Circularly permuted fluorogenic proteins for the design of modular biosensors

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Circularly permuted fluorogenic proteins for the design of modular biosensors, ACS Chem. Biol. 2018

Cells respond to external and internal stimuli by a series of biochemical events that are tightly controlled in space and time. These downstream processes consist of interactions between macromolecules as well as between macromolecules and analytes. Detecting these interactions involves coupling a measurable output to a nanometer-scale molecular recognition event, typically by linking a reporting module to a sensing domain that responds to an input by undergoing a conformational change. Fluorescent proteins (FP) such as the green fluorescent protein (GFP) and its variants have been essential in the development of genetically encoded optical biosensors. FP-based biosensors typically function either by F.rster resonance energy transfer (FRET), in which an analyte-binding domain is flanked with two FPs acting as donor and acceptor for FRET studies, or by fluorescence intensity change, in which conformational changes induced by analyte binding change the local environment of the chromophore of circularly permuted FPs.

 

 

Although conformationally coupling of FAST and the sensing unit allows one to condition fluorogen binding to analyte recognition, it also makes the dynamic range of the sensor and the affinity for the analyte dependent on the fluorogen concentration. Consequently, the fluorogen concentration is a control parameter that must be chosen with care to optimize measurements with the sensors. Despite this, the use of FAST for sensor design opens exciting perspectives. Its robustness and tolerance to insertions make FAST a particularly interesting scaffold for sensor design at once taking advantage of its relatively small size and the ability to change its color at-will. The use of cpFASTs could prove useful in the screening of new sensing modules for which current development is stifled by intrinsic limitations of FPs such as maturation time, tendency to oligomerize, and oxygen sensitivity. The simplicity and tractability of such an approach should allow the design of new plug-and-play biosensors for imaging in real-time analytes, endogenous biomolecules and cellular processes for which no systems currently exist.

 

 

Résumé: 

ACS Chem. Biol. 2018

Fluorescent reporters are essential components for the design of optical biosensors 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 Ca2+in living mammalian cells in real-time.

Equipe de rattachement 8640: 
Chimie Biophysique
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Références: 

Circularly permuted fluorogenic proteins for the design of modular biosensors

 

Alison G Tebo, Frederico M Pimenta, Martha Zoumpoulaki, Carlos Kikuti, Helena Sirkia, Marie-Aude Plamont, Ann Houdusse, and Arnaud Gautier

 

ACS Chem. Biol. 2018

 

DOI: 10.1021/acschembio.8b00417