Fluorescent proteins are members of a class of proteins that are structurally homologous. They have unique characteristics that they are self-sufficient enough to form a chromophore of visible wavelength from the three amino acid sequences in their own polypeptide sequence. For biologists, the usual research practice is to introduce a gene (or gene chimera) encoding an engineered fluorescent protein into living cells, and then use a fluorescence microscope to observe the location and dynamics of the gene product.
Figure 1. he molecular structure of the green fluorescent protein.
The earliest green fluorescent protein (GFP) was discovered by Shimomura and others in a jellyfish with the scientific name Aequorea victoria in 1962, and the second GFP was isolated from marine corals. Among them, jellyfish GFP is a monomer protein composed of 238 amino acids with a molecular weight of about 27KD. GFP fluorescence is mainly generated by the nucleophilic attack of glycine amide at position 67 on the carboxyl group of serine at position 65 in the presence of oxygen. After the dehydrogenation reaction of the imidazolyl group at the carbon atom at position 5 and the α-2β bond of the tyrosine at position 66, the aromatic group is bound to the imidazolyl group. In this way, the p-carboxybenzoate cyclone chromophore is formed in the GFP molecule to emit fluorescence. After clarifying this principle, GFP was widely used in biological research. Various manufacturers such as Promega, Stratagene (including orange protein preparation technology from the Chinese University of Hong Kong), Clontech (now Takara) And so on have produced the corresponding products.
In bioluminescent organisms such as jellyfish luminophores and Renilla, fluorescent proteins were found to bind to the bioluminescent protein aequorin and luciferase, respectively. The function of the fluorescent protein is to act as a bioluminescent resonance energy transfer (BRET) receptor, converting the original blue emission of the bioluminescent protein into a longer wavelength green emission. The possible role of bioluminescence in jellyfish luminescence may be to attract secondary predators when attacked. The so-called "theft alert" hypothesis. This is consistent with the observation that jellyfish rarely show bioluminescence without stimulation. Fluorescent proteins (and non-fluorescent chromoproteins) in amoeba seem to be able to function with one or more species-dependent capabilities. An important biological function may be to provide light protection for symbiotic photosynthetic algae in strong light environments. However, this feature alone cannot explain the different colors observed in the coral reef Anthozoans. The color provided by fluorescent proteins may also be important in the identification of coral fish species. These are just two of the many speculative assumptions about the biological role of fluorescent proteins. It is worth noting that, although coral reefs play a key role in supporting a wide range of marine life and providing food to humans, few studies have focused on understanding the role of color and fluorescence in coral reef biology and ecology.
The most popular applications of fluorescent proteins include their use in the localization and dynamics imaging of specific organelles (or recombinant proteins) in living cells. For imaging of specific organelles, standard molecular biology techniques are used to map genes encoding fluorescent proteins to known locations. CDNA fusion of a protein or peptide for that particular organelle.