| Use of fluorescent proteins in studying in vivo processes in mammalian systems requires development of near-infrared biomarkers due to clear signals unimpeded by absorption or auto fluorescence of biomolecules. Bacteriophytochromes that use biliverdin as their chromophore have been engineered to form monomeric near-infrared biomarkers. The original design template for a fluorescent BphP was the chromophore binding domain of Deinococcus radiodurans (DrCBD), with a D207H substitution. This variant proved to be the hallmark for the next generation of phytochrome biomarkers including IFP1.4 (D207H+11 substitutions), WiPhy (D207HY263F) and the iRFP family based on Rhodopseudomonas palustris BphPs. We solved the x-ray crystal structures of IFP1.4 and several monomeric DrCBD variants, to explain the origins of fluorescence in derived BphPs. A comparative study revealed two main themes. First, hydrophobic packing around the D-ring increases fluorescence by limiting D-ring motion. Second, while the 207th residue is critical to photochemistry it certainly does not need to be His in order to maximize fluorescence. In fact, the highest fluorescence quantum yield to date in this family belongs to a D207L variant in which waters are excluded from the chromophore vicinity (WiPhy2). Higher quantum yields and longer excited state lifetimes than in the parent suggest the loss of a de-excitation pathway via proton transfer. Continuing our structural analysis, we have turned to the formation of the deep figure of eight knot in the protein structure and uncovered the kinetic dependence of knot formation on cis-trans isomerization of Pro236 in the lasso loop of the GAF domain, which is essential for threading of the knot. We have also engineered a smaller phytochrome variant without the characteristic figure of eight knot topology. Our engineered knotless CBD folds and binds bilin. Combining structural insights with protein engineering will help us understand assembly of holophytochrome and design better near-infrared fluorophores. |
