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

Titelangaben

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. Bd. 1408 (2016) . - S. 93-105.
ISSN 1940-6029
DOI: https://doi.org/10.1007/978-1-4939-3512-3_7

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.

Weitere Angaben

Publikationsform: Artikel in einer Zeitschrift
Begutachteter Beitrag: Ja
Zusätzliche Informationen: PubMed-ID: 26965118
Institutionen der Universität: Fakultäten > Fakultät für Biologie, Chemie und Geowissenschaften
Fakultäten > Fakultät für Biologie, Chemie und Geowissenschaften > Fachgruppe Chemie
Fakultäten > Fakultät für Biologie, Chemie und Geowissenschaften > Fachgruppe Chemie > Lehrstuhl Biochemie > Lehrstuhl Biochemie II - Photobiochemie - Univ.-Prof. Dr. Andreas Möglich
Fakultäten
Fakultäten > Fakultät für Biologie, Chemie und Geowissenschaften > Fachgruppe Chemie > Professur Biochemie
Fakultäten > Fakultät für Biologie, Chemie und Geowissenschaften > Fachgruppe Chemie > Lehrstuhl Biochemie
Titel an der UBT entstanden: Nein
Themengebiete aus DDC: 500 Naturwissenschaften und Mathematik > 540 Chemie
Eingestellt am: 21 Sep 2016 06:41
Letzte Änderung: 03 Mai 2017 07:21
URI: https://eref.uni-bayreuth.de/id/eprint/34756