Biochemistry, mutagenesis, and oligomerization of DsRed, a red fluorescent protein from coral

TLDR

DsRed is a 28‑kDa coral protein that imparts red coloration and is of interest as a complementary fluorescent tracer to GFP, but its biochemical properties are poorly understood. The study aimed to characterize the biochemical properties of DsRed, including its photophysical behavior and oligomerization. DsRed exhibits a higher extinction coefficient and quantum yield, excellent pH and photobleaching resistance, and its 583‑nm emission can be shifted to 602 nm by Lys‑83 → Met, yet it forms obligate tetramers, matures slowly, and Lys‑83 → Arg blocks color conversion.

Abstract

DsRed is a recently cloned 28-kDa fluorescent protein responsible for the red coloration around the oral disk of a coral of the Discosoma genus. DsRed has attracted tremendous interest as a potential expression tracer and fusion partner that would be complementary to the homologous green fluorescent protein from Aequorea , but very little is known of the biochemistry of DsRed. We now show that DsRed has a much higher extinction coefficient and quantum yield than previously reported, plus excellent resistance to pH extremes and photobleaching. In addition, its 583-nm emission maximum can be further shifted to 602 nm by mutation of Lys-83 to Met. However, DsRed has major drawbacks, such as strong oligomerization and slow maturation. Analytical ultracentrifugation proves DsRed to be an obligate tetramer in vitro , and fluorescence resonance energy transfer measurements and yeast two-hybrid assays verify oligomerization in live cells. Also, DsRed takes days to ripen fully from green to red in vitro or in vivo , and mutations such as Lys-83 to Arg prevent the color change. Many potential cell biological applications of DsRed will require suppression of the tetramerization and acceleration of the maturation.