The goal of this proposal is to optimize the Discosoma red fluorescent protein DsRed for use as a reporter protein and fusion tag. Our previous work has generated rapidly maturing variants of DsRed. The current challenge is that DsRed forms a stable tetramer, and as a result, many DsRed fusion proteins are nonfunctional or even toxic. We will therefore attempt to generate a monomeric DsRed. This project has two Specific Aims.
Specific Aim #1 : To develop a monomeric variant of DsRed. To weaken the tetramerization, we will mutate key residues at the two tetramerization interfaces. Further disruption of the tetramer will be accomplished through genetic screens. The oligomeric status of the resulting DsRed variants will be assessed by fusion protein production in yeast, nondenaturing SDS-PAGE, and analytical ultracentrifugation. Although initial attempts to eliminate the tetramerization abolished the fluorescence, we have obtained new DsRed variants that develop strong fluorescence even after mutagenesis of both tetramerization interfaces. These new variants should pave the way for the creation of a DsRed monomer.
Specific Aim #2 : To optimize the spectral properties of monomeric DsRed. A monomeric DsRed variant will only be useful if it retains bright red fluorescence with minimal green emission. Converting DsRed to a monomer may diminish the red fluorescence, and is likely to boost the green emission by preventing FRET between the red and green species that are present in mature DsRed. We will address these deficiencies by random mutagenesis of monomeric DsRed. The anticipated end result is a DsRed variant that will be as versatile as GFP. Fluorescent proteins have become key experimental tools for basic and applied biomedical research. The studies described here will lead to new applications of this technology.
| Strongin, Daniel E; Bevis, Brooke; Khuong, Nhi et al. (2007) Structural rearrangements near the chromophore influence the maturation speed and brightness of DsRed variants. Protein Eng Des Sel 20:525-34 |
| Genove, Guillem; Glick, Benjamin S; Barth, Alison L (2005) Brighter reporter genes from multimerized fluorescent proteins. Biotechniques 39:814, 816, 818 passim |
