Green fluorescent protein

Aequorea victoria

Neural progenitors in the olfactory bulb.tif

FPbeachTsien

174-GFPLikeProteins GFP-like Proteins.tif

Lancelet GFP GIF

Green Fluorescent Protein (GFP) is a protein that exhibits bright green fluorescence when exposed to light in the blue to ultraviolet range. Originally isolated from the jellyfish Aequorea victoria in the early 1960s, GFP has become an invaluable tool in biotechnology, molecular biology, and biochemistry for its use as a molecular marker.

History

The discovery of GFP can be traced back to 1962 when Osamu Shimomura extracted a protein from the jellyfish Aequorea victoria that glowed bright green under ultraviolet light. Shimomura, along with Martin Chalfie and Roger Y. Tsien, was awarded the Nobel Prize in Chemistry in 2008 for their work on GFP, highlighting its impact on the scientific community.

Structure

GFP is composed of 238 amino acids, forming a barrel-shaped structure known as a β-barrel. Inside this barrel, there is a chromophore, which is responsible for the protein's fluorescent properties. The chromophore is formed by a post-translational modification of the protein's own amino acids, specifically through a reaction involving Ser65, Tyr66, and Gly67.

Mechanism of Fluorescence

The fluorescence of GFP occurs when the chromophore absorbs light of a specific wavelength and then emits light at a longer wavelength. The absorption peak of GFP is around 395 nm (ultraviolet), with a secondary peak at 475 nm (blue light), and it emits green light at around 509 nm.

Applications

GFP has revolutionized many areas of research and biotechnology. Its applications include:

• Gene expression monitoring: GFP can be fused to a protein of interest to monitor the expression and localization of the protein within a cell in real-time.
• Cell biology: Used as a marker to track cellular components and processes.
• Microscopy: Enhances the visualization of cells and cellular components under a fluorescence microscope.
• Genetic engineering: Acts as a reporter gene to study promoter activity and gene expression.
• Bioluminescence research: Studies on bioluminescent organisms and the mechanisms of light production.

Advantages and Limitations

The use of GFP has several advantages, including its ability to be expressed in a wide range of organisms, its stability under various conditions, and the non-invasive method of detection by fluorescence. However, there are limitations, such as the potential for photobleaching, the need for specific wavelengths of light for excitation, and the possibility of affecting the function of the protein to which it is attached.

Variants

To overcome some of the limitations of wild-type GFP and to expand its utility, numerous variants have been developed with altered fluorescence spectra, improved brightness, and increased photostability. These include enhanced GFP (eGFP), yellow fluorescent protein (YFP), and cyan fluorescent protein (CFP), among others.

Conclusion

GFP has become a cornerstone in the fields of molecular and cellular biology, allowing for the visualization and tracking of proteins, cells, and their functions in unprecedented detail. Its discovery and the development of its variants have opened new avenues for research and have had a profound impact on the study of life sciences.