Directed molecular evolution to design advanced red fluorescent proteins

In this Perspective the authors discuss strategies for the development of improved fluorescent proteins, with a focus on probes at the red end of the spectrum. They synthesize the literature on chromophore photochemistry and protein structure to identify residues for targeted mutagenesis, and consider improvements in molecular evolution methodologies to enable improved screening for desired probes. Fluorescent proteins have become indispensable imaging tools for biomedical research. Continuing progress in fluorescence imaging, however, requires probes with additional colors and properties optimized for emerging techniques. Here we summarize strategies for development of red-shifted fluorescent proteins. We discuss possibilities for knowledge-based rational design based on the photochemistry of fluorescent proteins and the position of the chromophore in protein structure. We consider advances in library design by mutagenesis, protein expression systems and instrumentation for high-throughput screening that should yield improved fluorescent proteins for advanced imaging applications.