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.
Highlights
At present, autofluorescent proteins have become indispensable tools in many biological and medical studies
This review does not discuss these indicators in detail, we need to specify that all of them work on the principle that is extremely analogous to that of circularly permuted FPs (cpFPs)-based probes wherein effective conformational coupling is achieved by fluorescent proteins (FPs) structure destabilization and locating a molecular switch close to the chromophore with a mobile microenvironment
The drawbacks of the already available Forster resonance energy transfer (FRET)-based biosensors for cGMP visualization were overcome by Nausch et al, who engineered cpEGFP-based probes by inserting the latter into the regulatory domains of protein kinases G (PKG) I α or β [87]
Summary
Autofluorescent proteins (aFPs) have become indispensable tools in many biological and medical studies. Many modern analytical techniques exploit the unique nature of these macromolecules in order to directly visualize structures and processes in living cells and organisms [10] Owing to their increasing scientific demand, different types of aFPs with optimized parameters—such as fluorescence intensity, maturation rate, phototoxicity, and oligomeric state—have been engineered using molecular biology methods [10]. Development of GEFIs for other cellular parameters usually requires considerably more genetic engineering efforts, often implying the creation of chimeric proteins with additional domains resulting from natural ‘sensors’ In such probes, the sensory unit is responsible for the detection of the tested parameter and provides a molecular switch that affects the FP structure, and the optical properties. We describe how circularly permuted (cp)FP-based sensors help to overcome these obstacles, highlight the general principles underlying their development, and provide a classification of these probes by analytes measured and color
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