A structural basis for the pH-dependent increase in fluorescence efficiency of chromoproteins.

Within the fluorescent protein and chromoprotein family, the phenomenon of photoswitching is both intriguing and biotechnologically useful. Illumination of particular chromoproteins with intense light results in dramatic increases in fluorescence efficiency (termed kindling) and involves cis-trans isomerization of the chromophore. Here we report that chromophore isomerization can also be driven via alteration in pH. Specifically, we demonstrate that a number of naturally occurring chromoproteins, and their engineered variants, undergo a dramatic 20-100-fold increase in fluorescence efficiency at alkaline pH (>pH9.0). We have determined to 1.8 A resolution the structure of one such chromoprotein, Rtms5(H146S), in its highly far-red fluorescent form (Phi(F), 0.11 at pH 10.7) and compared it to the structure of the non-fluorescent form (Phi(F), 0.002 at pH 8.0). At high pH, the cyclic tri-peptide chromophore was observed to be mobile and distributed between a trans non-coplanar and a cis coplanar conformation, whereas at the lower pH, only a trans non-coplanar chromophore was observed. Calculation of pK(a) values suggested that titration of the side-chain of the conserved Glu215 close to the chromophore is involved in promoting the cis-coplanar conformation. Collectively, our data establish that isomerization to form a coplanar chromophore is a basis of the increased fluorescence efficiency at high pH. The phenomenon of pH-induced fluorescence gain has similarities with photoswitching, thereby providing a model to study the mechanism of kindling.

The 1.7 A crystal structure of Dronpa: a photoswitchable green fluorescent protein.

The green fluorescent protein (GFP), its variants, and the closely related GFP-like proteins possess a wide variety of spectral properties that are of widespread interest as biological tools. One desirable spectral property, termed photoswitching, involves the light-induced alteration of the optical properties of certain GFP members. Although the structural basis of both reversible and irreversible photoswitching events have begun to be unraveled, the mechanisms resulting in reversible photoswitching are less clear. A novel GFP-like protein, Dronpa, was identified to have remarkable light-induced photoswitching properties, maintaining an almost perfect reversible photochromic behavior with a high fluorescence to dark state ratio. We have crystallized and subsequently determined to 1.7 A resolution the crystal structure of the fluorescent state of Dronpa. The chromophore was observed to be in its anionic form, adopting a cis co-planar conformation. Comparative structural analysis of non-photoactivatable and photoactivatable GFPs, together with site-directed mutagenesis of a position (Cys62) within the Dronpa chromophore, has provided a basis for understanding Dronpa photoactivation. Specifically, we propose a model of reversible photoactivation whereby irradiation with light leads to subtle conformational changes within and around the environment of the chromophore that promotes proton transfer along an intricate polar network.

Amino acid substitutions around the chromophore of the chromoprotein Rtms5 influence polypeptide cleavage.

Extension of the conjugated pi-system of many all-protein chromophores with an acylimine bond is the basis for their red-shifted optical properties. The presence of this post-translational modification is evident in crystal structures of these proteins. Harsh denaturation of proteins containing an acylimine bond results in partial polypeptide cleavage. For the red fluorescent protein DsRed, the extent of cleavage is quantitative. However, this is not the case for the blue non-fluorescent chromoprotein Rtms5, even though all chromophores in tetrameric Rtms5 contain an acylimine bond. We have identified two positions around the chromophore of Rtms5 where substitutions can promote or suppress the extent of cleavage on harsh denaturation. We propose a model in which cleavage of Rtms5 is facilitated by a trans to cis isomerisation of the chromophore.