Exploration of Green Fluorescent Protein (GFP) of Aequorea

Alexander PolidoreBIOL 44207

Background:

The green fluorescent protein is a very interesting and widely used protein in microscopy and other research methods. The protein originates in certain types of jellyfish living in cold waters of North Pacific including Aequorea, Obelia, Phialidium, and Renilla. The GFP of Aequorea and Renilla have been well characterized, and have been found to have the same fluorophore structure indicating evolutionary significance. In this paper, GFP refers to the green fluorescent protein from Aeqorea. It was first discovered in 1962 as a companion protein to aequorin after the researchers observed slightly green solutions instead of blue. They found that the green fluorescent protein (GFP) converted blue light emitted by the protein aequorin to green light. It is still unknown why these marine organisms exhibit such a fluorescent glow or why it is ecologically superior to express green fluorescence. However, GFP proves to be very useful in research methods.

The unique structure of GFP contains a beta-barrel with a fluorophore region inside the barrel structure. The function of the protein is reflected by its structure in which the barrel structure shields the fluorophore from the aqueous environment giving this region the ability to absorb light and produce a light-emitting proton. Without the barrel portion of the protein the fluorophore would not function to emit light. Furthermore, the fluorophore region is a p-hydroxybenzylideneimidazolinone composed of a glycine, tyrosine, and threonine or serine in which the serine or threonine form a unique bond with glycine to make a five membered ring structure. When UV light strikes the protein, the energy in the UV light gets absorbed by the bonds of this amino acid consortium producing high energy protons in the green light frequency.

There are many widely used applications of the GFB, for example, one can monitor the infection of bacteria by bacteriophage by labeling the phage with GFP. Other applications rely on the ability of the GFP to produce light. Attaching the GFP to other proteins allows one to view how the target protein moves within a cell, for example, observing microtubules movement during mitosis. Also, scientists have been able to modify the GFP to produce various colors to compare different proteins relative to each other. This modification involves the changing of the amino acid sequence in order to produce the desired color. The use of GFP even expands into the art and commerce world as GFP is used to create microscopy art or to bioengineer animals to express certain colors. This protein has very unique abilities as well as structure that can be utilized in many ways in industry or research.

Structure and Function:

The amino acid sequence reflects the function of the protein; therefore, it very important to discuss in analyzing the function of a protein. Figure 1 shows the GFP’s 238 amino acid sequence along with the regions that form alpha helices and beta sheets.

The GFP consists of two important structural features, the beta-barrel and the fluorophore as seen in Figure 2. Normally GFP functions as a homodimer, which serves to further stabilize its structure and function. In addition, the dimerization of GFP serves to increase the fluorescent capacity for each molecule of the protein; in other words, each molecule of GFP will emit a higher concentration of green light due to the fact that the protein exists in a homodimer form as compared to a non-dimerized GFP.

Figure 1 – The GFP Primary and Secondary Structure

Figure 2 – GFP and It’s Amino Acid Sequence

ASKGEELFTGVVPVLVELDGDVNGQKFSVSGEGEGDATYGKLTLNFICTTGKLPVPWPTLVTTFSYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFYKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKMEYNYNSHNVYIMGDKPKNGIKVNFKIRHNIKDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMILLEFVTAARITHGMDELYK

In Figure 2, the blue portion of the amino acid sequence represents the internal domain of the protein and the three green amino acids are the fluorophore, which is involved in the emitting of light upon excitation. One of the main properties that allows for the function of this protein is the beta-barrel structure (the red portions in the above sequence), which protects the fluorophore from the surrounding aqueous environment. In further analysis of the sequence of amino acids in the beta-barrel structure, a majority of the beta-barrel structure consists of hydrophilic amino acids, but some apolar amino acids as well. The coordination of the charged amino acids, apolar, and polar residues help to protect the fluorophore from polar, reactive species in the outside environment.

There are advantages to the dimer form of GFP. The protein concentration changes the excitation spectrum in which aggregation is favored. This is the reason for the dimerization of wild-type GFP. The interface between each subunit contains charged, hydrophilic contacts in addition to hydrophobic amino acids. Aggregation occurs due to hydrogen bonding between the amino acids at the interface and due to the hydrophobic effect, in which hydrophobic amino acids come together to shield each other from the polar effects of the water molecules. Figure 3 (left) shows an aerial view of the region between each subunit. There is a high potential for hydrogen bonding in this region, which helps stabilize the dimer. Figure 3 (right) also shows the coordination of hydrophobic residues in the dimerization involving tyrosine39, phenylalanine223, and leucine221 of one subunit and tyrosine39, leucine221, and phenylalanine223 of the second subunit. This type of interaction further stabilizes the dimer based on the hydrophobic effect of amino acids.

Molecular Structure of GFP Fluorophore:

Figure 4 - Detailed Structure of Fluorophore

Tyrosine

Glycine

Serine

Figure 3 – Aerial View of Dimer Interface (left) and Hydrophobic Region of Dimer Interface (right)

The fluorophore consists of the amino acids serine (or threonine), glycine, and tyrosine in which the serine and glycine coordinate to form a unique cyclic structure characteristic of this protein and can be distinguished as the green portion in Figure 2 and Figure 4. Without this unique bonding between these amino acids, the fluorescent ability of GFP would not exist indicating the importance of this unique bond. Figure 4 shows the detailed bonding of these three amino acids. The line shown in red indicates the unique bond that occurs between the serine and glycine. This bond creates a very rare cyclic structure only found in GFP. The aromatic amino acid structures within the fluorophore indicate its ability to absorb UV light. Many chemical changes take place upon excitation by the energy transfer from light, which involve other amino acids neighboring the fluorophore.

