Computational redesign of a fluorogen activating protein with Rosetta

The use of unnatural fluorogenic molecules widely expands the pallet of available genetically encoded fluorescent imaging tools through the design of fluorogen activating proteins (FAPs). While there is already a handful of such probes available, each of them went through laborious cycles of in vitr...

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Veröffentlicht in:PLoS computational biology 2021-11, Vol.17 (11), p.e1009555-e1009555
Hauptverfasser: Bozhanova, Nina G, Harp, Joel M, Bender, Brian J, Gavrikov, Alexey S, Gorbachev, Dmitry A, Baranov, Mikhail S, Mercado, Christina B, Zhang, Xuan, Lukyanov, Konstantin A, Mishin, Alexander S, Meiler, Jens
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container_title PLoS computational biology
container_volume 17
creator Bozhanova, Nina G
Harp, Joel M
Bender, Brian J
Gavrikov, Alexey S
Gorbachev, Dmitry A
Baranov, Mikhail S
Mercado, Christina B
Zhang, Xuan
Lukyanov, Konstantin A
Mishin, Alexander S
Meiler, Jens
description The use of unnatural fluorogenic molecules widely expands the pallet of available genetically encoded fluorescent imaging tools through the design of fluorogen activating proteins (FAPs). While there is already a handful of such probes available, each of them went through laborious cycles of in vitro screening and selection. Computational modeling approaches are evolving incredibly fast right now and are demonstrating great results in many applications, including de novo protein design. It suggests that the easier task of fine-tuning the fluorogen-binding properties of an already functional protein in silico should be readily achievable. To test this hypothesis, we used Rosetta for computational ligand docking followed by protein binding pocket redesign to further improve the previously described FAP DiB1 that is capable of binding to a BODIPY-like dye M739. Despite an inaccurate initial docking of the chromophore, the incorporated mutations nevertheless improved multiple photophysical parameters as well as the overall performance of the tag. The designed protein, DiB-RM, shows higher brightness, localization precision, and apparent photostability in protein-PAINT super-resolution imaging compared to its parental variant DiB1. Moreover, DiB-RM can be cleaved to obtain an efficient split system with enhanced performance compared to a parental DiB-split system. The possible reasons for the inaccurate ligand binding pose prediction and its consequence on the outcome of the design experiment are further discussed.
doi_str_mv 10.1371/journal.pcbi.1009555
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subjects Amino Acid Sequence
Amino acids
Binding
Binding proteins
Binding sites
Biology and Life Sciences
Boron Compounds - chemistry
Chromophores
Computational Biology
Computer applications
Crystallography, X-Ray
Design
Docking
Drug Design
Fluorescence
Fluorescence microscopy
Fluorescent Dyes - chemistry
Fluorescent proteins
Fluoroscopic imaging
Genetic code
HEK293 Cells
Humans
Image resolution
Ligands
Localization
Luminescent Proteins - chemistry
Luminescent Proteins - genetics
Methods
Microscopy, Fluorescence
Models, Molecular
Molecular Docking Simulation
Mutation
Performance enhancement
Physical Sciences
Protein Conformation
Protein Engineering - methods
Protein Engineering - statistics & numerical data
Proteins
Recombinant Proteins - chemistry
Recombinant Proteins - genetics
Redesign
Research and Analysis Methods
Software
title Computational redesign of a fluorogen activating protein with Rosetta
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