Origins of Fluorescence in Evolved Bacteriophytochromes [Molecular Biophysics]

September 24th, 2014 by Bhattacharya, S., Auldridge, M. E., Lehtivuori, H., Ihalainen, J. A., Forest, K. T.

Use of fluorescent proteins to study in vivo processes in mammals requires near-infrared (NIR) biomarkers that exploit the ability of light in this range to penetrate tissue. Bacteriophytochromes (BphPs) are photoreceptors that couple absorbance of NIR light to photoisomerization, protein conformational changes and signal transduction. BphPs have been engineered to form NIR fluorophores, including IFP1.4, Wi-Phy, and iRFP, initially by replacement of Asp207 by His. This position was suggestive because its main chain carbonyl is within hydrogen-bonding distance to pyrrole ring nitrogens of the biliverdin chromophore, thus potentially functioning as a crucial proton sink during photoconversion. In order to explain the origin of fluorescence in these phytofluors, we solved the crystal structures of IFP1.4 and a comparison non-fluorescent monomeric phytochrome DrCBDmon. Met186 and Val288 in IFP1.4 are responsible for the formation of a tightly packed hydrophobic hub around the biliverdin D ring. Met186 is also responsible for the blue-shifted IFP1.4 excitation maximum relative to the parent BphP. The structure of IFP1.4 revealed decreased structural heterogeneity and a contraction of two surface regions as direct consequences of side chain substitutions. Unexpectedly, IFP1.4 with Asp207 reinstalled (IFPrev) has a higher fluorescence quantum yield (~9%) than any NIR phytofluor published to date. In agreement, fluorescence lifetime measurements confirm the exceptionally long excited state lifetimes, up to 815 ps, in IFP1.4 and IFPrev. Our research helps delineate the origin of fluorescence in engineered BphPs, and will facilitate the widespread adoption of phytofluors as biomarkers.