USING THE GREEN FLUORESCENT PROTEIN

 TO TEACH MOLECULAR STRUCTURE AND FUNCTION AND THE CENTRAL DOGMA

BACKGROUND

     Bioluminescence is the production of visible light by living organism. Probably the best known example is that due to fireflies as they lend a sense of magic to summer evenings. But bioluminescent organisms on land are nowhere as plentiful as they are in the oceans; indeed, it would be very difficult to find any place in the ocean where bioluminescence doesn't exist. In particular, it is a property of many marine coelenterates.radially symmetric invertebrates, such as jellyfish. At depths where visible light intensity is too low, bioluminescence is a means of visual communication. There are many uses for this communication: predation, mating, camouflage, symbiosis, etc, but many think that the main use is in defense (a message that says: "stay away.I taste horrible".or, "I can hurt you".)

     The genus, Aequoria, is one of the most studied bioluminescent organisms. It is a transparent animal about 7-10 cm in diameter with a hemispherical umbrella. The mouth is at the underside of the body and the transparent nature of the jellyfish functions as a kind of magnifier lens when the mouth is fully open. The light organs consist of about 10 dozen tiny granules distributed evenly along the edge of the umbrella making a full circle.

     In 1971, it was discovered that the green color derives from an intrinsically green fluorescent protein, the three-dimensional structure of which was determined in 1996. The protein has 238 amino acids and looks like a barrel with a light bulb inside.

     The model on the left displays the protein as a wireframe model, showing all the amino acids and their side chains; in the interior is buried a green ball and stick representation of the part of the protein that fluoresces (called the chromophore or fluorophore). The overall plan of the protein, showing its barrel-like nature, is better visualized by showing just the backbone of the protein, without all the amino acid atoms, along with a green ball and stick representation of the fluorophore. One of the intriguing aspects of this protein is that the fluorophore is an integral part of the protein: amino acids at positions 65, 66, and 67 (ser-dehydrotyr-gly) undergo a relatively slow, oxygen-dependent cyclization just after the protein is synthesized. This spontaneous chemical transformation results in formation of the fluorophore.

     In the laboratory, fluorescence is easily achieved by exposing the protein to "black" light. That is, when imbedded in the protein interior the fluorophore absorbs light in the UV-B region (395 nm.. plus a smaller absorbance peak at 470 nm) and emits light (fluoresces) at 509 nm, which is in the green part of the visible spectrum¹

     In the intact organism, however, the process is more complicated.and more interesting. The mechanism of fluorescence involves two proteins: an influx of Ca⁺² causes the first protein, aequorin, to become excited and transfer the energy to the second protein, GFP, which loses the energy by emitting a photon of green light. Aequorin has been isolated and shown to fluoresce at 469 nm in vitro; however, it does not transfer its energy by fluorescence in vivo but rather by a quantum chemical (Förster transfer) process.

     The brilliant, intrinsic fluorescence of GFP has made it useful to molecular biologists as a non-invasive indicator of gene expression. For example, before the advent of "GFP biotechnology", if a researcher was interested in studying the expression of a particular gene.when it is turned on and where does the protein end up.he or she was severely limited in how they could approach the problem. With the cloning and sequencing of the GFP gene, however, an extremely useful tool was made available not only to the researcher interested in basic research but also to those interested in solving problems in medicine and agriculture at the molecular and cellular level. The reason is that through genetic engineering, it is possible to literally attach the GFP gene to the gene of interest. This puts the GFP gene under the control of the temporal and spatial regulation of the particular gene AND can result in the creation of a "fusion" protein in which the GFP is attached to the protein of interest without affecting its function. The value in this is that by simply exposing the cells, tissues, or organism to blue light will result in GFP fluorescence "reporting" on the presence and location of the particular protein. Many different fusion proteins have been made and expressed in organisms ranging from yeast to soybeans to mice.

THE TEACHING UNIT

This teaching unit consists of three related sections. It is adapted for this website from a more extensive curriculum of the same name which can be obtained as a CD from the Center for BioMolecular Modeling.

The first exercise uses the BioRad Explorer Kit to genetically engineer E. coli to express the GFP gene. This illustrates in very concrete steps step the molecular flow of genetic information.

The second exercise focuses on DNA as an information-encoding macromolecule. A gene database (GenBank) is accessed via the Internet and the exact nucleotide sequence encoding GFP is downloaded. Students can trace the "flow of information" from this sequence of deoxyribonucleotides in DNA, through the sequence of ribonucleotides that comprise the messenger RNA , and finally, to a sequence of amino acids that comprise the GFP .

The third exercise emphasizes the protein -- and how the unique three-dimensional structure of GFP allows it to carry out its function. This exercise begins with the BioRad Kit No. 2, in which students purify and analyze the GFP. The structure of the protein is then studied via the molecular visualization software, RasMol; in addition, an accurate three-dimensional model of GFP is used as a tactile learning tool.

¹The green part of the visible spectrum extends from about 470 (blue-green) nm to about 520 nm (yellow green).