In 1961, the scientist Osamu Shimomura was hard at work in Puget Sound, catching sea jellies. During his time in the San Juan Islands, he would catch more than 1 million sea jellies. Little did he know that he was to spark/ignite a revolution in science, that would benefit biological research forever. Shimomura had come to Puget Sound for his postdoctoral research studying luciferins, a group of proteins responsible for bioluminescence in living organisms; his goal was to isolate this protein from Aequorea Victoria, the crystal jelly. He tried various techniques to isolate the protein, in vain. The story goes that, at the end of one day, discouraged by the lack of success in his experiments,, he dumped the remains of his experiment into the laboratory sink. To his surprise, the mixture started glowing green. Following this accidental experiment, he was able to figure out that this protein from the sea jelly had reacted with calcium ions in the laboratory sink. From here, Shimomura then identified the two proteins responsible for Aequorea bioluminescence: aequorin, which generates blue light (in the presence of calcium ions), and green fluorescent protein, which absorbs and converts this light to green. Since its discovery, green fluorescent protein (or GFP) has revolutionized biological imaging, contributing to numerous fields of study. Little did he know what an impact his discovery, or that he would eventually win a Nobel prize for discovering this protein.
Green Fluorescent Protein has revolutionized biological research. It is incredibly versatile, and has been used from anything from HIV to cancer. It has allowed for discoveries that were previously unobtainable to now be in full reach. Using GFP, scientists can fluorescently tag a given protein, then watch as the fluorescent protein moves throughout an organism. In a way, GFP has illuminated not just proteins , but a whole new world of scientific discovery. Scientists are able to use GFP to track proteins (and genes). Scientists are able to see cancer as it matastisizes in a body, and explore the inner neuronal workings of the brain. This research brings hope that diseases such as Alzheimer’s, could be eventually left behind. In all, GFP provides an important tool for scientific research and medicine, which has expanded our horizon for understanding life.
(The gene for producing GFP was first sequenced in 1991. From this advancement, GFP could be now manufactured by bacteria to create widespread GFP) (Scientist do not know exactly why the jelly emits light; they are indebted to it for the introduction of GFP. When Shimomura discovered GFP, he thought that thought that it might be a contaminant in the jelly’s bioluminescence.
Following the work of Shimomura, GFP went through a series of developments, which improved the quality, and versatility in research. When Aequorea is frightened in the water, which presumably serves as defense, scaring away predators in the water. Thirty years later, Douglas Prasher, at the Woods Hole Institution Oceanographic Institution in Falmouth, Massachusetts, sequenced the genetic code required to make GFP. From this, plasmids, are circular pieces of DNA found in bacteria were developed. To manufacture a given protein, scientists can insert protein plasmids into bacteria. these bacteria replicate quickly, and manufacture a large amount of that protein. In 1994, Martin Chalfie became the first person to express GFP in a multicellular organism, the nematode C. elegans. In addition, scientists have chemically engineered/altered GFP to improve the quality and versatility of the protein, creating a diverse toolbox of fluorescent proteins to use in research. The original Aequorea GFP was designed for the frigid water of Puget Sound, where the jelly lives(other places too?). At higher temperatures, it broke down, and loses its fluorescence or “photostability”. In addition, it gave off a relatively weak green fluorescence. In response, scientists have engineered more environmentally stable fluorescent proteins, such as Enhanced GFP (EGFP). Scientist have created different colored Fps, in order to diversify the spectrum (of these tools). By modifying the amino acids in the center chromophore, new FPS, such as Blue, Yellow, and Cyan Fluorescent Protein were created. A group of scientist headed by Dr. Robert Tsien engineered a new palette of fluorescent proteins, derived from Discosoma, a group of coral. His lab named these the mFruits, a diverse new group of fluorescent proteins.There are advantages to having a spectrum of fluorescent proteins in research. Researchers can now use different colored FPs to track multiple proteins simultaneously. In addition, different colored proteins allow for use in various experiments, and in general, greater versatility. In 2008, Osamu Shimomura, Martin Chalfie, and Roger Tsien, three pioneers in the development of GFP, shared a nobel prize for their work.
