Chromophore Structure of Photochromic Fluorescent Protein Dronpa: Acid–Base Equilibrium of Two Cis Configurations

Dronpa is a novel photochromic fluorescent protein that exhibits fast response to light. The present article is the first report of the resonance and preresonance Raman spectra of Dronpa. We used the intensity and frequency of Raman bands to determine the structure of the Dronpa chromophore in two thermally stable photochromic states. The acid–base equilibrium in one photochromic state was observed by spectroscopic pH titration. The Raman spectra revealed that the chromophore in this state shows a protonation/deprotonation transition with a pK_a of 5.2 ± 0.3 and maintains the cis configuration. The observed resonance Raman bands showed that the other photochromic state of the chromophore is in a trans configuration. The results demonstrate that Raman bands selectively enhanced for the chromophore yield valuable information on the molecular structure of the chromophore in photochromic fluorescent proteins after careful elimination of the fluorescence background.