The directed evolution of Cyanobacterial phytochrome 1

The phytochrome family of red/far-red photoreceptors has been optimized by nature to harness light energy to drive the photochemical isomerization of a covalently bound linear tetrapyrrole (bilin) chromophore. Photoactivation of the pigment molecule initiates conformational changes within the surrounding protein scaffold that alter its kinase activity and its ability to interact with other proteins necessary for signal transduction. Directed evolution of the cyanobacterial phytochrome 1 (Cph) was undertaken to elucidate the structural basis of its light sensory activity by remodeling the chemical environment of its linear tetrapyrrole prosthetic group. Apophytochrome mutant libraries generated using random mutagenesis were expressed in Escherichia coli strains engineered to synthesize different bilin precursors, and screened for altered absorbance using digital imaging spectroscopy (DIS) and for gain-of-function fluorescence using fluorescence activated cell sorting (FAGS). In addition to identifying a small region of the apoprotein critical for maintaining Cph1's native spectroscopic properties, our studies revealed a tyrosine-to-histidine mutation that transformed phytochrome into an intensely red fluorescent biliprotein. This tyrosine (i.e. Tyr176 in Cph1) is conserved in all members of the phytochrome superfamily, implicating direct participation in the primary photoprocess of phytochromes.; To more fully probe the role of Tyr176, saturation mutagenesis of Cphl was performed. All Tyr176 mutants, with one exception i.e. Tyr176Pro, yielded soluble Cphl chromoproteins - none of which retained wild type photochemical properties. Substitution of Tyr176 with H-bonding donors His, Gln and Glu produced intensely fluorescent holoproteins with extended bilin chromophores, while other mutants possessed more cyclic, less fluorescent prosthetic groups. One of these mutants (i.e. Tyr176 Arg) altered the specificity of the bilin-binding pocket, enabling a porphyrin to covalently bind to the apoprotein. These results support a dual role for Tyr176 i.e. to direct both conformation and protonation of the bilin prosthetic group. A homology model of the GAF domain implicated Glu189 to be the direct proton donor to the bilin chromophore. Site-directed mutagenesis of Glu189 indicated that this residue was critical for the formation of an extended, protonated bilin chromophore, supporting the hypothesis that Glu189 plays a direct role in chromophore protonation.