cNMP actuators, we have developed an efficient fluorometric assay. Both the formation and the hydrolysis of cNMPs are accompanied by proton release which can be quantified with the fluorescent pH indicator 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF). This assay equally applies to nucleotide cyclases, e.g., blue-light-activated bPAC, and to cNMP phosphodiesterases, e.g., red-light-activated LAPD. Key benefits include potential for parallelization and automation, as well as suitability for both purified enzymes and crude cell lysates. The BCECF assay hence stands to accelerate discovery and characterization of light-regulated actuators of cNMP metabolism.

KEYWORDS:

BCECF; Cyclic nucleotide monophosphate; LAPD; Microtiter plate; Nucleotide cyclase; Optogenetics; Sensory photoreceptor; bPAC; cNMP phosphodiesterase

PMID:
26965118
DOI:
10.1007/978-1-4939-3512-3_7
[Indexed for MEDLINE]

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