Development of monomeric fluorescent proteins and fluorescent protein-based biosensors.

摘要

Fluorescent protein (FP) technology is now an indispensible tool of biomedical research. Nevertheless, only a few members of the hundreds of existing FPs are generally regarded as the preferred options for most imaging applications. Accordingly, new FPs with ever-improved properties are in demand and worth pursuing, as these efforts may lead to variants that are closer to the "ideal" FP. Also there remain a tremendous number of opportunities for developing FP-based biosensors for probing biological process in vivo. This thesis describes our effort on engineering new FPs with improved properties, and further modifying them to create novel biosensors. Directed evolution and semi-rational protein engineering are the main techniques used to develop these new FPs and FP-based biosensors.;The first class of FPs addressed in this thesis are the green-to-red photoconvertible FPs (pcFPs). In an effort to overcome the limitations imposed by the oligomeric structure of natural pcFPs, we created a new monomeric pcFP based on consensus design. Subsequent optimization yielded mClavGR2 and mMaple, two monomeric pcFPs displaying superior performance in folding and maturation, brightness, photoconversion efficiency and photostability. We demonstrate the application of mClavGR2 for dynamic monitoring of protein trafficking. Furthermore, in collaboration with researchers from several other groups, mMaple was demonstrated to be a multi-model probe that is suitable for use in several conventional and super-resolution fluorescence imaging modalities.;Using mMaple as a template for single-FP biosensor design, we successfully combined the two most important implementations of FPs, the "highlightable" trait and the Ca2+ sensing capability, into one construct. Optimization, characterization and live cell imaging of the resulting green-to-red highlightable Ca2+ indicators are described.;Another class of FPs that are of interest in this thesis are the true yellow emitting FPs that fill the spectral gap between monomeric greenish-yellow FPs and monomeric orange FPs. By disrupting the inter-subunit interfaces of zFP538, a FP with a distinct three-ring chromophore and an emission maximum at 538 nm, we successfully obtained its monomeric version and named it as mPapaya1. Again, characterization and live cell imaging application of mPapaya1 are described.