Core Concepts
In this article, you will learn about green fluorescent protein (GFP)’s functions and variations, and its ever-expanding biotechnological applications. You will also explore the biochemical mechanisms behind bioluminescence.
What is green fluorescent protein?
With the majority of Earth’s oceans still unexplored, there’s a lot of science waiting to be discovered under the sea. A rather illuminating finding came in the form of a jellyfish’s colorful glow. The more that researchers learned about this captivating phenomenon, the more its biochemical inner workings were brought to light.
Amazingly, the jellyfish species Aequorea victoria internally produces its own radiant glow. This phenomenon, called bioluminescence, wouldn’t be possible without the role of green fluorescent protein.
Green fluorescent protein (GFP) is a special protein. In response to specific wavelengths of light, it undergoes chemical reactions that result in bioluminescence. This bioluminescence emits light whose wavelengths fall into the green region of the visible light spectrum, so the glow takes on a bright green color – a striking contrast from the darker, duller colors of the seascape. But where does GFP come from, and how does it cause such distinctive hues in an otherwise ordinary creature?
The Origins of GFP
GFP provides us with insight into the fascinating realm of photochemistry, the study of how light impacts chemical reactions. In 1962, marine biologist Osamu Shimomura identified and purified GFP from A. victoria. This eastern Pacific jellyfish species is abundant in Washington state, USA, where Shimomura’s research team studied thousands of individual jellyfish.
Across their research, Shimomura and his team found that A. victoria uses GFP to bioluminesce. But how? Let’s investigate this through the impressive biochemical mechanisms that underlie GFP.
The Biochemistry behind GFP
GFP’s chemical structure embodies both form and function. Most distinctive is its tertiary structure’s β barrel (beta barrel) arrangement. The β barrel involves a β sheet (itself comprised of eleven β-strands) which folds into a cylindrical shape. The β barrel is highly stable due to hydrogen bonds and van der Waals forces contributing to the molecular unit. Then, in the interior of the β barrel, is an α-helix (alpha helix). Ultimately, this combination of barrel and helix is crucial to GFP’s fluorescent ability. The α-helix contains the molecule that makes it all happen: 4-(p-hydroxybenzylidene)imidazolidin-5-one (HBI).
Keep in mind that molecules can exist in various electronic states. In an excited electronic state, a molecule has an electron in a higher energy orbital compared to when the molecule is in its ground state. When a molecule is in an excited state, it can emit energy in the form of light or heat. There are several ways a molecule can become excited: for example, by absorbing a photon or as the result of a chemical reaction (chemiluminescence).
HBI is always present in GFP, but not always in an excited state. When GFP absorbs photons from othersources, a chemical rearrangement occurs that excites HBI. This chemical rearrangement changes HBI into its mature form, which is a slightly different molecular configuration than its structure at rest. Later, we’ll see how a series of cyclization, dehydration, and oxidation reactions achieve the chemical rearrangement that gives rise to HBI’s fluorescent mature form. Only in HBI’s mature form is it capable of emitting fluorescent green light – and only within the fluorescent protein’s β barrel.
GFP’s β barrel structure shields HBI from water, which would quench the reaction that causes HBI to fluorescence. If isolated from GFP’s protective molecular structure, HBI would be unable to create its unique glow, even when exposed to blue light. Furthermore, denaturing GFP would mean HBI can no longer work properly, since HBI would no longer be shielded from water, so the GFP would lose its fluorescent capability. It’s important to understand that HBI is not just floating in the space inside the β barrel. Rather, covalent bonds tether HBI to the α-helix. This covalent connection is supplemented by the hydrogen bonds and van der Waals forces throughout the GFP barrel. This way, the β strands making up the barrel serve as a physical barrier preventing the otherwise-vulnerable HBI from damage or unwanted side reactions arising from the protein’s solvent.
How does GFP work?
The bioluminescence process begins when calcium (Ca2+) ions activate a different protein, aequorin. We will learn about aequorin in more detail soon, but for now, you should know that activated aequorin uses energy from ATP hydrolysis to emit blue light. Like all light, this blue light is a form of energy. Through a process called Förster resonance energy transfer, activated aequorin (the donor fluorophore) donates this energy (blue light, as photons) to GFP (the acceptor fluorophore).
After aequorin transfers energy to GFP, GFP is in a higher-energy, excited state. To stabilize itself and return to its ground energy state, GFP releases energy in two rapid steps. First, vibrational relaxation occurs, where the GFP molecule’s vibrational modes cause GFP to dissipate some energy as heat. After vibrational relaxation, GFP is still in an excited electronic state, but with slightly less energy than when the process started.
