The Role of Individual Cysteine Substitutions in the Fast Photoswitching and Photoconversion of the Biphotochromic Fluorescent Protein SAASoti
Alexandra GAVSHINA1, Nadezhda MARYNICH1 and Alexander SAVITSKY1, 2
1 A.N. Bach Institute of Biochemistry, Research Center of Biotechnology of the Russian Academy of Sciences, Russia,
2 Department of Chemistry, M.V. Lomonosov Moscow State University, Russia
Abstract
Biphotochromic fluorescent protein (FP) SAASoti is a unique representative of GFP-family as its wild type gene can be irreversibly photoconverted (λ=400 nm) and reversibly photoswitched (λ=470 nm). In the course of this work, we obtained a series of SAASoti mutant forms with introduced single amino acid substitutions of cysteine residues (C21N, C117S, C117T, C72V, C106V, and C176A). Corresponding recombinant proteins were expressed in E. coli BL21 (DE3) cells, isolated and purified by chromatographic methods (combination of HIC and anion-exchange chromatography). Different physicochemical and fluorescent parameters (excitation/emission maxima, pKa values of the chromophore, molar extinction coefficient and quantum yield) of the obtained V127T SAASoti mutant variants were measured. C21 residue was found to be involved in the intermolecular dimerization of SAASoti at high concentrations, C72V SAASoti variant has an increased photoconversion (green-to-red) rate during 400 nm illumination, while C106V SAASoti demonstrates the maximum photoswitching rate between green fluorescent and dark states under 470 nm illumination. The photoswitching kinetics of C176A SAASoti follows a distinct mathematical model, indicating on different processes.
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Alexander Savitsky
FRC Biotechnology of the RAS
Russia
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