Computational redesign of a fluorogen activating protein with Rosetta

• Main Authors: Baranov, M.S (Author), Bender, B.J (Author), Bozhanova, N.G (Author), Gavrikov, A.S (Author), Gorbachev, D.A (Author), Harp, J.M (Author), Lukyanov, K.A (Author), Meiler, J. (Author), Mercado, C.B (Author), Mishin, A.S (Author), Zhang, X. (Author)
• Format: Article
• Language: English
• Published: Public Library of Science 2021
• Subjects: 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; amino acid sequence; Amino Acid Sequence; Article; biology; Boron Compounds; boron derivative; chemistry; chromatophore; Computational Biology; computer analysis; computer model; controlled study; Crystallography, X-Ray; drug design; Drug Design; fluorescence; Fluorescence; fluorescence imaging; fluorescence microscopy; fluorescent dye; Fluorescent Dyes; fluorogen activating protein; genetics; HEK293 cell line; HEK293 Cells; human; Humans; in vitro study; ligand binding; Luminescent Proteins; macromolecule; Microscopy, Fluorescence; Models, Molecular; molecular docking; Molecular Docking Simulation; molecular model; mutation; photoprotein; prediction; procedures; protein analysis; protein binding; protein conformation; Protein Conformation; protein engineering; Protein Engineering; protein stability; recombinant protein; Recombinant Proteins; Rosetta modeling; software; Software; unclassified drug; X ray crystallography
• Online Access: View Fulltext in Publisher

 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 finetuning 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. Copyright © 2021 Bozhanova et al.