Cyan Thermal Proteins Derived From Thermal Green Protein

Thermal green protein (TGP) is a consensus derived green fluorescent protein designed with extreme thermostability, high pH and chemical stability, as well as high quantum yield for use in more severe conditions. Our goal is to design a cyan version of TGP that maintains these characteristics. We were able to shift the fluorescence wavelength of TGP from green to cyan creating CTP 0.0 by incorporating a single chromophore mutation, Y67W, but this mutation also decreased the quantum yield to 0.056. Further mutations were incorporated to increase the quantum yield through incorporating hydrogen bonding interactions to the chromophore and to remove a kink present in beta strand seven. These proteins, CTP 0.5 (Y67W I199T) and CTP 1.0 (Y67W I199T W143L E144I P145D S146A), increased the quantum yield to 0.07 and 0.37, respectively and improved stability characteristics. CTP 0.75 incorporated another chromophore mutation into CTP 1.0 (Q66E) to increase the stability characteristics but decreased the quantum yield to 0.22. The CTP 1.0 cyan protein was also compared to mTurquoise2, one of the current best cyan fluorescent proteins based on GFP. CTP 1.0 had comparable chemical stability and improved acid stability. Crystal structures were solved for CTP 0.5 at pH 6.5 (2.00 Å), CTP 1.0 at pH 6.5 (1.70 Å), CTP 1.0 at pH 8.5 (1.60 Å), and CTP 0.75 at pH 7.4 (1.70 Å). Structural analysis of the proteins showed that while improvement to beta strand seven was unsuccessful, the increase in quantum yield is likely due to the incorporation of the T199 residue and subsequent hydrogen bonding interaction improvements with the chromophore.