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.

在由细菌植物色素设计的二聚体近红外（NIR）生物标志物中，BV与蛋白质的一个单体中的半胱氨酸残基共价结合会变构地阻止嵌入另一种单体口袋中的发色团与半胱氨酸残基共价结合。在这项工作中，我们分析了在位置204（在这些蛋白质的进化过程中诱变的目标残基之一）上的取代的二聚NIR生物标记物对单体间变构效应的影响。基于Rp开发的iRFP713中的T204A替代BphP2及其突变体iRFP713 / C15S / V256C中的配体共价连接位点发生了变化，导致氢键网络的重排，使发色团与相邻的氨基酸结合，并在其局部环境中结合了水分子。由T204A取代引起的iRFP713 / C15S / V256C中发色团与其蛋白环境之间分子内接触的变化降低了辅因子结合的负协同作用。我们讨论了单体之间的串扰对全长植物色素的作用的可能影响。