To release the rest of its energy, GFP undergoes the second step in its relaxation process, emission. In emission, GFP falls from the excited state back to the ground state, emitting a photon in the process. This photon carries with it the energy from the excited state. Because vibrational relaxation occurred, the energy in the photon is less than the initial energy GFP absorbed from aequorin. Therefore, the light that GFP emits is green light instead of blue light.
Förster Resonance Energy Transfer
During bioluminescence, aequorin transfers energy to GFP through a process known as Förster resonance energy transfer (FRET).
Let’s consider a scenario involving two fluorophores, compounds that emit light as a result of electronic excitation. Many types of fluorophores exist, some of which you may have already encountered elsewhere: crystal violet, bilirubin, and quinine, to name a few.
In our scenario, one of the fluorophores, the donor, is in an electronically excited state. This excited state is usually created when light interacts with a molecule’s transition dipole. Meanwhile, the second fluorophore, the acceptor, is in the ground state, but still has a transition dipole connecting it to its own excited state. The two transition dipoles can interact with each other electrostatically. If the two fluorophores are close enough in space and their two excited states are close enough in energy, the energy contained in the donor’s excited state can transfer to the acceptor molecule. The transfer of this energy is FRET. After FRET occurs, the donor molecule is in its ground state and the acceptor molecule is in its excited state.
Because FRET requires two molecules to be close to one another in space, scientists can use this process to understand how proteins interact with each other. We assess energy transfer between fluorophores in terms of efficiency, on a scale of zero percent to 100% efficiency. Zero percent efficiency indicates that no energy was transferred from the donor fluorophore to the acceptor fluorophore, whereas 100% efficiency represents a complete energy transfer. In general, energy transfer is more efficient when the two fluorophores are closer in space and closer in energy.
With regard to bioluminescence, light-producing reactions are more efficient when more of the energy input successfully transforms into light energy. The output of light energy can be measured by how much fluorescent light gets emitted.
After emitting light as fluorescence, GFP is now back in its electronic ground state, with the same baseline amount of energy as it had before the FRET happened.
From Blue to Green: The Role of Aequorin
As alluded to earlier, aequorin is the donor fluorophore molecule that donates its blue light to GFP. Just as he isolated GFP, Shimomura also isolated aequorin from A. victoria.
Aequorin is a holoenzyme, an enzyme consisting of an inactive protein and the cofactor(s) required to activate that protein. In aequorin, the inactive protein portion is apoaequorin and the cofactor is coelenterazine. With both of these components bonded together to make its holoenzyme format, aequorin works closely with GFP to create bioluminescence.
Let’s examine this chain of events more closely. Inside the jellyfish, GFP and aequorin proteins are located close to one another. Ca2+ ions in the cytosol activate an aequorin molecule. Once activated by Ca2+, the aequorin is in a higher (excited) energy state than before because it has absorbed energy from the Ca2+ ions. Since activated aequorin’s energy state is relatively high, let’s call activated aequorin the donor fluorophore. Activated aequorin also appears blue in color, because it’s emitting energy as blue light, at a wavelength of approximately 400 nanometers.
Similarly, we’ll identify GFP, in a more relaxed state, as the acceptor fluorophore. GFP accepts the energy from the donor aequorin. The energy has transferred from aequorin to GFP, meaning that aequorin has fallen to its ground state energy state while GFP has ascended to an excited energy state. Now excited, GFP releases the energy by emitting its own fluorescent, green light.
There are a few interesting points to note here. First, the FRET process has shifted the activated aequorin’s light, originally blue, to a green color. This is an example of the Stokes shift, where the emitted photon has lower energy than the absorbed photon had. When it comes to photons (which carry light energy), lower energy indicates a longer wavelength on the electromagnetic spectrum. On the electromagnetic spectrum, green has a longer wavelength than blue. Therefore, the Stokes shift explains why GFP emits green light despite having absorbed blue light. GFP’s light is green because, when GFP became activated, its HBI was excited too. HBI is a chromophore that produces a green color, so the GFP emitted green light rather than any other color.
The diagram above depict a FRET scenario in which the energy transfer between these particular molecules of aequorin and GFP is 100% efficient. We can deduce this because the amount of energy emitted by aequorin is identical to the amount of energy absorbed by GFP. In other words, all of aequorin’s energy output has transferred to GFP. Note that, individually, both aequorin and GFP perform the same three-step process of absorbing energy, releasing a small portion of that energy via vibrational relaxation, then emitting the rest of that energy as fluorescent light. As we’ll see next, a compound called luciferin is responsible for creating the light.
