Engineering Novel Fluorescent Proteins
Fluorescent proteins are intrinsically fluorescent, genetically encodable tags that can be expressed in many heterologous organisms. Originally cloned from jellyfish and corals, these proteins and their engineered derivatives have become ubiquitous tools in molecular and cell biology. While wild-type fluorescent proteins sometimes possess sufficiently beneficial properties to be used unmodified, many applications require improvements in brightness or photostability, reduction of oligomerization, or other specific properties that require additional engineering of the wild-type protein. This dissertation presents experiments drawn from the entire spectrum of fluorescent protein science, from the cloning of novel wild-type fluorescent proteins to the engineering of wavelength-shifted, photostable, and photoactivatable variants of existing fluorescent proteins. The previously engineered monomeric red fluorescent protein, mRFP1, was engineered through a combination of rational design and directed evolution into a set of monomeric fluorescent proteins spanning from yellow-green through far-red. Far-red fluorescent proteins were studied in further detail after the discovery that certain variants exhibited the novel property of reversible photoactivation. A systematic analysis of the photostability and spectral properties of the most commonly used fluorescent proteins led to greater insight into the best proteins to use for specific types of experiment. Novel screening methods were developed to select for highly photostable variants, resulting in the evolution of substantially more photostable red and orange monomers. Finally, novel fluorescent proteins were cloned from a cold-water anemone and a tropical large-polyp stony coral, providing additional insights into the evolution of color in wild-type fluorescent proteins. The central issue in all of these studies is the examination of the origin of the widely varied photophysical properties of these fluorescent proteins. In the course of this work, the relationship between chromophore chemistry and interaction with the protein scaffold and the photophysical properties of the fluorescent protein have been further elucidated, and several novel tools with wide applicability to cell and molecular biology research have been developed.