Computational redesign of a fluorogen activating protein with Rosetta.

Nina G Bozhanova,Mikhail S Baranov,Dmitry A Gorbachev,Alexander S Mishin,Xuan Zhang,Brian J Bender,Konstantin A Lukyanov,Joel M Harp,Christina B Mercado,Alexey S Gavrikov,Jens Meiler,Marco Punta

doi:10.1371/journal.pcbi.1009555

Abstract

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.

Highlights

Fluorogenic molecules are compounds whose ability to fluoresce can be modulated, for example, by a chemical modification, change in the environment, or electronic structure [1]
The identity of two amino acids in positions 36 and 141 of the wild type Blc was converted to the corresponding amino acids in DiB1, and a total of 50 structures were generated using Rosetta Relax application [18]
The best scoring docking pose was further used for the ligand binding pocket design. In this model the ligand was located within an interaction distance from amino acids at positions 141 and 74, mutations in which were shown to influence the properties of the ligand:protein complex in our previous study [12]

Summary

Introduction

Fluorogenic molecules are compounds whose ability to fluoresce can be modulated, for example, by a chemical modification, change in the environment, or electronic structure [1]. A number of fluorogenic molecules have been discovered in living organisms, among them are retinal, flavin mononucleotide, tetrapyrroles such as biliverdin and bilirubin, etc. The biological role of many natural fluorogens is often directly connected to their ability to absorb light. The fluorescence of natural fluorogenic molecules appears to be only a side effect or its function is not yet understood. A fatty-acid-binding protein UnaG has been discovered and cloned from Japanese eel. UnaG binds bilirubin, which allows for its bright fluorescence, but the biological role of the observed fluorescence is not known [4]

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Discussion

Conclusion