GFP Emission: How Green Fluorescent Protein Glows

Green fluorescent protein, or GFP, is a protein discovered in the jellyfish Aequorea victoria that glows green when exposed to certain types of light. Its discovery was a byproduct of research on a different light-emitting protein from the same jellyfish. Scientists Osamu Shimomura, Martin Chalfie, and Roger Tsien were awarded the Nobel Prize in Chemistry in 2008 for their work on the protein. In its natural environment, GFP absorbs blue light from another protein and re-emits it as a green glow. This natural curiosity has become a valuable tool for illuminating the inner workings of cells.

The Process of Fluorescence

The glow of Green Fluorescent Protein is a process called fluorescence, where light is absorbed at one energy level and emitted at a lower one. This is distinct from the chemical light production of bioluminescence. The process relies on a structure at the heart of the GFP molecule called the chromophore. This light-absorbing component is not an external molecule but is created by the protein itself as it folds into its final shape.

The protein is composed of 238 amino acids, but only three of them—serine, tyrosine, and glycine—are responsible for the glow. As the protein folds, it forms a barrel-shaped structure made of eleven beta-sheets, which creates a protected central core. Inside this protective barrel, the three amino acids undergo a self-catalyzed series of chemical reactions, including cyclization and oxidation, that link them together to form the chromophore. This process happens automatically, requiring only oxygen.

Once formed inside its protein barrel, the chromophore is ready to fluoresce. The process begins when the protein is struck by blue or ultraviolet (UV) light. The chromophore absorbs this high-energy light, causing its electrons to jump to an unstable, excited state. The electrons quickly fall back to their ground state, releasing the absorbed energy as a photon of lower-energy green light. The rigid barrel structure shields the chromophore from the environment, which helps maintain its fluorescence and contributes to its stability.

Visualizing Biology with GFP

GFP’s primary use in scientific research is as a visual marker to watch biological processes in real time. Using genetic engineering, researchers can attach the gene for GFP to the gene of a protein they wish to study. This creates a “fusion gene.” When expressed by a cell, this gene produces the target protein with a GFP molecule attached to it.

This fusion tag allows scientists to track the location and movement of a protein within a living cell. For example, if a researcher fuses an enzyme to GFP, the green glow will reveal the enzyme’s precise location when viewed under a fluorescence microscope. This method can be used in living cells, unlike older techniques that required killing and fixing cells, which only provided a static snapshot.

The applications of this technology are widespread. In cancer research, scientists can use GFP to track the spread of cancer cells in a living animal model. Neurobiologists can watch individual neurons form connections in the brain, and developmental biologists can follow cells during embryonic development.

GFP also serves as a “reporter gene.” In this use, the GFP gene is placed under the control of a specific genetic switch, or promoter. When that switch is activated, the cell produces GFP and glows green, reporting that the gene of interest is active.

A Palette of Glowing Proteins

The versatility of GFP was expanded when scientists realized they could modify it. By understanding the protein’s structure and the chemistry of its chromophore, researchers began to introduce mutations into the GFP gene. These targeted changes to the amino acid sequence resulted in new fluorescent proteins with different properties, most notably a variety of different colors.

This engineering effort led to the creation of a full palette of glowing proteins. A mutation changing the chromophore’s tyrosine amino acid to a histidine resulted in Blue Fluorescent Protein (BFP). Another mutation created Cyan Fluorescent Protein (CFP), while a different change led to Yellow Fluorescent Protein (YFP). Further research on proteins from other organisms, like corals, yielded a range of red fluorescent proteins (RFPs).

The ability to use multiple colors at once is a significant advantage in research. Scientists can tag several different proteins or cellular structures simultaneously within the same living cell. For instance, one protein can be labeled with GFP (green) and another with RFP (red). By observing if the green and red signals move together or overlap, a researcher can determine if the two proteins are interacting. This multicolor imaging allows for the study of complex cellular events.