Single-point substitution F97M leads to in cellulo crystallization of the biphotochromic protein moxSAASoti

To enhance the photoconversion performance of biphotochromic moxSAASoti protein, a substitution F97 M was introduced. In addition to enhancing the target properties, this substitution also resulted in the crystallization of the recombinant protein within living HeLa cells (also referred to as in cellulo crystallization). The phenomenon of protein crystallization in living cells is not unique, yet the mechanisms and application of in cellulo crystallization remain significant for further research. However, in cellulo crystallization is atypical for fluorescent proteins and detrimental for their biotechnological application. The objective of this study was to elucidate the underlying mechanisms responsible for the crystallization of moxSAASotiF97Min cellulo. For this purpose, the crystal structure of the green form of biphotochromic protein moxSAASotiF97M was determined at high resolution, which surprisingly has a space group, different from those of parent mSAASotiC21N. The analysis provided allowed to propose a mechanism of new crystal contacts formation, which might be a cause of in cellulo protein crystallization. Single-point substitution F97 M leads to in cellulo crystallization of the biphotochromic protein moxSAASoti in HeLa cells. The crystal structure of the moxSAASotiF97M was determined at 1.9 Å. We conducted a comprehensive analysis of the moxSAASotiF97M and mSAASotiC21N proteins in order to investigate the structural changes that result from the F97 M mutation.