Circularly Permuted Fluorescent Protein-Based Indicators: History, Principles, and Classification

Abstract

Genetically encoded biosensors based on fluorescent proteins (FPs) are a reliable tool for studying the various biological processes in living systems. The circular permutation of single FPs led to the development of an extensive class of biosensors that allow the monitoring of many intracellular events. In circularly permuted FPs (cpFPs), the original N- and C-termini are fused using a peptide linker, while new termini are formed near the chromophore. Such a structure imparts greater mobility to the FP than that of the native variant, allowing greater lability of the spectral characteristics. One of the common principles of creating genetically encoded biosensors is based on the integration of a cpFP into a flexible region of a sensory domain or between two interacting domains, which are selected according to certain characteristics. Conformational rearrangements of the sensory domain associated with ligand interaction or changes in the cellular parameter are transferred to the cpFP, changing the chromophore environment. In this review, we highlight the basic principles of such sensors, the history of their creation, and a complete classification of the available biosensors.

Genetically Encoded Biosensors Based on Fluorescent Proteins

“…Various biosensors have been developed based on cpFP [ 83 ]. For example, the first cpFP-based biosensors, GCaMP and pericam [ 84 , 85 ], were developed to detect the intracellular Ca 2+ levels.…”

Genetically encoded thiol redox-sensors in the zebrafish model: lessons for embryonic development and regeneration

“…One broadly used type of genetically encoded sensor for redox state alterations is based on circularly permutated fluorescent proteins (cpFPs) (Kostyuk et al 2019). In cpFPs, the original protein ends are fused and new termini generated at another place in the sequence while maintaining the overall structure.…”

A Bright and Colorful Future for G-Protein Coupled Receptor Sensors

“…These tools purposely combine the high ligand-binding affinity and molecular specificity which were fine-tuned in the receptor by natural evolution, with the large sensitivity typical of intensity-based probes engineered from cpGFP (Marvin et al, 2011;Chen et al, 2013b;Marvin et al, 2013;Kostyuk et al, 2019). Because GPCRs are a very large family of receptors (class-A alone comprises approximately 350 members without including odorant receptors (Pándy-Szekeres et al, 2018), for an overview see Figures 1A,B), in principle they represent a largely unexplored pool from which novel fluorescent sensors could be developed to probe a vast amount of endogenous neurochemicals.…”