Role of Ser65, His148 and Thr203 in the Organic Solvent‐dependent Spectral Shift in Green Fluorescent Protein

The photophysics of green fluorescent protein (GFP) is remarkable because of its exceptional property of excited state proton transfer (ESPT) and the presence of a functional proton wire. Another interesting property of wild‐type GFP is that its absorption and fluorescence excitation spectra are sensitive to the presence of polar organic solvents even at very low concentrations. Here, we use a combination of methodologies including site‐specific mutagenesis, absorption spectroscopy, steady‐state and time‐resolved fluorescence measurements and all‐atom molecular dynamics simulations in explicit solvent, to uncover the mechanism behind the unique spectral sensitivity of GFP toward organic solvents. Based on the evidences provided herein, we suggest that organic solvent‐induced changes in the proton wire prevent ground state movement of a proton through the wire and thus bring about the spectral changes observed. The present study can not only help to understand the mechanism of proton transfer by further dissecting the intricate steps in GFP photophysics but also encourages to develop GFP‐based organic solvent biosensors. Absorption spectra of GFP are displayed in the presence of aqueous and polar organic solvents. The equilibrium between neutral (395 nm) and anionic (475 nm) chromophore (p‐hydroxybenzylidene imidazolidin‐5‐one) species under aqueous environment is altered toward anionic population in the organic solvent environment.