LAUSR

In dimeric near-infrared (NIR) biomarkers engineered from bacterial phytochromes the covalent binding of BV to the cysteine residue in one monomer of a protein allosterically prevents the chromophore embedded into the pocket of the other monomer from the covalent binding to the cysteine residue. In this work, we analyzed the impact on inter-monomeric allosteric effects in dimeric NIR biomarkers of substitutions at position 204, one of the target residues of mutagenesis at the evolution of these proteins. The T204A substitution in iRFP713, developed on the basis of RpBphP2, and in its mutant variant iRFP713/C15S/V256C, in which the ligand covalent attachment site was changed, resulted in the rearrangement of the hydrogen bond network joining the chromophore with the adjacent amino acids and bound water molecules in its local environment. The change in the intramolecular contacts between the chromophore and its protein environment in iRFP713/C15S/V256C caused by the T204A substitution reduced the negative cooperativity of cofactor binding. We discuss the possible influence of cross-talk between monomers the functioning of full-length phytochromes.