A thermostable flavin-based fluorescent protein from Chloroflexus aggregans: a framework for ultra-high resolution structural studies.

Autor: Nazarenko VV; Moscow Institute of Physics and Technology, Dolgoprudny, Russia. ivan.gushchin@phystech.edu., Remeeva A; Moscow Institute of Physics and Technology, Dolgoprudny, Russia. ivan.gushchin@phystech.edu., Yudenko A; Moscow Institute of Physics and Technology, Dolgoprudny, Russia. ivan.gushchin@phystech.edu., Kovalev K; Moscow Institute of Physics and Technology, Dolgoprudny, Russia. ivan.gushchin@phystech.edu and ICS-6: Structural Biochemistry, Forschungszentrum Jülich GmbH, Jülich, Germany and Institut de Biologie Structurale Jean-Pierre Ebel, Université Grenoble Alpes-Commissariat à l'Energie Atomique et aux Energies Alternatives-CNRS, Grenoble, France and Institute of Crystallography, RWTH Aachen University, Aachen, Germany., Dubenko A; Moscow Institute of Physics and Technology, Dolgoprudny, Russia. ivan.gushchin@phystech.edu., Goncharov IM; Moscow Institute of Physics and Technology, Dolgoprudny, Russia. ivan.gushchin@phystech.edu., Kuzmichev P; Moscow Institute of Physics and Technology, Dolgoprudny, Russia. ivan.gushchin@phystech.edu., Rogachev AV; Moscow Institute of Physics and Technology, Dolgoprudny, Russia. ivan.gushchin@phystech.edu and Joint Institute for Nuclear Research, Dubna, Russia., Buslaev P; Moscow Institute of Physics and Technology, Dolgoprudny, Russia. ivan.gushchin@phystech.edu., Borshchevskiy V; Moscow Institute of Physics and Technology, Dolgoprudny, Russia. ivan.gushchin@phystech.edu., Mishin A; Moscow Institute of Physics and Technology, Dolgoprudny, Russia. ivan.gushchin@phystech.edu., Dhoke GV; Institute of Biotechnology, RWTH Aachen University, Aachen, Germany., Schwaneberg U; Institute of Biotechnology, RWTH Aachen University, Aachen, Germany and DWI-Leibniz Institut für Interaktive Materialien, Aachen, Germany., Davari MD; Institute of Biotechnology, RWTH Aachen University, Aachen, Germany., Jaeger KE; Institute of Molecular Enzyme Technology, Heinrich Heine University Düsseldorf, Forschungszentrum Jülich, Jülich, Germany and IBG-1: Biotechnology, Forschungszentrum Jülich GmbH, Jülich, Germany., Krauss U; Institute of Molecular Enzyme Technology, Heinrich Heine University Düsseldorf, Forschungszentrum Jülich, Jülich, Germany., Gordeliy V; Moscow Institute of Physics and Technology, Dolgoprudny, Russia. ivan.gushchin@phystech.edu and ICS-6: Structural Biochemistry, Forschungszentrum Jülich GmbH, Jülich, Germany and Institut de Biologie Structurale Jean-Pierre Ebel, Université Grenoble Alpes-Commissariat à l'Energie Atomique et aux Energies Alternatives-CNRS, Grenoble, France., Gushchin I; Moscow Institute of Physics and Technology, Dolgoprudny, Russia. ivan.gushchin@phystech.edu.
Jazyk: angličtina
Zdroj: Photochemical & photobiological sciences : Official journal of the European Photochemistry Association and the European Society for Photobiology [Photochem Photobiol Sci] 2019 Jul 10; Vol. 18 (7), pp. 1793-1805.
DOI: 10.1039/c9pp00067d
Abstrakt: Light-Oxygen-Voltage (LOV) domains are conserved parts of photoreceptors in plants, bacteria and fungi that bind flavins as chromophores and detect blue light. In the past, LOV domain variants have been developed as fluorescent reporter proteins (called flavin-based fluorescent proteins; FbFPs), which due to their ability to fluoresce under anaerobic conditions, fast folding kinetics and a small size of ∼12-16 kDa are a promising reporter system for quantitative real-time analysis of biological processes. Here, we present a small thermostable flavin-based fluorescent protein CagFbFP derived from a soluble LOV domain-containing histidine kinase from the thermophilic bacterium Chloroflexus aggregans. CagFbFP is composed of 107 amino acids with a molecular weight of 11.6 kDa and consists only of the conserved LOV core domain. The protein is thermostable with a melting point of about 68 °C. It crystallizes easily and its crystals diffract to 1.07 Å. Both the crystal structure and small angle scattering data show that the protein is a dimer. Unexpectedly, glutamine 148, which in LOV photoreceptor proteins is the key residue responsible for signal transduction, occupies two conformations. Molecular dynamics simulations show that the two conformations interconvert rapidly. The crystal structure of the wild-type Chloroflexus aggregans LOV domain determined at 1.22 Å resolution confirmed the presence of two alternative conformations of the glutamine 148 side chain. Overall, this protein, due to its stability and ease of crystallization, appears to be a promising model for ultra-high resolution structural studies of LOV domains and for application as a fluorescent reporter.
Databáze: MEDLINE
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