A coveted mainstay in any life-sciences toolbox, green fluorescent protein (GFP) has replaced many dyes in biological studies because it is non-toxic and can function as a reporter of expression when attached to a gene of interest. Though first isolated in the 1960s from a bioluminescent jellyfish, GFP’s utility in the lab was not demonstrated until the early 1990s. Since then, GFP has profoundly impacted the field of fluorescence microscopy. It continues to be central to a vast array of studies of gene expression and protein tagging, and more recently has earned aesthetic praise in “Brainbow” imaging (see the cover of our January 2009 issue). The 2008 Nobel Prize in Chemistry was awarded for the discovery and development of GFP to Osamu Shimomura, professor emeritus at Marine Biological Laboratory (Woods Hole, MA); Martin Chalfie, professor of biological sciences at Columbia University (New York, NY); and Roger Tsien, an investigator at the Howard Hughes Medical Institute and professor at the University of California, San Diego (UC San Diego).
As indicated by its appearance in several papers this month, scientists are still busy uncovering further uses for GFP. In one paper, vibration was found to be the key in the GFP fluorescence mechanism, while in another, a method for synthetic GFP assembly was developed that produced high-yield samples with robust fluorescent output. Also of interest is a study of a photoconvertible fluorescent protein that visualized real-time assembly of HIV particles and their release.
Good vibrations
Scientists at the University of California, Berkeley (UC Berkeley) have determined how GFP gets its glow. Using laser pulses as short as 20 femtoseconds, the researchers imaged GFP to determine the structural changes it undergoes upon fluorescence. Using femtosecond stimulated Raman spectroscopy—which takes laser “snapshots” fast enough to freeze vibrating molecules—they were able to distinguish the chemical and atomic steps involved in GFP’s fluorescence.
"With a femtosecond laser and Raman spectroscopy, we can see all the steps in the proton transfer reaction in the excited state of GFP," said Richard Mathies, UC Berkeley professor of chemistry and dean of the College of Chemistry, in a press release. “This is something I’ve wanted to do for 40 years.”
The laser snapshots show that when GFP’s light absorber, or chromophore, absorbs an incoming photon of blue light, it starts vibrating while its electrons move around until they align. At that point, a proton is transferred via a water molecule to a nearby amino acid. This continues down a reaction chain, leaving a negatively charged chromophore that emits green light.
One of the goals of the study was to understand the processes of light absorption and emission in such detail that molecules can be redesigned to more efficiently capture sunlight. This work may influence the development of photovoltaics—or solar cells—that better harness solar energy. The findings, “Mapping GFP structure evolution during proton transfer with femtosecond Raman spectroscopy,” were published in the Nov. 12 issue of Nature.
Semi-synthetic GFP
Scientists at Stanford University are assembling semisynthetic GFP that promises to improve the strength of the reporter for interpretation of in vivo studies. The study, titled “Synthetic control of green fluorescent protein” and published in the Journal of the American Chemical Society on Nov. 11, describes a method for replacing any of the 11 β-strands or replacing the interior α-helix (which contains the chromophore) by adding a synthetic peptide to a truncated form of the protein produced recombinantly.
This in vitro assembly method mimicked the in vivo assembly of “split GFP.” In 2004, Nobel laureate Chalfie described how GFP could be split into two polypeptides that each could be bound to a target protein or protein-interaction domain. Upon colocalization, the parts would bind and then fluoresce. Split GFP is used for protein solubility assays and protein colocalization studies.
The Stanford team assembled several high-yield constructs with a mature chromophore, overcoming past limitations where sample yield was low and the chromophore was either not formed or only partially formed. Synthetic control over GFP has wide applications for robust reporting of labeled biomolecules.
Photoconvertible fluorescent protein helps HIV research
Researchers have developed a photoconvertible fluorescent protein to visualize how HIV is assembled and released from infected cells. This photoconvertible protein, named EosFP, emits strong green fluorescence that changes to red when exposed to UV radiation. Focused UV light changes the emission wavelength of the protein by inducing a break in the peptide backbone next to the chromophore. This makes EosFP a superb marker for tracking movements of molecules within a cell.
Don C. Lamb, professor at the Ludwig-Maximilians-Universität (LMU) in Munich’s Department of Chemistry and Biochemistry, together with colleagues in Heidelberg, Germany, described how HIV particles assemble at the membrane of infected cells and are released. By attaching EosFP to the Gag protein—a structural protein found in HIV—the team could track individual viruses in real time and was able to follow the processes of viral assembly and release. “Dynamics of HIV-1 assembly and release ” was published Nov. 6 in Public Library of Science’s Pathogens.
The photoconvertible fluorescent labeling technique and emergence of synthetic GFP methods represent two more additions to the toolbox researchers have to report expression and track biomolecules . Furthermore, the demonstration of GFP’s fluorescence mechanism is a hallmark discovery by the researchers at UC Berkeley. The widespread use of GFP and its recognition by the Nobel Committee in 2008 reinforce how important this molecule is for science, and its focus in current research shows that researchers are far from finished exploring its usefulness for future novel applications.
