Structure-based rational design of an enhanced fluorogen-activating protein for fluorogens based on GFP chromophore

• DOI: 10.1038/s42003-022-03662-9
• Publication Date: 2022-07-15T04:02:57Z
• Authors: Marina V. Goncharuk; Nadezhda S. Baleeva; Dmitry E. Nolde; Alexey S. Gavrikov; Alexey V. Mishin; Alexander S. Mishin; Andrey Y. Sosorev; Alexander S. Arse...; Sergey A. Goncharuk; Valentin I. Borsh...; Roman G. Efremov; Konstantin S. Mineev; Mikhail S. Baranov
• Keywords: Rational design; Chromophore

"Fluorescence-Activating and absorption-Shifting Tag" (FAST) is a well-studied fluorogen-activating protein with high brightness low size, able to activate wide range of fluorogens. This makes FAST promising target for both fluorogen optimization. Here, we describe the structure-based rational design enhanced mutants, optimized N871b fluorogen. Using spatial structure FAST/N871b complex, NMR relaxation analysis, computer simulations, identify mobile regions in complex suggest mutations that could stabilize ligand. Two our mutants appear brighter than wild-type FAST, these provide up 35% enhancement several other fluorogens similar structure, vitro vivo. Analysis by reveals demonstrate highest stability lowest length intermolecular H-bonds. Computer simulations structural basis such stabilization.