NanoFAST: structure-based design of a small fluorogen-activating protein with only 98 amino acids.
Konstantin S. Mineev,Konstantin S. Mineev,Sergey A. Goncharuk,Sergey A. Goncharuk,Marina V. Goncharuk,Natalia V. Povarova,Anatolii I. Sokolov,Nadezhda S. Baleeva,Alexander Yu. Smirnov,Ivan N. Myasnyanko,Dmitry A. Ruchkin,Sergey Bukhdruker,Alina Remeeva,Alexey Mishin,Valentin Borshchevskiy,Valentin Borshchevskiy,Valentin Gordeliy,Alexander S. Arseniev,Dmitriy A. Gorbachev,Alexey S. Gavrikov,Alexander S. Mishin,Mikhail S. Baranov,Mikhail S. Baranov +22 more
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TLDR
The shortened FAST is designed, which is composed of only 98 amino acids, the shortest genetically encoded tag among all known fluorescent and fluorogen-activating proteins, by truncating 26 N-terminal residues.Abstract:
One of the essential characteristics of any tag used in bioscience and medical applications is its size. The larger the label, the more it may affect the studied object, and the more it may distort its behavior. In this paper, using NMR spectroscopy and X-ray crystallography, we have studied the structure of fluorogen-activating protein FAST both in the apo form and in complex with the fluorogen. We showed that significant change in the protein occurs upon interaction with the ligand. While the protein is completely ordered in the complex, its apo form is characterized by higher mobility and disordering of its N-terminus. We used structural information to design the shortened FAST (which we named nanoFAST) by truncating 26 N-terminal residues. Thus, we created the shortest genetically encoded tag among all known fluorescent and fluorogen-activating proteins, which is composed of only 98 amino acids.read more
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Engineering of a fluorescent chemogenetic reporter with tunable color for advanced live-cell imaging
Hela Benaissa,Karim Ounoughi,Isabelle Aujard,Evelyne Fischer,Rosette Goïame,Julie Nguyen,Alison G. Tebo,Alison G. Tebo,Chenge Li,Chenge Li,Thomas Le Saux,Giulia Bertolin,Marc Tramier,Lydia Danglot,Nicolas Pietrancosta,Nicolas Pietrancosta,Xavier Morin,Ludovic Jullien,Arnaud Gautier,Arnaud Gautier +19 more
TL;DR: In this paper, a collection of fluorogenic chromophores with various electronic properties enables to generate bimolecular fluorescent assemblies that cover the visible spectrum from blue to red using a single protein tag engineered and optimized by directed evolution and rational design.
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Transient Fluorescence Labeling: Low Affinity-High Benefits.
TL;DR: In this article, the authors highlight the pros and cons of a wide variety of transiently interacting labels and discuss the state of the art and future perspectives of low-affinity labeling methods.
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Recent Advancements in Tracking Bacterial Effector Protein Translocation
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A unique photochromic UV-A sensor protein, Rc-PYP, interacting with the PYP-binding protein.
Suhyang Kim,Yusuke Nakasone,Akira Takakado,Yoichi Yamazaki,Hironari Kamikubo,Masahide Terazima +5 more
TL;DR: In this paper, the intermolecular interaction dynamics observed between PYP from Rhodobacter capsulatus (Rc-PYP) and a possible downstream protein, PYP-binding protein (PBP), were investigated.
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Structure-based rational design of an enhanced fluorogen-activating protein for fluorogens based on GFP chromophore
Marina V. Goncharuk,Nadezhda S. Baleeva,Dmitry E. Nolde,Alexey S. Gavrikov,Alexey Mishin,Alexander S. Mishin,Andrey Yu. Sosorev,Alexander S. Arseniev,Sergey A. Goncharuk,Valentin Borshchevskiy,Roman G. Efremov,Konstantin S. Mineev,Mikhail S. Baranov +12 more
TL;DR: In this paper , the structure-based rational design of the enhanced FAST mutants, optimized for the N871b fluorogen, was described, and two mutants appeared brighter than the wild-type FAST, and these mutants provided up to 35% enhancement for several other fluorogens of similar structure, both in vitro and in vivo.
References
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Journal ArticleDOI
Next-Generation Fluorogen-Based Reporters and Biosensors for Advanced Bioimaging.
Tiphaine Péresse,Arnaud Gautier +1 more
TL;DR: This review presents the toolbox of fluorogen-based reporters and biosensors available to biologists and various applications are detailed to illustrate the possible uses and opportunities offered by this new generation of fluorescent probes and sensors for advanced bioimaging.
Journal ArticleDOI
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TL;DR: A pair of orthogonal tags based on the fluorescence-activating and absorption shifting tag (FAST) that are compatible with two-color, live-cell imaging and display orthogonality not only in their fluorogen recognition capabilities, but also in their one- and two-photon absorption profiles.
Journal ArticleDOI
ReAsH as a Quantitative Probe of In-Cell Protein Dynamics.
TL;DR: Fast relaxation imaging (FReI), combining millisecond temperature jump kinetics with fluorescence microscopy detection, circumvents many of the difficulties encountered working with the ReAsH system, allowing us to obtain quantitative FRET measurements of protein stability and kinetics both in vitro and in cells.
Journal ArticleDOI
Red-Shifted Substrates for FAST Fluorogen-Activating Protein Based on the GFP-Like Chromophores
Natalia V. Povarova,Snizhana O. Zaitseva,Nadezhda S. Baleeva,Alexander Yu. Smirnov,Ivan N. Myasnyanko,Marina B. Zagudaylova,Nina G. Bozhanova,Dmitriy A. Gorbachev,Dmitriy A. Gorbachev,Kseniya K. Malyshevskaya,Kseniya K. Malyshevskaya,Alexey S. Gavrikov,Alexander S. Mishin,Mikhail S. Baranov,Mikhail S. Baranov +14 more
TL;DR: The proposed dye instantly stains target cellular proteins fused with FAST, washes out in a minute timescale, and exhibits higher photostability of the fluorescence signal in confocal and widefield microscopy, in contrast with previously published fluorogen:FAST complexes.
Journal ArticleDOI
Docking-guided identification of protein hosts for GFP chromophore-like ligands
Natalia V. Povarova,Nina G. Bozhanova,Karen S. Sarkisyan,Roman Gritcenko,Mikhail S. Baranov,Ilia V. Yampolsky,Konstantin A. Lukyanov,Alexander S. Mishin +7 more
TL;DR: The computational identification of protein hosts capable of binding to and enhancing fluorescence of GFP chromophore derivatives is reported, and two proteins were found to possess sub-micromolar affinity for some Kaede-like chromophores and activatefluorescence of these fluorogens.