Rational design of a red fluorescent protein displaying increased quantum yield
The longer emission wavelengths of red fluorescent proteins (RFPs) make them useful for whole-body imaging because cells are more transparent to red light. However, RFPs are typically less bright than other types of fluorescent proteins that emit at shorter wavelengths due to their lower quantum yields. The goal of this project is to increase the quantum yield of RFPs by rational design. We hypothesized that restricting the conformational flexibility of the RFP chromophore would decrease radiationless decay, thereby resulting in
higher quantum yields. To test this hypothesis, we will increase the packing around the RFP chromophore by sandwiching it between two aromatic residues in a triple decker motif. In order to predict which mutations are needed to introduce the desired pi-stacking interactions, we have used computational protein design (CPD)
algorithms to identify mutations in six residues surrounding the chromophore of mCherry, a widely used monomeric RFP. These mutations, which are predicted to stabilize the chromophore while not destabilizing the protein fold, will be introduced into mCherry using combinatorial mutagenesis, resulting in a mutant library containing ~200 variants, a compromise between ease of screening and sufficient sequence diversity. This library will be expressed in E. coli and screened for fluorescence intensity using a 96-well plate fluorimetric
assay that we have developed. Variants displaying increased brightness will be expressed and purified, and their spectral properties, including quantum yield, will de determined.