Beyond GFP

COURTESY NATHAN SHANER, PAUL. STEINBACH, AND ROGER TSIEN

The paper:

N.C. Shaner, et al. "Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein," Nat Biotechnol, 22:1567-72, 2004. (cited in 103 papers) | [PubMed]

The finding:

Roger Tsien's group at the University of California San Diego has a knack for tinkering with fluorescent proteins. In 2004, they published a follow-up to a 2002 paper reporting the first monomeric red fluorescent protein, mRFP1, this time using molecular evolution to improve the protein's properties and create new red, orange, and yellow color varieties.

The challenge:

Improving qualities such as maturation rate, brightness, and tolerance to terminal fusions required careful balance. Attempts to brighten the new orange monomer, for example, reduced its pH-stability.

The surprise:

The team found that its mOrange protein had a different chromophore structure from that of the green or red varieties. Another color, mCherry, turned out to be more photostable than expected.

The follow-up:

Recently, Tsien's group and James Remington's team at the University of Oregon solved the crystal structures of several fluorescent proteins, providing insight for further tinkering. "Because we know where everything is, we can direct mutations where they are going to make a difference," Shaner says.

The verdict:

"Before this paper, there was only one not-so-good choice in the red category," says Michael Davidson, who uses fluorescent proteins for multicolor live cell imaging at the National High Magnetic Field Laboratory in Tallahassee, Fla. Tsien et al. "opened up the entire spectrum from the mid-500s to more than 600 nanometers."