Exploring cofactor-binding fluorescent proteins as novel platforms for oxygen-independent metal ion sensors

Fluorescent protein-based metal ion sensors have played central roles in elucidating the dynamics, trafficking, and homeostasis of metals across biological systems. Many of these platforms have combined proteins from the green fluorescent protein (GFP) family and mFruits with metal-sensitive binding domains like calmodulin or zinc fingers. These sensors have often been well-optimized and widely used but given their reliance on GFPs they cannot be applied in living systems under low oxygen conditions. GFPs and mFruits contain an intrinsic fluorophore that requires oxygen to form. Alternative oxygen-independent cofactor-binding fluorescent proteins utilize extrinsic chromophores that do not require oxygen to bind and impart fluorescence. This review will focus on using flavin- and bilin-based fluorescent proteins as platforms for metal ion sensors that do not require oxygen. We organize these oxygen-independent metal sensors into three categories, those based on green-emitting flavin-based fluorescent proteins, far-red/near-infrared-emitting biliverdin IXα-based fluorescent proteins, and orange fluorescent phycoerythrobilin-binding cyanobacteriochrome-based fluorescent proteins. The aim of this review is to provide a detailed compilation and analysis of the utility of each of these groups of fluorescent proteins for developing metal ion biosensors. We describe the background and highlight protein engineering efforts underlying each category of proteins and then examine metal ion sensors that have been developed, in our work and by others, using these flavin- and bilin-based fluorescent proteins. We also discuss challenges and directions for future studies, drawing inspiration from the GFP toolbox of metal ion sensors and including routes for developing and applying advanced cofactor-binding fluorescent protein-based metal sensors.