The role of cysteine residues in the allosteric modulation of the chromophore phototransformations of biphotochromic fluorescent protein SAASoti.

Biphotochromic fluorescent protein SAASoti contains five cysteine residues in its sequence and a V127T point mutation transforms it to the monomeric form, mSAASoti. These cysteine residues are located far from the chromophore and might control its properties only allosterically. The influence of individual, double and triple cysteine substitutions of mSAASoti on fluorescent parameters and phototransformation reactions (irreversible green-to-red photoconversion and reversible photoswitching) is studied. A set of mSAASoti mutant forms (C21N, C117S, C71V, C105V, C175A, C21N/C71V, C21N/C175A, C21N/C71G/C175A) is obtained by site-directed mutagenesis. We demonstrate that the C21N variant exists in a monomeric form up to high concentrations, the C71V substitution accelerates photoconversion to the red form and the C105V variant has the maximum photoswitching rate. All C175A-containing variants demonstrate different photoswitching kinetics and decreased photostability during subsequent switching cycles compared with other considered systems. Classical molecular dynamic simulations reveal that the F177 side chain located in the vicinity of the chromophore is considerably more flexible in the mSAASoti compared with its C175A variant. This might be the explanation of the experimentally observed slowdown the thermal relaxation rate, i.e., trans-cis isomerization of the chromophore in mSAASoti upon C175A substitution.