Green Fluorescent Protein (GFP) has become a transformative tool in biological research, enabling scientists to observe previously invisible cellular processes. This remarkable protein possesses a natural ability to glow with a vibrant green light when exposed to certain wavelengths of light. Its surprising origin in a marine organism and subsequent development as a scientific instrument have profoundly impacted various fields, from cell biology to medical diagnostics.
Understanding Green Fluorescent Protein
Green Fluorescent Protein is a protein isolated from the Pacific Northwest jellyfish, Aequorea victoria. This jellyfish exhibits a distinctive green glow. In 1962, Osamu Shimomura identified and purified GFP from these jellyfish, observing its green fluorescence under ultraviolet light. His work revealed that the jellyfish’s bioluminescence involved aequorin, which emits blue light, and GFP then absorbs this blue light and re-emits it as green light.
The potential of GFP as a biological tool was recognized in the early 1990s when Douglas Prasher cloned its gene in 1992. Martin Chalfie demonstrated in 1994 that the GFP gene could be expressed in other organisms, such as the nematode Caenorhabditis elegans, causing their cells to glow green without additional substances. This showed that GFP could function as a genetic marker. Roger Tsien improved GFP’s properties through genetic engineering, enhancing its brightness and creating variants with different colors. For their contributions to the discovery and development of GFP, Shimomura, Chalfie, and Tsien were jointly awarded the Nobel Prize in Chemistry in 2008.
The Science Behind GFP’s Glow
GFP glows due to fluorescence, where a molecule absorbs light at one wavelength and then emits it at a longer wavelength. Its fluorescence comes from a chromophore, a chemical structure formed post-translationally by three amino acids: serine (65), tyrosine (66), and glycine (67). These amino acids undergo spontaneous chemical reactions within the protein.
The process begins with the cyclization of these amino acids, forming a five-membered ring. This is followed by dehydration and oxidation, creating the conjugated system for light absorption and emission. The chromophore is encased within a beta-barrel structure (an 11-stranded antiparallel beta-sheet), shielding it from the cellular environment and maintaining its fluorescent properties. When blue to ultraviolet light excites this chromophore, it absorbs and releases energy as green light, typically peaking at 509 nm.
Revolutionizing Biology with GFP
GFP transformed biological research by providing a means to visualize dynamic processes within living cells and organisms. Scientists can genetically attach the GFP gene to the gene of a protein they wish to study. This creates a “fusion protein” where the target protein carries the GFP tag, making its location and movement visible. This technique allows researchers to track proteins in real-time, observing their localization within cells, their transport pathways, and how they interact with other molecules.
GFP also serves as a powerful reporter gene, indicating when and where a gene is active. By placing the GFP gene under a gene’s regulatory elements, scientists observe green fluorescence whenever that gene is expressed. This application is useful for studying gene regulation during development, in response to environmental cues, or in disease progression.
For example, GFP tracks cancer cell spread in experimental models, monitoring tumor growth and metastasis non-invasively. It also aids in studying developmental processes, like cell migration and differentiation, by marking cell lineages. Modified forms of GFP have also been engineered into biosensors to detect changes in cellular conditions like pH, calcium levels, or specific ions.
Expanding the Spectrum: Other Fluorescent Proteins
Following GFP’s success, scientists engineered variants of the original protein, expanding the palette of fluorescent colors. Through targeted mutations in the GFP gene, researchers created proteins that fluoresce in blue, cyan, yellow, and red hues. These different colored fluorescent proteins, such as BFP, CFP, and YFP, have distinct excitation and emission spectra. Red fluorescent proteins (RFPs) were often derived from other marine organisms, like corals, broadening the spectrum.
Having a range of colors provides significant advantages in biological imaging. Multicolor labeling allows scientists to observe several proteins or cellular processes simultaneously within the same cell or organism. This enables more complex and nuanced studies of cellular interactions and pathways. For instance, researchers can use two different colored fluorescent proteins to study protein-protein interactions through techniques like Förster Resonance Energy Transfer (FRET). The development of these fluorescent proteins continues to enhance imaging capabilities, providing greater detail into the living world.