Circularly Permuted Fluorogenic Proteins for the Design of Modular Biosensors

Abstract

Fluorescent reporters are essential components for the design of optical biosensors that are able to image intracellular analytes in living cells. Herein, we describe the development of circularly permuted variants of Fluorescence-Activating and absorption-Shifting Tag (FAST) and demonstrate their potential as reporting module in biosensors. Circularly permutated FAST (cpFAST) variants allow one to condition the binding and activation of a fluorogenic ligand (and thus fluorescence) to analyte recognition by coupling them with analyte-binding domains. We demonstrated their use for biosensor design by generating multicolor plug-and-play fluorogenic biosensors for imaging the intracellular levels of Ca in living mammalian cells in real time.

A Far‐Red Emitting Fluorescent Chemogenetic Reporter for In Vivo Molecular Imaging

“…We demonstrated in this work that frFAST could be turned, using the design previously used for the generation of splitFAST, into a far‐red split fluorescent reporter with rapid and reversible complementation for the detection of protein‐protein interactions with no additional engineering. We anticipate that frFAST could be used for the design of far‐red fluorescent biosensors through allosteric coupling of frFAST (or circularly permuted versions) and analyte binding domains, in the same way FAST was previously used for the design of biosensors …”

A far-red fluorescent chemogenetic reporter for in vivo molecular imaging

“…We demonstrated in this work that frFAST could be turned, using the design previously used for the generation of splitFAST 20 , into a far-red split fluorescent reporter with rapid and reversible complementation for the detection of protein-protein interactions with no additional engineering. We anticipate that frFAST could be used for the design of far-red fluorescent biosensors through allosteric coupling of frFAST (or circularly permuted versions) and analyte binding domains, in the same way FAST was previously used for the design of biosensors 19 .…”

“…Imaging of proteins below the diffraction limit using super-resolution imaging by radial fluctuations 17 or single-molecule localization microscopy (SMLM) 18 could moreover be achieved thanks to the ability to label only a subset of FAST-tagged proteins by adjusting the chromophore concentration. Because of its modular nature, FAST allowed also the design of a Ca 2+ biosensor 19 and a fluorescent split reporter, splitFAST, with rapid and reversible complementation for the detection of transient protein-protein interactions 20 .…”

Engineering Glowing Chemogenetic Hybrids for Spying on Cells

“…In this sensor, calcium binding promotes fluorogen binding, and thus fluorescence increase, enabling to image Ca 2+ levels in cells. [99] Sensors can also be built by conditioning the fluorogen binding to a given signal. An infrared fluorescent protease reporter was engineered from a circularly permuted IFP1.4 so that the biliverdin chromophore incorporation is regulated by protease activity.…”