Novel biphotoswitchable forms of photoconvertible fluorescent proteins SAASoti: the role of the correlated motion(s) of the chromophore

The wild type SAASoti and its monomeric variant mSAASoti can undergo phototransformations including reversible photoswitching of the green form to a non-fluorescent state and irreversible photoconversion from green to red form. In this study we extend the photochemistry of mSAASoti variants bringing the ability of reversible photoswitching of the red form. This result is achieved by rational and site-saturated mutagenesis of the M163 and F177 residues that are known to be important for the flexibility of the phenyl part of the chromophore. Thus, novel mSAASoti variants are biphotochromic fluorescent proteins with efficient green-to-red photoconversion and photoswitching of both green and red forms. We got a 30-fold increase in the switching efficiency of the green form and the appearance of a comparable switching of the red form for the C21N/M163T mSAASoti variant. Dynamic network analysis reveals that efficient photoswitching occurs if motions of the H68 residue and phenyl fragment of chromophore are correlated and these moieties belong to the same community.