Lighting It Up with Luciferin
Aequorin is a photoprotein: an enzyme that helps catalyze the breakdown of another compound called luciferin. Found in several bioluminescent species besides A. victoria (such as fireflies and glowworms), luciferins actually represent a family of compounds. When a photoprotein breaks down an excited luciferin molecule, the luciferin returns to its ground state, emitting light during this reaction.
Recall that two molecules are necessary to produce aequorin: apoaequorin (a protein that is inactive by itself) and coelenterazine (apoaequorin’s cofactor). Coelenterazine is a type of luciferin. Apoaequorin and its substrate coelenterazine bind together to form the holoenzyme aequorin, a stable and functional enzyme. At this stage, the aequorin is in its energy ground state.
Then, when Ca2+ ions bind to aequorin, aequorin activates and becomes less stable. As previously described, activating aequorin means it absorbs energy, transitions to a higher energy state, and emits blue light. This blue light then can transfer to GFP through FRET, allowing GFP to fluoresce green due to activated HBI’s influence. Through this mechanism, the combination of GFP, aequorin, and luciferin in A. victoria is what induces the jellyfish’s characteristic glow.
In the previous section, we saw two side-by-side Jablonski diagrams showing the qualitative energy transfer between aequorin and GFP during FRET. Since aequorin encompasses coelenterazine (a type of luciferin), the Jablonski diagram for aequorin also pertains to luciferin. Luciferin itself returns to its ground state when aequorin breaks it down. This breakdown is what produces light, allowing aequorin’s energy emission to be blue and fluorescent rather than simply blue.
At this stage, we’ve discussed two central points: aequorin activation is a prerequisite to FRET happening to GFP, and GFP fluoresces upon being excited. Next, we will uncover the remarkable biochemical underpinnings of how HBI makes GFP’s fluorescence possible.
How does HBI work?
Remember that GFP is a fluorophore, so when FRET excites it, it changes energy states and emits fluorescent light. Like how an excited fluorophore emits energy as fluorescent light, an excited chromophore emits energy as color. Color is simply light energy with wavelengths in the visible range of the electromagnetic spectrum, which allows us to perceive it visually.
HBI is a chromophore. When aequorin donates energy to GFP, the GFP’s HBI absorbs that energy, becomes excited, then emits that energy as color. Since the HBI emits a green color, we can deduce that it is emitted at a wavelength somewhere between 495 and 570 nanometers (the green portion of the visible range of the electromagnetic spectrum). Other fluorescent proteins instead emit this energy at other wavelengths that align with their respective color. For example, yellow fluorescent protein (YFP) emits at roughly 580 nanometers, and cyan fluorescent protein (CFP) at about 480 nanometers.
Aequorin has a chromophore, too! This is what gives activated aequorin the ability to emit blue-colored light. When aequorin absorbs energy from Ca2+ ions, coelenterazine and another cofactor, molecular oxygen (O2), combine to make the excited form of aequorin’s chromophore. Then, during the final steps of FRET, the activated aequorin releases that energy as photons carrying blue light energy.
HBI represents an important tripeptide chain along the GFP’s protein backbone: the very chain that gives GFP its glow. To put this sequence of events into context, first we have to examine the sequence of events that turns a tripeptide into a chromophore.
HBI Formation
FRET creates the conditions necessary for GFP to glow by exciting the GFP’s HBI. Prior to excitation, the GFP’s β barrel contains a three-peptide chain consisting of serine, tyrosine, and glycine residues (Ser65-Tyr66-Gly67). This chain is the basis of what will later become the excited, mature HBI fluorophore. To form the fluorophore, a series of three reactions must happen: cyclization, dehydration, and oxidation. These reactions, representing protein maturation, occur spontaneously in the GFP molecule in response to light activation.
Step 1: Cyclization
We begin with this tripeptide chain of serine, tyrosine, and glycine within a GFP molecule. These amino acid residues naturally rotate around one another in space. As the serine’s carbonyl carbon approaches the glycine’s nitrogen atom, the nitrogen performs nucleophilic attack on the carbon. The resulting intermediate has a 5-membered imidazolidinone ring containing two nitrogen atoms (one from the glycine and one from the peptide backbone).
Step 2: Dehydration
Very shortly after the ring forms, a dehydration reaction converts one of the ring’s single bonds into a double bond. As a byproduct of this step, we lose the equivalent of one water molecule. What remains of the five-membered ring is an imidazoline ring.
