Engineering hydrogen bonding at tyrosine-201 in the orange carotenoid protein using halogenated analogues.

The Orange Carotenoid Protein (OCP) is a unique water-soluble photoactive protein that plays a critical role in regulating the balance between light harvesting and photoprotective responses in cyanobacteria. The challenge in understanding OCP´s photoactivation mechanism stems from the heterogeneity of the initial configurations of its embedded ketocarotenoid, which in the dark-adapted state can form up to two hydrogen bonds to critical amino acids in the protein's C-terminal domain, and the extremely low quantum yield of primary photoproduct formation. While a series of experiments involving point mutations within these contacts helped us to identify these challenges, they did not resolve them. To overcome this, we shifted from classical mutagenesis to the translational introduction of non-canonical amino acid residues into the OCP structure. In this work, we demonstrate that replacing a single meta-hydrogen in tyrosine-201 with a halogen atom (chlorine, bromine, or iodine) leads to targeted modifications in the keto-carotenoid-protein matrix interaction network, both in the dark-adapted state and upon photoactivation. We found that such atomic substitutions allow us to effectively weaken key hydrogen bonds without disrupting protein folding, thereby increasing the yield of OCP photoactivation products. Such genetically encoded chemical modification of individual atoms and their systematic in situ variation in complex protein structures establishes a foundation for transforming OCP into a practical tool for optogenetics and other applications.