Genetically encoded biosensors based on engineered fluorescent proteins

Abstract

Fluorescent proteins have revolutionized cell biology by allowing researchers to non-invasively peer into the inner workings of cells and organisms. While the most common applications of fluorescent proteins are to image expression, localization, and dynamics of protein chimeras, there is a growing interest in using fluorescent proteins to create biosensors for minimally invasive imaging of concentrations of ions and small molecules, the activity of enzymes, and changes in the conformation of proteins in living cells. This tutorial review provides an overview of the progress made in the development of fluorescent protein-based biosensors to date.

Fig. 1

The protein and chromophore structure of avGFP. Two orthogonal views of the avGFP structure (PDB ID. 1EMA) with the intrinsic chromophore shown in stick representation. Also shown is the chromophore formation mechanism.

Fig. 2

Schematic representation of the mechanism of a translocation-based biosensor. At the top of the figure is shown the expected result when imaging a cell expressing a translocation-based biosensor.

Fig. 3

Methods of detecting protein–protein interactions using FPs. (a) FP complementation. (b) FRET for detecting protein–protein interactions.

Fig. 4

FRET-based biosensors. (a) Biosensors based on a ligand-dependent protein–protein interaction. Cameleons (based on a fusion of calmodulin and M13) and GTPase biosensors (based on a fusion of the GTPase and its effector) fall into this category. (b) Post-translational modification biosensor (i.e., for a kinase). (c) Protease substrate-type biosensor. (d) Biosensor based on conformational change of a single protein (i.e., based on periplasmic binding proteins).

Fig. 5

Single FP-based biosensors. (a) Single FP biosensor based on intrinsic (i.e., pH) sensitivity. (b) Single FP biosensor based on the extrinsic sensitivity (i.e., Ca²⁺) of a genetically fused domain (i.e., calmodulin). (c) GCaMP X-ray crystal structure. Linker regions that were not visible in the crystal structure are represented with dashed lines.