First biphotochromic fluorescent protein moxSAASoti stabilized for oxidizing environment.

Biphotochromic proteins simultaneously possess reversible photoswitching (on-to-off) and irreversible photoconversion (green-to-red). High photochemical reactivity of cysteine residues is one of the reasons for the development of "mox"-monomeric and oxidation resistant proteins. Based on site-saturated simultaneous two-point C105 and C117 mutagenesis, we chose C21N/C71G/C105G/C117T/C175A as the moxSAASoti variant. Since its on-to-off photoswitching rate is higher, off-to-on recovery is more complete and photoconversion rates are higher than those of mSAASoti. We analyzed the conformational behavior of the F177 side chain by classical MD simulations. The conformational flexibility of the F177 side chain is mainly responsible for the off-to-on conversion rate changes and can be further utilized as a measure of the conversion rate. Point mutations in mSAASoti mainly affect the pK values of the red form and off-to-on switching. We demonstrate that the microscopic measure of the observed pK value is the C-O bond length in the phenyl fragment of the neutral chromophore. According to molecular dynamics simulations with QM/MM potentials, larger C-O bond lengths are found for proteins with larger pK. This feature can be utilized for prediction of the pK values of red fluorescent proteins.