| Fluorescent proteins (FPs) have had a tremendous impact on biomedical research. Since the discovery of the green fluorescent protein from Aequoria victoria (GFP), FPs have been discovered and engineered to span the visible spectrum. However, not all of these FPs are equally well suited for experimental use. To be generally useful, FPs must have desirable photophysical properties such as high brightness and photostability. In addition, they must function as inert labels to avoid artefactual results.;Applications for FPs can be divided into two broad classes: those involving fusion to a protein of interest, and those involving whole-cell, tissue, or organism labeling. Monomeric FPs are versatile because they can be used as fusion partners or for whole-cell labeling. Fusion to an oligomeric FP is generally undesirable due to crosslinking of the target protein, but oligomeric FPs can be preferable to their monomeric counterparts for whole-cell labeling because the oligomeric variants are often brighter and more photostable.;Despite an abundance of available monomeric and oligomeric red FPs, those proteins were of limited utility for whole-cell labeling due to cytotoxicity. We overcame this limitation by developing DsRed-Express2, a noncytotoxic tetrameric DsRed derivative. Because cytotoxicity was proposed to stem from higher order aggregation, we started with DsRed-Express and then optimized the surface of the tetramer to eliminate higher-order aggregation. DsRed-Express2 retains the brightness, photostability, and fast maturation of DsRed-Express, and is noncytotoxic in bacterial and mammalian systems. It is now the red FP of choice for whole-cell labeling.;I then decided to use the surface of DsRed-Express2 as a starting point for mutating residues in the interior of the protein, with the goal of generating noncytotoxic FPs with different spectral properties. The first DsRed-Express2 derivative was E2-Orange, which is blue-shifted relative to DsRed-Express2 due to changes in the chromophore structure. E2-Orange is bright, relatively fast-maturing, and extremely photostable, making it the orange FP of choice for wholecell labeling. In addition, I generated E2-Red/Green, which is a dual red and green FP. E2-Red/Green was designed for use in three-color labeling with green and red FPs. When three separate populations of cells are labeled with a green FP, a red FP, and E2-Red/Green, they are clearly resolvable by flow cytometry. Both E2-Orange and E2-Red/Green retain the low cytotoxicity of DsRed-Express2, making them generally useful tools.;I also developed E2-Crimson, a far-red DsRed-Express2 derivative. E2-Crimson is among the farthest red-shifted GFP homologs and has the farthest red-shifted excitation of any GFP homolog, with excitation and emission maxima at 611 and 646 nm, respectively. In addition to its unique spectral properties, E2-Crimson is both bright and photostable. Furthermore, E2-Crimson is the most rapidly maturing red or far-red FP, making it uniquely suited to experiments requiring high time sensitivity. These properties combined with low cytotoxicity make E2-Crimson an important tool for whole-cell and live animal imaging.;In addition to the creation of useful FPs, a long-standing interest of our laboratories is the mechanism of chromophore formation in DsRed. A central mystery regarding DsRed maturation is that fully mature DsRed is an approximately 1:1 mixture of green and red chromophores. This result is not explained by the conventional idea that the green chromophore is a precursor to the red chromophore. We propose that chromophore formation actually occurs by a novel branched pathway, in which the green and red chromophores are alternate endpoints that share a common intermediate. A kinetic competition between oxidation and dehydration of this common intermediate determines the final ratio of red to green chromophores. Several aspects of this model have been thoroughly tested and are presented in Chapter 6 of this dissertation.;Overall, my engineering work has generated useful tools for biomedical research and has emphasized the importance of in vivo assays for FP behavior. DsRed-Express2, E2-Orange, E2-Red/Green, and E2-Crimson should facilitate many experiments and serve as benchmarks for the next generation of FPs. In addition, my mechanistic work allows for a more complete understanding of chromophore formation in DsRed. More broadly, this work provides a conceptual and experimental framework for studying chromophore chemistry in a variety of FPs. |
