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Green Fluorescent Proteins
Jul 1, 2016

Many animals, including squirrels and birds, bury their excess acorns or nuts in summer. When winter arrives and everything is buried under thick snow, they are still able to find the food they had hidden.

Just as animals can find their food, GPS seems like it can find almost any spot on Earth. Thanks to this technology, it is now possible to find people lost on mountains, in caves, or at sea. GPS can instantly detect the whereabouts of a car in a city; parents worried about their children are able to track them with ease and precision.

Though we’re able to track people with shocking degrees of accuracy, it’s harder for us to follow the miraculous activities of the micro-structures and complex molecules in our cells. We do not yet have instruments which can accurately track objects that are so small. Though we can see the holes in our skin with the naked eye, we can only see molecules, and smaller structures, with the help of an electron microscope.

Green fluorescent proteins

Taking the minute activities that occur inside an organism and transferring these processes into a visible format, and photographing intracellular chemical changes, are among the major struggles scientists face. Many researches have tried to develop ways to transfer the micro and nano worlds into a visible form.

One way of doing this is called molecular tracking. To do this, scientists track one molecule, which is easy to spot or photograph because of the light it has been conditioned to emit, and connect it to the item they’re going to examine. It is possible to make minute things observable by means of this light-marking method. The question is: how to make the molecule emit light in the first place?

The answer lies in nature. Certain sea creatures emit colorful lights. Producing light as a result of biochemical activity is called “bioluminescence”. Many creatures living in the depths of the sea, as well as fireflies and a few other species, possess bioluminescence. 

Bioluminescent creatures live with special proteins that absorb light energy. While they absorb a little of this energy, they reflect the rest, emitting light. For a long time, scientists did not understand how these proteins worked. But in the 1960s, scientists managed to isolate green fluorescent proteins. This was a breakthrough in terms of marking and viewing human cells. The researchers who decoded the chemical structure of these molecules won the 2008 Nobel Prize in chemistry.

One of the most important bioluminescent creatures is the jellyfish aequorea victoria.  Shimomura et al first obtained this creature’s proteins through chemical means in 1962.  These green fluorescent proteins are now widely used in molecular biology and biochemistry. They are used as gene and cell markers, and thus help scientists determine the amounts of these genes in different organisms.

One of the proteins scientists track is GAD67, a protein which has important duties, particularly in the brain. GAD67 produces some messenger molecules, which function in the communication activities of the nervous system. When scientists want to know if there is GAD67 in the brain or somewhere in the nervous system, they first inject green fluorescent proteins into the cell. Later, they study the cell by using special antibodies attached to the green fluorescent proteins. If there is GAD67 in the tissue, scientists recognize it from the green color. What they are seeing is not actually GAD67, but the green fluorescent proteins attached to it. 

These proteins, which are complex structured giant molecules, have a very special geometry and pattern. While even modeling the mathematical structures of a molecule takes much effort, another molecule of a different color produced by another creature makes things easy for researchers. This “marking” makes it possible to understand whether genes work or not, and whether they are active in protein production or not. It is possible to trace connections between cells, observe reactions between proteins, and to find mechanisms that form signals.

During the diffusion of light from the green fluorescent protein, no substance other than oxygen is needed (such as metal ion, phosphate, etc.). Such research can thus be simply conducted by observing the green-lit protein under ultraviolet light. This protein absorbs light from a spectrum between 395 nm and 470 nm. This range is within the wavelengths of light visible to the human eye. Normal proteins diffuse light at 300 nm, and are not visible to humans. Here, we witness another miracle in the way the world are created. It is a blessing that this special protein diffuses light of 450 nm, unlike other proteins. 

Uses of green fluorescent proteins

Since the 1970s, fluorescent proteins have been used as markers in researches studying cell biology, biochemistry, and material sciences.  The discovery of these proteins marked the beginning of a new era for intracellular (in vivo) research. Scientists have used this method to discover the biochemical workings of bacteria, nematodes (round worms), insects, and mammalian cells. For example, green fluorescent proteins can be used to observe the development of embryos, the unfolding of genes, or to monitor cancer metastasis in the human liver. Since a series of cancerous cells generates a very bright green light, they can easily be spotted. Hopefully, the wider use of such proteins will make identifying cancer easier in the future.

In addition to these discoveries, the emergence of some plant viruses has been observed thanks to these proteins. They’ve also been used to monitor the development of pollen. Lastly, the proteins have been useful when tracking gene transfers from plants to animals.

All of this has been possible because sea creatures absorb light differently than creatures on land. Organisms living underwater diffuse light best at a wavelength of 450-490 nm. The jellyfish who provide the necessary green proteins are perfectly adapted to their environment, and this enables them to hide from their enemies. It is this light that scientists are now using to understand nature – and to potentially save lives.