Researchers from the Russian Academy of Sciences and Russian biotech firm Evrogen have developed a fluorescent protein that can change its emission from green to red following activation with blue light, the first known fluorescent protein to display such properties.
The discovery is notable because it may allow researchers to conduct so-called pulse-chase experiments to track the movement of organelles and proteins over longer periods in living cells due to the minimal toxicity effects of blue light on living organisms.
Furthermore, the fluorescent dyes, called photo-activatable fluorescent proteins, or PAFPs, may be a boon to researchers who wish to conduct such trafficking experiments, but who may not be able to afford the relatively expensive imaging equipment necessitated by prior fluorescent tags.
The Russian scientists' discovery was serendipitous. A group led by brothers Konstantin and Sergey Lukyanov of the Russian Academy of Sciences in Moscow developed the PAFP, called Dendra, from a natural green fluorescent protein they cloned several years ago from the sea coral Dendronephthya as part of a project to characterize evolution diversity of fluorescent proteins in corals.
"In some cases [the] UV-violet light required for PAFP activation is phototoxic for cells or can induce drastic changes in cell biochemistry. Also, a real problem for many cell biologists who wish to use PAFPs for their studies is a shortage of laser equipment."
They published this fluorescent protein evolution research in 2002, but subsequently discovered that the particular green fluorescent protein, called dendGFP, could be converted into a red fluorescent state by activating it with UV light.
The scientists then went about converting the tetrameric dendGFP to a monomeric form that would be suitable for labeling proteins in live cells, and eventually developed Dendra, which, as they expected, could be irreversibly converted to fluoresce red instead of green after being expressed in bacteria cells and irradiated with UV light.
However, when the Lukyanovs and colleagues began evaluating expression of the PAFP in mammalian cells, they stumbled across an unexpected result: Several seconds of irradiation with high-intensity blue light in the range of 460 nanometers to 500 nanometers caused the protein to fluoresce bright red light.
As detailed in the April 2006 issue of Nature Biotechnology, the scientists were able to determine the exact range of blue light intensity and exposure time that causes Dendra to convert from green to red, while noting that irradiation outside this range did not result in photoconversion. According to the researchers, this feature may afford researchers the versatility of visualizing green fluorescence with a light source, and then simply changing the intensity settings to cause red fluorescence.
Furthermore, the scientists compared the photostability, or how long the protein fluoresces under constant irradiation, of photoconverted Dendra with that of DsRed, a commonly used monomeric red fluorescent protein developed by Sergey Lukyanov and later developed into a monomeric version by Roger Tsien and colleagues at the University of California, San Diego. The Russian scientists found that DsRed reached 50-percent bleaching 3.3 times as fast as Dendra under similar conditions.
According to Sergey Lukyanov, who is a Howard Hughes Medical Institute international research scholar, the discovery is important because all other known PAFP's require UV or near-UV light for activation.
"In some cases [the] UV-violet light required for PAFP activation is phototoxic for cells or can induce drastic changes in cell biochemistry," Lukyanov wrote in an e-mail to CBA News. "Also, a real problem for many cell biologists who wish to use PAFPs for their studies is a shortage of laser equipment.
"Indeed, UV-violet lasers are quite expensive and require a UV-adapted whole set of optics, [because] many confocals can not be simply upgraded with a UV laser," Lukyanov wrote. "Multi-photon lasers are even more expensive, and as a result, confocal microscopes with UV-violet or multiphoton lasers still represent quite rare equipment.
In contrast, Lukyanov said, virtually all confocal microscopes carry 488- and 543-nm laser lines, which would be sufficient to work with Dendra.
Lukyanov's lab has an ongoing collaboration with Russian biotech Evrogen, and as such, Evrogen offers several of the fluorescent proteins developed by the Russian Academy of Science researchers over the past several years. Similarly, Lukyanov said, Dendra will eventually be commercially available from Evrogen, "hopefully no later than April."
The laboratory and Evrogen also have had an ongoing collaboration since 1998 with Clontech, the former Becton Dickinson unit now owned by Japanese biotech Takara Bio. According to Lukyanov, the researchers believe dendGFP and Dendra are protected by a broad patent application on fluorescent proteins submitted by Clontech.
The next step for the researchers besides commercialization efforts is to learn exactly why Dendra converts from green to red under irradiation with blue light when all other PAFPs from the same family do not.
"The structural basis for Dendra sensitivity to blue light remains yet unclear," the researchers wrote in the Nature Biotechnology paper. "Dendra possesses some distinct structural features ensuring photoconversion in response to blue light … and further studies are required to clarify the mechanism."
— Ben Butkus ([email protected])