(ii) The ESIPT process is also sensitive to environmental factors. Enol emission tends to dominate in polar-protic solvents, presumably due to hydrogen bonding that disrupts the intramolecular isomerization from 1-E* to 1-K*. Alternatively, non-polar solvents or local desolvation from polar-protic solvents allows the excited-state isomerization to proceed-effectively turning ON keto emission. The environmental dependence of the excited state isomerization process allows for a dual-emission readout. This multichannel output adds ratiometric characteristics to ESIPT capable dyes which enhance assay sensitivity. The ratiometric difference between enol and keto emission was originally demonstrated by Syntik and Kasha 10 to interrogate the local desolvation effects upon nonspecific binding to human serum albumin. More recent studies have expanded on this work in various albumin proteins. ESIPT fluorophores have also been incorporated into non-natural amino acids to detect peptide/nucleic acid interactions, protein secondary structure, and detection of α-synuclein aggregation. Fig. (A) X-ray crystal structure of WT-transthyretin (WT-TTR) with bound tafamidis (PDB 3tct). A limitation of the above studies was that only relatively subtle changes in the enol and keto fluorescence ratios were observed. This general lack of sensitivity arose from a common theme of ESIPT schemes reported to date: (i) the assay relied on non-specific binding of the EISPT chromophore to the target of interest. (ii) When the ESIPT reporter was conjugated to a molecule that provides selectivity for the intended target, this construct meant that the environmentally sensitive ESIPT fluorophore did not directly interact with the target. Thus, any environmental changes experienced by the ESIPT fluorophore were largely an outer-sphere effect. Furthermore, conjugating a chromophore onto a specific binding ligand can often lead to a decrease in binding affinity, selectivity and sensitivity to the target. The goal of this study was to design a protein that could bind an ESIPT fluorophore and selectively alter the enol and keto fluorescence signals through the specific interactions that take place between the small molecule and protein binding partner.