Fluorescence Decay Analysis of the Model Compounds as an Approach to Photophysical Engineering of Fluorescent Proteins

The study of structure-function relationships between a chromophore and its protein environment plays a key role in photophysical engineering of fluorescent proteins (FPs), specifically, in the guided design of their new variants with a higher fluorescence quantum yield (FQY). The known approaches to FQY increasing mostly rely on suppression of the excited state nonradiative processes, but no tools have been suggested for the tuning of the radiative rate constant (kr), which is also a potentially " adjustable" value. Here, we propose an experimental approach, where the synthetic chromophore of FP models the "fixation" of the most important radiationless constants and allows monitoring of the fluorescence lifetime flexibility (as an indicator of the kr value). As a proof-of-concept, we studied the time-resolved fluorescence behavior of the green and blue FP chromophore analogs in diverse chemical environments. The conformationally locked analog of the GFP chromophore in most cases showed monophasic fluorescence decay kinetics with a lifetime of 2.7-3.0 ns, thus adequately modeling the typical behavior of GFPs with the highest FQYs. Under the conditions of stimulated ionization of this chromophore, we observed the increased (up to 4.3-4.6 ns) fluorescence lifetimes, which can be interpreted in terms of an increase in the radiative constant (kr). The conformationally locked analog of the Sirius chromophore showed biexponential fluorescence decay kinetics, partly simulating the properties of the blue FPs. In an acetic acid solution, this compound exhibited distinct fluorescent properties (elevated fluorescence intensity with a major lifetime population of similar to 4 ns), which can be interpreted as emission of an unusual cationic form of the chromophore.