Lab Members |
Research Fluorescent proteins have found widespread application in cell biology as reporters for the cellular localization of various proteins. In the last decade, many fluorescent proteins with emission spectra spanning the entire visible region have been isolated in nature or have been developed in the laboratory. Those that emit in the red portion of the visible spectrum are of particular interest because longer wavelengths are less toxic to cells and because cells are more transparent to red light. DsRed, a tetrameric red fluorescent protein found in the sea anemone Discosoma striata, has been extensively studied and has been evolved to yield monomeric red fluorescent proteins with red-shifted emission maxima. One such DsRed mutant, mCherry, has a very red-shifted emission maximum of 610 nm. Our goal is to further evolve mCherry to yield mutants with improved spectral properties such as an even more red-shifted emission maximum, higher quantum yield, and increased brightness. To achieve this, we will use a computational design method to generate “smart” combinatorial libraries of mCherry. The protein sequences in these libraries will have a higher probability of folding properly since our method eliminates mutations that would destabilize the protein fold. The designed libraries will contain ~500 mutants, a size which can easily be screened for the desired properties using a 96-well plate-based assay. In addition, exhaustive sequencing of all the fluorescent mutants from the library will provide information on structure/function relationships and will facilitate optimization of the computational method. |
