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Sea coral's trick helps scientists tag proteins

ed the DNA sequence that encodes the fluorescent protein, then expressed the resulting mutant proteins in bacteria until they found one that could be irreversibly converted from green to red.

As with GFP, the sequence for Dendra and an accompanying short amino acid tail can be inserted into the gene encoding the protein a scientist wants to study. In living cells, the fluorescent tail is produced along with the protein, acting like a beacon to researchers who want to learn what the main protein does and where it goes.

To test how well the new tag labeled and tracked target proteins, the team fused Dendra to proteins that form the cytoskeleton of the cell, including actin and tubulin filaments and found the expected patterns of intracellular protein distribution.

Dendra belongs to a small family of green-to-red PAFPs. The first protein in this family, Kaede--which is Japanese for maple leaf--was discovered by Japanese researcher Atsushi Miyawaki four years ago. "All known Kaede-like proteins are sensitive to ultraviolet light irradiation, which results in immediate conversion from green to red fluorescence, but they are not sensitive to blue light," Konstantin Lukyanov said.

So the researchers did not expect Dendra to be activated by low-toxic visible light. "It was very surprising for us to observe an efficient Dendra photoconversion upon illumination with intense blue light," Konstantin Lukyanov said. "We still have no clear explanation for why Dendra differs from other Kaede-like fluorescent proteins."

Sergey Lukyanov pointed out that the ultraviolet light required to activate many PAFPs can be toxic to cells, and can dramatically alter their biochemistry. Further, the equipment needed to generate ultraviolet light can be expensive and in short supply. "We anticipate that Dendra will broaden considerably the circle of PAFP users," he said.


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Source:Howard Hughes Medical Institute


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