Colorful Proteins Express Themselves as Tags

More than 150 fluorescent proteins--all genetically related--are now known, each one containing at least 200 amino acids. These proteins compose a veritable rainbow, each one fluorescing at a different wavelength.

In their natural form, many of the proteins clump together as tetramers. Such aggregation can ruin them as tags, Tsien said. A lot of protein engineering goes into adapting the naturally fluorescent proteins so that they don't oligomerize but retain their fluorescence. He uses the proteins' crystal structures as a guide to change the amino acids that cause oligomerization. Finding the right changes can be tricky, because changing amino acids initially eliminates the fluorescence as well as the oligomerization.

Tsien's lab also has engineered monomeric VFPs with better photostability and a shift in their fluorescence wavelength toward the infrared. Emission wavelengths in the infrared would make them better suited to imaging inside intact animals and patients.

However, VFPs are full-length proteins with molecular weights on the order of 27 kilodaltons. For applications requiring smaller labels, Tsien has made available smaller genetically encoded options. He has developed a type of synthetic label known as FlAsH, which stands for fluorescein-based arsenical hairpin binder [J. Am. Chem. Soc., 124, 6063 (2002)]. Each of the fluorophores contains two arsenic atoms.

In live cells, FlAsH and similar synthetic fluorophores home in on tetracysteine motifs genetically engineered into the protein of interest. The tetracysteine motifs are much smaller (six to 12 amino acids) than a full-sized protein and therefore less perturbing. The dyes, which are membrane permeable and nonfluorescent by themselves, become fluorescent when they bind to the tetracysteine motif.

At the symposium, Horst Vogel, a professor in the Institute of Chemical Sciences & Engineering at the Swiss Federal Institute of Technology, Lausanne, described two other approaches for adding synthetic fluorophores to proteins. In the first one, a chromophore and metal-ion-chelating nitrilotriacetate bind reversibly and specifically to engineered oligohistidine sequences on the protein of interest [Nat. Biotechnol., 22, 440 (2004)].

Vogel's second approach is based on the formation of fusion proteins with human alkylguanine transferase for specific in vivo labeling. The protein itself is not fluorescent, but synthetic fluorophores can easily be added to it. Fusion proteins expressed in different parts of a cell thus can be labeled with different fluorophores [Proc. Natl. Acad. Sci. USA, 101, 9955 (2004)].