Advances in engineering of fluorescent proteins and photoactivatable proteins with red emission

Abstract

Monomeric fluorescent proteins of different colors are widely used to study behavior and targeting of proteins in living cells. Fluorescent proteins that irreversibly change their spectral properties in response to light irradiation of a specific wavelength, or photoactivate, have become increasingly popular to image intracellular dynamics and superresolution protein localization. Until recently, however, no optimized monomeric red fluorescent proteins and red photoactivatable proteins have been available. Furthermore, monomeric fluorescent proteins, which change emission from blue to red simply with time, so-called fluorescent timers, were developed to study protein age and turnover. Understanding of chemical mechanisms of the chromophore maturation or photoactivation into a red form will further advance engineering of fluorescent timers and photoactivatable proteins with enhanced and novel properties.

Figure 1

Mechanisms of chromophore conversion from a neutral (protonated) to anionic (deprotonated) forms are illustrated for three key subgroups of fluorescent proteins such as PA-GFP, PS-CFP and PS-CFP2 (left), PA-mCherry1 and PA-mRFP1 (middle), and Fluorescent Timers (right). Chemical structures are shown for chromophores of the representative proteins before (top) and after (bottom) the conversion reactions. Colors of the chemical structures correspond to the spectral range of the chromophore emission except for the gray color, which indicates the non-fluorescent chromophore. UV-violet light-induced decarboxylation of the Glu222 residue is followed by the reorganization of the hydrogen bond network around the GFP-like chromophore that results in the chromophore deprotonation (left). Photoactivation by UV-violet light involves decarboxylation of the Glu222 residue and oxidation of the mTagBFP-like chromophore by molecular oxygen to the DsRed-like chromophore in the trans-configuration (middle). Slowed down oxidation of the mTagBFP-like chromophore by molecular oxygen without any light irradiation results in the formation of the DsRed-like chromophore in Fluorescent Timers (right).

Figure 2

Mechanisms of chromophore photoconversion from an anionic (deprotonated) green to anionic (deprotonated) red forms are illustrated for two key subgroups of fluorescent proteins such as Kaede, KikGR, EosFP, tdEosFP, mEos2 and Dendra2 (left), and EGFP, aceGFP, TagGFP, zFP506, amFP486 and ppluGFP2 (right). Chemical structures are shown for chromophores of the representative proteins before (top) and after (bottom) the photoconversion reactions. Colors of the chemical structures correspond to the spectral range of the chromophore emission. The Glu222 residue stabilizes a transition state of the UV-violet light-induced polypeptide backbone cleavage by forming the hydrogen bond network with the Gln42 residue and chromophore forming His65 residue via water molecules; protonation-deprotonation equilibrium shown for the green chromophore is important for the photochemical behavior (left). The oxidative redding of the GFP-like chromophore is a one-photon process, which requires two equivalents of the oxidant per molecule of the fluorescent protein and possibly goes via formation of a radical of the chromophore, resulting in the DsRed-like chromophore (right).