GFP has important chemical and physical properties, that allow it to be used as a universal genetic tag. GFP is a cylindrical protein, comprised of 11 strands of amino acids which crisscross the structure. Running through the middle of this is a single alpha helix; in the middle of this helix lies the chromophore the structure which is responsible for the protein’s fluorescence. The outer beta barrel protects the chromophore from being altered by outside chemical reactions. GFP is relativity stable, and is able to retain its barrel structure as the protein moves throughout a system. The original Aequorea GFP chromophore consists of three amino acids: Serine, Tyrosine, and Glycine. The physical properties of this chromophore allow for the protein’s fluorescence. This raises the question of what is fluorescence and how it works.
Fluorescence occurs when a matter absorbs light, and re-emits it at a longer, less energised wavelength. At the atomic level, an electron absorbs the incoming photon of light, and move to a higher energy level. Now, this electron, like all matter, inclines to seek the lowest energy level possible. To achieve this, the electron re-emits a lower energy photon, and falls back to its original position. When a lighter energy wavelength moves to a lower one, this is called redshifting, as it is moving in the direction of red on the visible light spectrum. Fluorescence is a rather common physical property in nature; for example, human teeth fluoresce under with UV light, also known as black light. However, fluorescent proteins like GFP are useful, because they can be used to tag specific genes and proteins, visually illuminating them. This protein has important properties that allow it to be used as a universal genetic marker. For example, GFP does not require any additional enzymes to fluoresce; it only requires diatomic oxygen, which is readily available in nature. Unlike bioluminescence, it does not rely on an organism’s metabolism to give off light. All fluorescence requires is an external light source. Since fluorescence is more of a physical property, rather than a chemical one, scientists can artificially activate it at will more easily; it is thereby more easily controlled. Scientists can use light to activate the fluorescence, than screen out this activation light to see the fluorescence. GFP will stop fluorescing if an input light is removed. These intrinsic physical properties can be harnessed by attaching GFP onto proteins of interest. This, in turn is accomplished through genetic modification.
Inside the cell of any organism lies DNA, the genetic code which is responsible for every new protein manufactured in an organism. The genetic code for GFP can be programmed onto the gene for a given protein. The cell’s machinery, or ribosomes, will make this protein with a GFP attached to it. The protein is now fluorescent, and can be tracked as it moves around the organism. This process has been used to illuminate a variety of cellular processes, from cell division, to gene expression. Scientists can track this protein in time and space throughout the organism. Likewise, they can use GFP to determine how a gene is being expressed in the organism. Dionne W. and researchers at the UW Genetic Sciences in Seattle, Washington are studying the expression of the MLTN-1 gene in Caenorhabditis elegans, the roundworm. Previously, this gene had been relatively unknown to science. They tagged this gene with fluorescent protein, which allows them to see where it is expressing in the organism. According to their findings, the gene expresses mainly in the the rectrum and the hypodermis of the worm. They hope to continue to explore this gene. Fluorescent proteins have allowed for these new discoveries to be made.
Scientists use a variety of techniques to study fluorescently tagged proteins. One important technique is FRET. FRET stands for Fluorescence Resonance Energy Transfer, and can occur when two fluorescent proteins are less than 8 to 10 nanometers from each other. When two fluorescent proteins come within proximity of each other, they experience an energy exchange, from one FP to the other, which can be measured. Scientists can use this information to determine the distance between two tagged proteins. This process can be used to monitor different kinds of cell activity from protein interactions.
Fluorescent proteins have many applications, resulting from widespread research, and their versatility. As such, it would take a book or more to discuss the full impact of GFP. It is best to split the applications into specific categories, and choose a sample of these findings.
