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

Title data

Schumacher, Charlotte Helene ; Körschen, Heinz G. ; Nicol, Christopher ; Gasser, Carlos ; Seifert, Reinhard ; Schwärzel, Martin ; Möglich, Andreas:
A Fluorometric Activity Assay for Light-Regulated Cyclic-Nucleotide-Monophosphate Actuators.
In: Methods in Molecular Biology. Vol. 1408 (2016) . - pp. 93-105.
ISSN 1940-6029
DOI: https://doi.org/10.1007/978-1-4939-3512-3_7

Abstract in another language

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.

Further data

Item Type: Article in a journal
Refereed: Yes
Additional notes: PubMed-ID: 26965118
Institutions of the University: Faculties > Faculty of Biology, Chemistry and Earth Sciences
Faculties > Faculty of Biology, Chemistry and Earth Sciences > Department of Chemistry
Faculties > Faculty of Biology, Chemistry and Earth Sciences > Department of Chemistry > Chair Biochemistry > Chair Biochemistry - Univ.-Prof. Dr. Andreas Möglich
Faculties
Faculties > Faculty of Biology, Chemistry and Earth Sciences > Department of Chemistry > Professorship Biochemistry
Faculties > Faculty of Biology, Chemistry and Earth Sciences > Department of Chemistry > Chair Biochemistry
Result of work at the UBT: No
DDC Subjects: 500 Science > 540 Chemistry
Date Deposited: 21 Sep 2016 06:41
Last Modified: 03 May 2017 07:21
URI: https://eref.uni-bayreuth.de/id/eprint/34756