Monitoring of post-translational modification dynamics with genetically encoded fluorescent reporters

Abstract

Post-translational modifications (PTMs) of proteins are essential mechanisms for virtually all dynamic processes within cellular signaling networks. Genetically encoded reporters based on fluorescent proteins (FPs) are powerful tools for spatiotemporal visualization of cellular parameters. Consequently, commonly used modular biosensor designs have been adapted to generate several protein-based indicators for monitoring various PTMs or the activity of corresponding enzymes in living cells, providing new biological insights into dynamics and regulatory functions of individual PTMs. In this review, we describe the application of general design strategies focusing on PTMs and discuss important considerations for engineering feasible indicators depending on the purpose. Moreover, we present developments and enhancements of PTM biosensors from selected studies and give an outlook on future perspectives of this versatile approach.

Keywords: FRET; biosensor; microscopy.

Figure 1

General design and operation of a FP-based biosensor. (A) Molecular toolbox for the generation of modular FP-based biosensors. A sensing unit comprised of an intrinsic or engineered molecular switch is combined with a FP-based reporting unit. Optionally, a targeting motif can be added. (B) Schematic example of the operation of a unimolecular FRET-based PTM sensor with an engineered molecular switch. The substrate peptide is modified resulting in a conformational change that increases FRET. (C) Representative schematic diagram of the dependence of the readout on the PTM activity or state, respectively, for a FP-based PTM reporter, also illustrating the general properties of a biosensor.