Step 3: Oxidation
So far, the tyrosine residue has remained largely unaffected by these reactions during the first two steps. However, through the next oxidation reaction, it takes on the vital role of finalizing our chromophore’s structure. By introducing molecular oxygen (O2), an oxidation reaction proceeds for the carbon-carbon bond that connects it to the imidazoline ring. As a result, this bond converts from a single to a double bond.
The double bond’s greater electron density allows for additional conjugation along the entire tyrosine residue. This newly-formed double bond bridges the imidazoline ring with the tyrosine’s benzene ring and terminal hydroxyl group. All of these functional groups have significant electron density that work together to create resonance and increase stability. In addition to this stabilizing force, the entire molecule is supported by hydrogen bonds and van der Waals forces that surround it within the β barrel structure.
We see the fully-formed, mature HBI chromophore within the structure of the final product. These three simple reactions, culminating in oxidation, have altered the chromophore greatly since beginning in its unexcited state.
Why is GFP important?
The introduction of GFP into new laboratory contexts has redesigned the way scientists approach biotechnology research. Using fluorescent proteins like GFP as an innovative tool, they can measure gene expression, visualize cellular activity, and even engineer new fluorescent products.
Synthetic GFP
Throughout this article, we’ve examined the groundbreaking wild-type form of GFP, the form that A. victoria produces in nature. Although the newly-discovered GFP warranted further research, studying this wild-type form in the laboratory proved challenging because GFP is somewhat sensitive to environmental conditions. Researching GFP in a lab setting had unwanted effects on its expression and folding behavior. Unfortunately, this made it difficult for scientists to artificially replicate how GFP works in nature.
To circumvent this obstacle, researchers engineered various mutant forms of GFP using directed mutagenesis. Building upon the work of Douglas Prasher and Martin Chelfie, who had already sequenced the wild-type GFP’s genetic code, the mutated GFP was tailored to thrive under laboratory conditions. For example, Roger Y. Tsien’s single point mutation gave GFP more photostability (less degradation when exposed to light) and stronger, more detectable fluorescence. A single point mutation is the process of changing, inserting, or removing one nucleotide from a DNA sequence. Ultimately, these mutant versions of GFP showed improved utility in lab applications.
Besides engineering GFP to function outside of A. victoria, other possible mutations reveal additional research avenues. Notably, scientists have mutated GFP to change the color it emits in response to excitation. Many mutant colors now exist in the fluorescent protein family, including YFP if the color emitted is yellow and CFP if the color emitted is light blue. Different colors of fluorescent protein are excited by, and emit, various different wavelengths of light.
HBI Modification
By making GFP more photostable, less sensitive to its environment, and different colors, Tsien and others demonstrated that mutating fluorescent protein can confer new properties on it. Changing GFP’s color, specifically, means changing the protein structure such that the HBI chromophore no longer cyclizes, dehydrates, or oxidizes like this.
A small adjustment may be all that it takes, and it doesn’t even require directly modifying the chromophore itself. For example, by substituting only four amino acids elsewhere in the GFP’s molecular structure, we obtain YFP and leave the chromophore untouched. Substituting the four residues influences the chromophore’s stability, but not its light-emitting function. Therefore, the protein continues to emit light, just at a wavelength tailored to the yellow region of visible light.
Why conceal HBI deep within the GFP’s β barrel? Exposure to certain solvents, such as water, would quench HBI, essentially deactivating it and lessening the intensity of its fluorescence. Encircled by β strands, HBI is protected from being exposed to water or similar solvents that could impede GFP’s function. Given how critical HBI is that function, when modifying GFP or its chromophore, scientists must ensure that the protective qualities of the protein’s secondary structure remain intact.
GFP as a Fluorescent Tag
So why do scientists undergo the effort of inventing new colors of fluorescent protein? Why utilize fluorescent protein in the lab at all? GFP’s versatility and diverse color range enable its application in new research contexts, taking biotechnological studies to new heights.
Since GFP responds to energetic excitation by producing a bright light, GFP is ideal as a fluorescent tag. Scientists customize fluorescent tags to selectively bind to a molecule that they want to detect, like an antibody or mRNA. When this molecule is tagged with GFP and then exposed to light energy at a wavelength of about 400 nanometers, the GFP will fluoresce, making the molecule appear bright green. Using this approach, GFP is a simple way for scientists to detect and visualize a molecule of interest.
