Genetically encoded fluorescent indicators require fluorescent protein moieties to report biological events as fluorescent signals. In the case of indicators that use a change in protein conformation to alter the degree of fluorescence resonance energy transfer (FRET) between two fluorophores, ideal fluorescent protein components should have no intrinsic environmental sensitivity, so that their FRET level indicates only the concentration of the analyte of interest. In the case of single fluorescent protein indicators, in which a protein conformational change can be directly tranduced into a fluorescence intensity change, an ideal fluorescent protein component should be maximally environmentally sensitive, but to just one analyte, to minimize interference and cross talk.; In this dissertation, I report what I learned about the biochemical and structural properties of fluorescent proteins, and how I used that knowledge to design fluorescent indicators. The first chapter is a biochemical and structural classification of an environmentally insensitive Yellow Fluorescent Protein useful in the construction of indicators. The second chapter is a report of a new, very environmentally sensitive class of fluorescent proteins that form the basis of a new type of fluorescent indicator. The third chapter outlines the design, testing, and analysis of a new type of calcium indicator based on the protozoan protein spasmin. The fourth and fifth chapters describe the biochemical, structural and chemical classification of the Discosoma red fluorescent protein, DsRed, and efforts made to improve DsRed's utility as a component of fluorescent indicators.
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