Chemigenetic tags are versatile labels for fluorescence microscopy that combine some of the advantages of genetically encoded tags with small molecule fluorophores. The Fluorescence Activating and absorbance Shifting Tags (FASTs) bind a series of highly fluorogenic and cell-permeable chromophores. Furthermore, FASTs can be used in complementation-based systems for detecting or inducing protein-protein interactions, depending on the exact FAST protein variant chosen. In this study, we systematically explore substitution patterns on FAST fluorogens and generate a series of fluorogens that bind to FAST variants, thereby activating their fluorescence. This effort led to the discovery of a novel fluorogen with superior properties, as well as a fluorogen that transforms splitFAST systems into a fluorogenic dimerizer, eliminating the need for additional protein engineering.

Members of the ArsR/SmtB family of transcriptional repressors, such as CadC, regulate the intracellular levels of heavy metals like Cd(II), Hg(II), and Pb(II). These metal sensing proteins bind their target metals with high specificity and affinity, however, a lack of structural information about these proteins makes defining the coordination sphere of the target metal difficult. Lingering questions as to the identity of Cd(II) coordination in CadC are addressed via protein design techniques. Two designed peptides with tetrathiolate metal binding sites were prepared and characterized, revealing fast exchange between CdS3O and CdS4 coordination spheres. Correlation of (111m)Cd PAC spectroscopy and (113)Cd NMR spectroscopy suggests that Cd(II) coordinated to CadC is in fast exchange between CdS3O and CdS4 forms, which may provide a mechanism for rapid sensing of heavy metal contaminants by this regulatory protein.

Observing the localization, the concentration, and the distribution of proteins in cells or organisms is essential to understand theirs functions. General and versatile methods allowing multiplexed imaging of proteins under a large variety of experimental conditions are thus essential for deciphering the inner workings of cells and organisms. Here, we present a general method based on the non-covalent labeling of a small protein tag, named FAST (fluorescence-activating and absorption-shifting tag), with various fluorogenic ligands that light up upon labeling, which makes the simple, robust, and versatile on-demand labeling of fusion proteins in a wide range of experimental systems possible.