Fluorescent Proteins have been used in medical research, to image infectious diseases such as malaria. Malaria is transmitted through Anopheles mosquito bites, and affects around 300 to 500 million people every year. (It can be a fatal disease.) Dr. Robert Menard, at the Pasteur Institute in Paris, has used fluorescent proteins to track and visualize the progression of malaria in mice. First, Menard tagged the mice’s arteries with RFP, providing a contrast for the GFP. Next, he inserted the malaria parasites into the mouse, tagged with GFP; now he could see a green fluorescent path, as the malaria progressed throughout the mouse’s body. From this experiment, Menard discovered that malaria first makes its way to the liver, where it attacks dying liver cells. It then replicates, to create more parasites, and thereby building strength. When ready, the Plasmodium exit into the bloodstream, where they prey on red blood cells, and further multiplies. The red blood cells burst, releasing new parasites and toxins into the bloodstream. The cycle repeats, and more red blood cells are overtaken. Before, it was not know that Plasmodium travels to and first infects the liver. Now, this process is illuminated. (Transition) By discovering the disease process of malaria, scientists are able to be one step closer to curing the disease.
Scientist have used fluorescent proteins to study neurodegenerative diseases such as Parkinson’s and Alzheimer’s. These diseases gradually break down brain cells, causing them not to function properly. This in term, causes symptoms/issues such as memory loss, speech difficulty, tremors, and muscle stiffness (Zimmers 51). One way of studying these diseases is to examine how the brain reacts to disability. From this, they hope that we might be able to understand and rewire the brain, curing Alzheimer’s and Parkinson’s. Currently, there are no cures for either of these diseases. In a research team headed by Karel Svoboda, scientists shaved one side of the whiskers on a mouse’s face. They then observed the mouse’s brain, which was tagged with fluorescent proteins. The team could see how the brain responded based on fluorescence. The mouse’s whiskers are an important sensory organ in their body, which help the it navigate throughout an environment. By removing part of this organ, scientists hope to see how the brain reacts to compromised senses, similar to those of an alzheimer’s patient . (They hope to imitate and study and the brain of an Alzheimer’s patient.) This research will be important in the quest to better understand Alzheimer’s.
Fluorescent Proteins have been used to illuminate cancer. Scientists can fluorescently tag cancer tumors in mice; they watch as the cancer moves and metastasizes throughout the organism. AntiCancer, Inc., a cancer research and pharmaceutical(is it technically?), has used this kind of research to develop new medicine for cancer. For example, they have used mice models to test angiogenesis inhibitors- anticancer drugs which prevent blood vessels from entering tumors. Fluorescent Proteins have aided in the creation of these new cancer therapeutics(look up definition.) Because scientists are able to image cancer, this opens up a wealth of discovery. This research gives us hope that, in the future, we may be able to eventually cure these diseases. This advancement would be beneficial to humanity.
GFP has contributed to research even outside of biology; In fact… it has happened with physics. Scientists continue to find innovative ways to use GFP. In 2017, a group of physicists headed by Prem Kemar, at Northwestern University, entangled (pairs of) fluorescent photons from GFP. GFP has become the first biological model for quantum entanglement. Dr. Kemar hopes that this research will lead to more___ quantum computers, the super-fast computers of the future. The physical properties of GFP including its structure and fluorescence allow it to be used. The physical properties of GFP allow for the photon to line up “perfectly,” which is important when conducting this experiment.
When Osamu Shimomura was fishing for sea jellies nearly 60 years ago, he had no idea what he was about to unfold. (he caught this protein) GFP has transformed biomedical and chemical research(find single adjective), providing a versatile tool for both biological imaging(microscopy), and gene expression. Because fluorescence is a physical property, rather than a chemical one, scientist can activate GFP at will, independent of an organism’s metabolism. In addition, GFP can be inserted into DNA, as a tracer molecule for virtually any protein. This has aided new discoveries, such as in the medical field with some cancer drugs/therapeutics, and malaria discoveries. We will nevertheless see more discoveries, and continue to be enlightened by these fascinating proteins. And that’s nothing to take lightly.