Assessment of Physiological Redox State with Novel FRET Protein Probes

Significance: Development of redox-sensing fluorescent proteins (redox probe proteins) have enabled live imaging of the physiological redox state within a cell, generating new strategies for detecting changes in the redox state during developmental, pathogenic, and aging processes. Several of the probe proteins utilize their characteristic redox-sensing segments as linkers in between two fluorophores, where structural alternations of the segments lead to changes in FRET efficiencies across the fluorophores. In this review we summarize two types of novel FRET-based redox probe proteins, namely redox linker (RL)-derived probes and Redoxfluor. Recent Advances: After these FRET-based redox probe proteins were generated, their responsiveness toward redox-related compounds as well as toward reactive oxygen species or reducing stimuli was investigated in vitro. Notably, both the RL-derived probe and Redoxfluor were found to directly respond to the redox state of glutathione, a main redox-formulating compound, showing a promising property for their use in subsequent in vivo analyses. Redoxfluor was not only used for redox sensing in the cytoplasm, but also utilized for assessing the redox state within peroxisomes. Critical Issues: In contrast to one-fluorophore redox probes such as roGFP and rxYFP proteins, whose usage has been established and widely expanded to various experimental systems, FRET-based redox probes were invented very recently and their applications to in vivo studies are still in their infancy. Future Directions: FRET-based redox probes provide novel approaches for redox sensing that are complementary to other methodologies. Antioxid. Redox Signal. 16, 698-704.