Red Radiance

PEERING DEEP into live animals may now be easier, thanks to a red sea anemone and some clever protein engineering. Russian researchers are reporting a new red fluorescent protein that is more than significantly brighter than any existing contender (Nat. Methods, DOI: 10.1038/nmeth1083).

Dmitriy M. Chudakov, Andrey G. Zaraisky, Sergey Lukyanov, and their colleagues at the Institute of Bioorganic Chemistry, in Moscow, have engineered what they hope will be "the protein of choice for whole-body imaging techniques," Chudakov says. It's a protein called Katushka.

Those who develop fluorescent tools to visualize living tissues battle several molecular adversaries. Hemoglobin and melanin proteins absorb enlightening photons having wavelengths below 650 nm, while water soaks up light beyond 1,100 nm. In between these two wavelengths is the sweet spot in the far-red spectrum for tissue visualization. But existing far-red fluorescent proteins are dimmer than desired.

Katushka "establishes the current state of the art in a rapidly moving field," comments Roger Y. Tsien, a biochemist at the University of California, San Diego, who engineers fluorescent proteins. While he's impressed with the deep penetration of the red fluorescence in frog tissues shown in the paper, Tsien would like to see how the protein fares in mammalian tissues, which contain more obfuscating hemoglobin absorbance.

To engineer Katushka, the researchers first isolated a red fluorescent protein from a sea anemone that Lukyanov bought from a Moscow pet shop. After looking at an X-ray crystal structure of a related protein, the researchers made strategic substitutions to three amino acids near the fluorescing chromophore. The team then gave fate a chance by introducing random amino acid mutations to the library of strategic mutations.

In total, more than 100,000 proteins were screened for fluorescent ability. The pay dirt was Katushka, which is a dimer protein. Although dimer proteins are useful for whole-animal imaging, they are too bulky for labeling single proteins in cells. So the team used a similar strategy to engineer a monomer version of Katushka called mKate.

"We have a winner here as a potential FRET [fluorescence resonance energy transfer] acceptor for live-cell imaging," comments Taekjip Ha, a physicist at the University of Illinois, Urbana-Champaign.