Development of Cyan and Blue Thermostable Fluorescent Proteins: Characterization and Structural Analysis

Fluorescent proteins are commonly used as cell markers in living organisms. Modifications by mutation can be used to improve the qualities of these proteins including fluorescence, thermostability, pH stability, and chemical stability. The goal of this project is to use mutagenesis to improve the fluorescence of thermostable cyan and blue proteins derived from the thermal green protein (TGP). The first cyan protein developed by the DeVore lab (CTP 0.0) shifted the fluorescence to cyan but decreased the quantum yield (ΦF) 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. The resulting 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. The first blue protein developed by the DeVore lab (BTP 0.0) shifted the fluorescence to cyan but decreased the quantum yield (ΦF) to 0.004. Three blue mutants BTP 0.1 (W143L E144I P145D S146A I199T), BTP 0.2 (W143L E144I P145F S146F I199T), and BTP 0.3 (W143L E144I P145D S146H I199T) all increased the quantum yield to 0.006, 0.014, and 0.017 respectively. Stability characterization showed varying improvements among the three mutants. Crystal optimization was done for BTP 0.1 for future structural analysis for the blue protein project.