Fine Tuning the Optical Properties of Green to Red Photoconvertible Fluorescent Proteins

Abstract

Dendra2 is an engineered, monomeric GFP-like protein that belongs to a sub-class of fluorescent proteins undergoing irreversible photoconversion from a green- to a red-emitting state upon exposure to purple-blue light. We have measured the X-ray structure of the green species of Dendra2 and performed a comprehensive characterization of the optical absorption and fluorescence properties of the protein in both its green and red forms. The structure, which is very similar to those reported for the closely related proteins EosFP and Kaede, revealed a local structural change next to the chromophore, involving mainly Arg66 and a water molecule. We propose that this structural change explains the blue shift of the absorption and emission bands, as well as the markedly higher pKs of the hydroxyphenyl moiety of the chromophore, which were determined as 7.1 and 7.5 for the green and red species, respectively. The 20-fold enhancement of the neutral species in Dendra2 at physiological pH accounts for the observed higher photoconversion yield of this protein in comparison to EosFP.