A Light When All Other Lights Go out

When I was a teenager, the trend was to frequent nightclubs wearing clothing that fluoresced under UV light. The glowing jeans that we wore were based on a chemical fluorophore. As I think back today, the signaling engendered by that attire parallels one of the purported roles of green fluorescent protein (GFP) in the jellyfish Aequorea victoria--as an attractant (or deterrent) to others of the same species.

Fluorescent proteins have intrinsic value to jellyfish, but for humans their use is quite different, giving us the ability to image events in living cells and organisms. This is due to the intramolecular formation of the GFP chromophore from three amino acids once the protein has folded. Thus, GFP is intrinsically fluorescent, and coexpression of the gene for GFP allows a target protein to be tagged with a fluorophore that enables imaging in living cells.

Marc Zimmer, a professor at Connecticut College, illuminates the events that led to the discovery of GFP and resulted in the prolific use of fluorescent proteins in cell and molecular biology in his engaging and highly readable book, Glowing Genes: A Revolution in Biotechnology. Particularly attractive aspects of Zimmer's account are the personal details he uses to bring the various characters of this scientific discovery to life.

The ability of organisms to generate light from lightlessness has been known for thousands of years, and reports of the application of bioluminescence are sprinkled throughout recorded history. Pliny the Elder, in his 1st-century work Naturalis Historia, recounts the use of bioluminescent clams at Roman glow-in-the-dark banquets, where they were used as cosmetics or maybe as a way of monitoring food intake and emesis.

Efforts to identify the molecular basis for bioluminescence, a property most commonly found in marine organisms, focused historically on fireflies, where an enzyme they produce, luciferase, catalyzes the light-emitting reaction of oxygen with adenosine triphosphate and luciferin. They also focused on the Aequorea jellyfish.

Osamu Shimomura at Princeton University, whom Zimmer calls the grandfather of GFP, collected and processed more than 1 million jellyfish over two decades starting in the 1960s in his successful attempts to identify the protein responsible for the green bioluminescence in Aequorea. This led to the discovery of aequorin, a bioluminescent protein in jellyfish that produces light in the presence of calcium, oxygen, and coelenterazine.

Aequorin, however, emits blue light, and GFP subsequently was identified as the protein that turns the blue light emitted by aequorin into green light. Shimomura purified 5 mg of aequorin from 10,000 jellyfish, a pertinent reminder of the heroic efforts required to purify enough protein for analysis in the pre-molecular biology era.

The GFP gene was subsequently cloned at Woods Hole Oceanographic Institution by Douglas Prasher, paving the way for initial experiments in protein tagging by Tulle Hazelrigg of Columbia University and expression in the nematode Caenorhabditis elegans by Martin Chalfie, also at Columbia.

Subsequently, Roger Y. Tsien at the University of California, San Diego, and others have pioneered in vivo labeling of proteins for monitoring the expression, localization, and trafficking of tagged proteins in living cells. The success of this approach is remarkable, given that GFP adds an additional 238 residues to the N or C terminus of the fusion protein.

After a detailed recounting of the discovery of GFP, Zimmer spends the remainder of his book documenting applications of fluorescent proteins. The color range of these proteins has been expanded by mutagenesis and the isolation of fluorescent proteins from other organisms. This has facilitated dual-color applications, such as fluorescence resonance energy transfer (FRET) experiments for monitoring protein-protein interactions.

Fluorescent proteins also are being used as markers of transgenic experiments and for imaging tumors in living animals. In addition, a range of sensors has been developed based on fluorescent proteins, one example being the construction of a sensor for detecting TNT by fusing GFP to a protein from Pseudomonas putida bacteria.

While fluorescent proteins have attracted much interest as useful imaging reagents, there is also a subset of devotees who use these proteins as model systems for understanding the interaction of light with chromoproteins. The 4-hydroxybenzylideneimidazolinone GFP chromophore is only fluorescent when contained within the folded protein known as a -can (which has a structure that looks like a soda can). Depending on the specific fluorescent protein, light-driven changes in emission have been documented over a wide range of timescales, from picoseconds to hours. It's important to understand these processes in case they lead to new applications and also to ensure that current observations are not misinterpreted.

In wild-type GFP, the chromophore has two absorption bands assigned to neutral and anionic forms of the chromophore. These forms are stabilized by two different conformations of the matrix that surrounds the chromophore. Excitation into the absorption band assigned to the neutral chromophore results in formation of a green fluorescent excited state by deprotonation of the neutral excited state on the picosecond timescale. The terminal proton acceptor for this excited-state proton transfer (ESPT) reaction is almost certainly E222, an acidic residue close to the chromophore.

In support of this proposal, my group has used ultrafast time-resolved infrared spectroscopy, in collaboration with Stephen R. Meech at the University of East Anglia, in England, to show that an acidic residue is protonated on the 20-picosecond timescale following photoexcitation.

Several GFP mutants also have been shown to blink following irradiation. In this case, a dark (nonfluorescent) state is populated with a lifetime of minutes to hours. Fluorescence can be recovered by irradiation with the appropriate wavelength, raising the possibility that fluorescent proteins can be used for information storage. Finally, a fluorescent protein known as Kaede has an observed emission spectrum that changes from green to red upon irradiation.

In addition to the photophysics of GFP, a second area of basic research, not heavily reviewed in Zimmer's book, concerns the mechanism of chromophore formation. Reaction of the three amino acids (S65, Y66, and G67 in wild-type GFP) to form the chromophore requires at least three steps involving cyclization, dehydration, and oxidation. Zimmer has devoted considerable computational effort to this area of research and has proposed that chromophore formation is promoted by a tight turn conformation in the folded immature protein.

An interesting twist to this story is the discovery of the red fluorescent protein DsRed from Discosoma sea coral. It also has a -can structure and a chromophore identical to that found in GFP, except that an additional oxidation event has extended the conjugation and hence red-shifted the excitation and emission spectra compared with that observed for GFP.

An enjoyable aspect of Glowing Genes is the constant barrage of analogies and parallels Zimmer draws to other systems. For example, he likens our ability to visualize events in living cells using GFP to the initial observation of bacteria using a microscope by Antoni van Leeuwenhoek some 340 years ago. In addition, Zimmer goes to considerable length to provide simple but clear explanations for common concepts, such as the structure and function of DNA, so that the book is accessible to nonscientists.

Zimmer makes an excellent point that Shimomura's exploits were not driven by the desire to create a valuable biological tool. Instead, they were undertaken to understand the molecular basis for green bioluminescence in jellyfish. At the time, it was impossible to predict whether a discovery like GFP would be useful. But as today's progress with GFP shows, curiosity-driven basic research leads to scientific and technological advances that have tremendous practical value and that play a critical role in maintaining and improving the health and wealth of our society.

Peter Tonge is a chemistry professor at the State University of New York, Stony Brook. His research includes studying the structure-function relationships of green and red fluorescent protein chromophores, as well as identifying and inhibiting enzyme drug targets from the tuberculosis bacterium.