In the jellyfish, no enzymes are required in order to form the fluorophore during the folding of GFP. The formation of the fluorophore consists of about four distinct steps. First, the protein folds into its native conformation, then a nucleophilic attack from the amide on Gly67 on the carbonyl of residue 65 forms an imidazolinone. Dehydration of the imidazolinone occurs followed by the dehydrogenation of the alpha-beta bond of residue 66 by molecular oxygen. This puts the aromatic group into conjunction with the imidazolinone. The protein now is capable of visible absorbance and fluorescence. The final product of this self-catalyzing reaction can be seen in Figure 4 in which the red bond of picture II (right) is the result of the nucleophilic attack by glycine.

BLAST comparison of GFP derivatives:

Recently discovered fluorescent proteins in coral species have allowed researches to engineer variants that are capable of producing wide ranges of colors. Some of these fluorescent proteins highly resemble the GFP, but have been engineered to withstand harsher conditions such as high pH or increased temperature. One of these fluorescent proteins was isolated from Clavularia coral, which is capable of emitting wavelengths in the cyan color range. Ai et. al. engineered a variant of this protein that is said to be the brightest and most photostable fluorescent protein to date (Ai, Henderson, Remington & Campbell, 2006). In an analysis of the structure of this variant protein, called mTFP1, the fluorophore region of this protein resembles that of the GFP with minor differences. These minor differences are significant enough to give the protein the ability to emit a different wavelength of color.

Figure 5 compares the fluorophore region of mTFP1 with the fluorophore region of GFP. There are a few structural differences with these regions due to the differences in the amino acid sequences surrounding the fluorophore region. For example, a glutamine residue has been replaced by a positively charged arginine residue in mTFP1, which appears to be causing an upward kink in the cyclic structure of the fluorophore perhaps due to steric hindrance as well as electronic effects. In addition, where GFP has a cysteine, mTFP1 has an alanine, which results in the absence of a bulky sulfur side chain group causing differences in conformation. These structural differences contribute to the overall differences in the phenotype of each protein. Even minor changes in amino acid sequence can have significant changes in the function of the protein. For example, sickle cell anemia results from a single amino acid change from a glutamic acid to a valine causing deformed red blood cells. This condition results in complications that can lead to death. Based on this knowledge, researchers are generating new variant fluorescent proteins with many different colors available already including blue, cyan, and yellow.Conclusion:

There are endless applications for GFP and its variants. Recently, these applications have been applied to consumerism and art. For example, Nathan Shaner created bacteria plate art using variants of GFP and the red-fluorescent coral protein dsRed. The colors include BFP, mTFP1, Emerald, Citrine, mOrange, mApple, mCherry and mGrape which make up a San Diego beach scene as seen in Figure 6. Another application involves glow in the dark zebrafish also known as Danio rerio. In this application, the genes for the fluorescent proteins are inserted into the genomes of the zebrafish causing them to glow under certain light conditions. However, more practical applications include use in the research lab, for example, to observe real time movement of phage or structural features on a membrane tagged with the protein.

Figure 6 - Artwork by Nathan Shaner, photography by Paul Steinbach, created in the lab of Roger Tsien in 2006.

Figure 5 - Cyan FP Fluorophore (left) Compared to GFP Fluorophore (right)

The green fluorescent protein is one of the most diverse and fascinating proteins. From its cyclic bonding characteristics and beta-barrel structure to its fluorophore, GFP has a very specific structure allowing for its fluorescence. The uniqueness of the structure of GFP gives rise to its unique ability to produce green fluorescent light. To understand the function of GFP, one must analyze its structure; these topics go hand in hand. Knowing a protein’s structure allows for an in depth understanding of the function of the protein. Without this knowledge, one can only attempt to try and fully understand the protein. Understanding protein function helps in understanding cellular processes and genetic makeup, and is essential in fighting certain human diseases and disorders.

References:Ai, H. W., Henderson, J. N., Remington, S. J., & Campbell, R. E. (2006). Directed evolution of a

monomeric, bright and photostable version of clavularia cyan fluorescent protein: structural characterization and applications in fluorescence imaging. The Biochemical Journal, 531-540. http://www.ncbi.nlm.nih.gov/pubmed/16859491?dopt=Abstract

Cody, C.W., Prasher, D.C., Westler, W.M., Prendergast, F.G. and Ward, W.W., (1993). Chemical structure of the hexapeptide chromophore of the Aequorea green-fluorescent protein. Biochemistry, 32: 1212-8

Roger Y. Tsien (1998). The Green Fluorescent Protein. Annual Review of Biochemistry, 67: 509-544

Yang, F., Moss, L., & Phillips, G. (1996). The molecular structure of green fluorescent protein. Nature Biotechnology, 1246-1251

Figure 7 - Photo courtesy of www.glofish.com