Since multiple colors of fluorescent protein have been developed, multiple types of molecules can be detected simultaneously. For instance, a researcher can visualize two completely different proteins by labeling one with GFP and the other with YFP. When exposed to light energy at their respective excitation wavelengths, the GFP-tagged protein will fluoresce green and the YFP-tagged protein will fluoresce yellow. This is helpful in easily distinguishing between different molecules or structures. What’s more, it allows scientists to observe how these different molecules interact with each other.
However, the fluorescent tagging procedure extends beyond GFP. Other molecule types, like individual amino acids, longer peptides, and synthetic fluorescent probes, can also serve as tags. GFP sets a powerful standard compared to these other fluorescent tag options, and the reason for this is threefold. First, fluorescent proteins don’t interfere with the tagged target molecule’s functions or behavior, and thus have no negative effects on the target molecule. Furthermore, fluorescent proteins’ sensitivity to light absorption makes it easy to readily see them even under small amounts of light. Less input (light energy) is necessary to yield a meaningful amount of fluorescence. Finally, scientists can employ multiple fluorescent protein colors simultaneously. This makes it possible to study several target molecules at once by tagging each with a different color. Alternatively, when trying to visualize multiple targets, the use of these other types of molecules can supplement the use of GFP.
As for why this jellyfish species bioluminesces in nature? This is a topic of interest for marine biologists, and the investigation is ongoing. Leading theories propose that A. victoria bioluminesces for intraspecific communication, or as a defense mechanism to deter, startle, or misdirect predators.
Other Applications of GFP
As we saw previously, the interactions between three particular amino acid residues (Ser65-Tyr66-Gly67) are responsible for the green fluorescent effect. The three-step chemical reaction that activates HBI is an autocatalytic reaction. Autocatalysis refers to a reaction where the product later serves as the catalyst for that same reaction to repeat. Molecular oxygen, as depicted in the oxidation step above, is the only cofactor or external substance necessary to induce this positive feedback loop in GFP. Apart from this, GFP’s iconic fluorescence is completely self-propagated, adding to GFP’s appeal as a research tool. The fewer reagents and materials required to set a laboratory assay in motion, the more cost-effective it is to perform the assay. Note, however, that in the A. victoria jellyfish, Ca2+ ions are necessary for bioluminescence to take place. In other words, bioluminescence entails a chemical reaction whereas fluorescence alone does not.
Importantly, when used as a fluorescent tag on a molecule, GFP serves as a reporter gene, measuring that molecule’s level of gene expression. In this assay, scientists are trying to determine if the molecule expresses a specific gene of interest. If so, then exposing the molecule to blue light makes its GFP tag fluoresce green. Fluorescence microscopy or confocal microscopy can visualize this green light, and more expression results in more intense fluorescence. This provides a qualitative and quantitative measurement of gene expression in that molecule, indicating whether or not the gene is expressed, and precisely how much it is expressed.
Along these lines, GFP can tag molecules, like proteins of interest, inside of cells in order to detect cellular density (how close together individual cells are) and cellular activity (to what extent the molecule of interest is active inside of a cell). Through cell imaging, we can assess these properties in cells (live or dead) that contain a GFP tag. The usage of GFP doesn’t interfere with the cells’ function at all, and the only tool necessary to visualize it is blue light. As a noninvasive, low-effort reporter gene, using GFP removes barriers in biotechnology research, streamlining experiments and maximizing lab efficiency.
Imagine you’re a cell biologist who wants to watch the dynamics of how different organelles interact with each other. You can do that by tagging specific molecules in each organelle with a different color of fluorescent protein. Or maybe you’re a genetic engineer who wants to study water pollution levels. All you’d need to do is tag fish living in that body of water with GFP! (This is known as transgenics, and was the original inspiration behind inventing GloFish, the fluorescent zebrafish commonly found in pet stores.)
It’s clear that GFP’s scientific impact reaches far beyond the depths of the ocean. Fluorescent protein enables researchers to build upon existing techniques, and new applications are just waiting to be developed.
Conclusion
Although it may appear desolate, there’s actually a lot of inspiring biochemistry to be found amid the undersea darkness. Aequorea victoria jellyfish and their characteristic bioluminescence have lent themselves to great advances in biotechnological and genetic research. The chromophore HBI is fundamental to green fluorescent protein’s ability to emit light, which it achieves when activated aequorin donates energy to it via Förster resonance energy transfer. Decades of further studies have led new scientific strategies to emerge, utilizing GFP as a highly-efficient technique for detecting molecules and investigating their behavior. With room to grow in the research field, GFP offers adaptable approaches to genomics, cell biology, and much more.