Probing the spectral signatures of orange carotenoid protein by orthogonal translation with aromatic non-canonical amino acids.

Orange Carotenoid Protein (OCP) is a water-soluble photoreceptor involved in photoprotection of cyanobacteria. The photoactive OCP contains a bound ketocarotenoid cofactor held in a protein matrix with a hydrogen bonding network. We have developed a system to replace essential residues of the photoactive OCP with non-canonical aromatic analogues that produce well-defined chemical or steric changes. Preliminary spectroscopic evaluation of the generated OCP variants demonstrates the potential of this "molecular surgery" to disentangle protein-chromophore interaction networks that are critical for photoreceptor function. In this way, the number and strength of key contacts with non-canonical amino acids could be controlled and manipulated. We have illustrated this principle here by replacing hydrogen bond donating residues with aromatic non-canonical amino acids that alter the state preference of OCP. • Orange Carotenoid Protein (OCP) is a water-soluble photoreceptor involved in photoprotection of cyanobacteria. • OCP contains a bound ketocarotenoid cofactor held in a protein matrix with a hydrogen bond network. • OCP expression with expanded genetic code enabled the study interactions between the ketocarotenoid and the protein matrix. • The number and strength of key contacts could be controlled and manipulated with non-canonical amino acids. • Replacement of hydrogen-bond donating residues by aromatic non-canonical amino acids changes state preference of OCP.. [ABSTRACT FROM AUTHOR]