GFP-like proteins:Diversity and determinants of color

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DESCRIPTION (provided by applicant): Proteins homologous to green fluorescent protein (GFP) from the jellyfish Aequorea victoria have become indispensable tools for in vivo imaging, and are widely used in basic biomedical research and drug discovery. Finding such proteins in non-bioluminescent Anthozoa offers an exciting opportunity to expand the technology by providing more colors to choose from. In addition, a number of novel features valuable for the engineering of advanced reporter systems have become available, such as precisely time-dependent changes of emission color or light-driven fluorescence activation/inhibition. However, since only a few limited-scale attempts have been undertaken thus far to clone GFP-like proteins from Anthozoa, it can be expected that many more proteins with novel spectral and structural features can be discovered during systematic survey of these organisms. In particular, these may include variants of existing proteins that are free of their drawbacks, such as oligomerization or slow chromophore maturation. Here we propose such a systematic study aimed at complete characterization of a diversity of GFP-like proteins in Anthozoa in order to create a large inventory of potential reporters for biomedical imaging applications. We intend to use the accumulated body of information on sequence/spectral features to pinpoint sequence determinants of coloration, and to verify their role by means of site-specific mutagenesis. This would greatly facilitate rational design of GFP-like reporters.