Green Fluorescence Protein

Green Fluorescent Protein (GFP) is a naturally occurring fluorescent protein that emits bright green light when exposed to ultraviolet or blue light. Originally discovered in the jellyfish Aequorea victoria, GFP has become one of the most powerful tools in modern molecular biology and biotechnology. It is widely used as a biological marker to study gene expression, protein localisation, and cellular processes in living organisms.
Its discovery and development revolutionised biological imaging and earned the 2008 Nobel Prize in Chemistry for Osamu Shimomura, Martin Chalfie, and Roger Y. Tsien.

Discovery and Origin

The history of GFP begins in the early 1960s, when Osamu Shimomura isolated two proteins from the jellyfish Aequorea victoria collected from the Pacific coast near Washington, USA. One protein, aequorin, emitted blue light through bioluminescence. The second protein, GFP, absorbed that blue light and re-emitted it as green fluorescence.
This natural fluorescence mechanism, where aequorin transfers energy to GFP, fascinated scientists because GFP required no additional enzymes or substrates to glow—it fluoresced simply in the presence of oxygen and light, making it self-sufficient for use in living cells.
Later, in the 1990s, Martin Chalfie demonstrated that the GFP gene could be expressed in other organisms such as bacteria and nematodes, producing the same green fluorescence. Roger Tsien expanded the research by creating modified versions of GFP with altered colours and improved brightness.

Structure and Chemical Composition

GFP is a small, barrel-shaped protein composed of 238 amino acids with a molecular weight of about 27 kilodaltons (kDa).

• The protein forms a β-barrel structure consisting of 11 β-strands with an α-helix running through its centre.
• Inside the barrel lies the chromophore, the light-emitting component responsible for fluorescence.
• The chromophore is formed from three specific amino acids — Serine (Ser65), Tyrosine (Tyr66), and Glycine (Gly67) — which spontaneously undergo chemical rearrangements after protein folding to create a conjugated ring system that absorbs and emits light.

This unique structure protects the chromophore from the surrounding environment, ensuring its stability and ability to fluoresce even within cells.

Fluorescence Mechanism

The fluorescence of GFP occurs through a simple two-step process:

1. Excitation: When exposed to ultraviolet or blue light (around 395 nm or 475 nm wavelengths), the chromophore absorbs energy.
2. Emission: The excited chromophore then releases part of that energy as visible green light with a peak emission at about 509 nanometres.

The intensity and stability of GFP fluorescence make it ideal for non-invasive observation of biological systems in real time.

Genetic and Molecular Applications

1. Reporter Gene: GFP is often used as a reporter gene in molecular biology. When attached to the promoter of a gene of interest, GFP expression indicates whether that gene is active. Researchers can visually track gene regulation by observing green fluorescence under a microscope.
2. Protein Tagging: The GFP gene can be fused to another gene encoding a target protein, producing a fusion protein. This allows scientists to monitor the location, movement, and interactions of the target protein inside living cells without disrupting its function.
3. Cell and Tissue Labelling: GFP is used to label specific cell types or tissues, helping visualise cellular structures and developmental processes in multicellular organisms such as zebrafish, mice, and plants.
4. Promoter Activity and Gene Regulation Studies: By coupling GFP to different promoters, researchers can study how environmental or chemical factors regulate gene expression.
5. Transgenic Organisms: GFP has been incorporated into the genomes of various model organisms—like Drosophila (fruit flies), C. elegans (nematodes), mice, and Arabidopsis (plants)—producing visibly fluorescent organisms.

Variants and Derivatives of GFP

Since its discovery, researchers have developed multiple variants of GFP to enhance brightness, stability, and colour diversity:

• EGFP (Enhanced GFP): A modified version with improved fluorescence intensity and folding efficiency.
• CFP (Cyan Fluorescent Protein) and YFP (Yellow Fluorescent Protein): Mutations that shift fluorescence colour, useful for multi-colour imaging.
• DsRed and mCherry: Red fluorescent proteins derived from coral species, allowing simultaneous imaging of different cellular components.
• Split GFP Systems: Used to study protein–protein interactions, where two non-fluorescent halves of GFP reassemble and fluoresce when interacting proteins come together.

These variants enable multi-channel fluorescence imaging, allowing scientists to track several biomolecules simultaneously within living systems.

Advantages of Using GFP

• Non-toxic and Self-contained: GFP does not require external substrates or cofactors to fluoresce, making it safe and convenient for use in living organisms.
• Real-time Observation: Enables direct visualisation of dynamic biological processes in living cells.
• Stability: The fluorescence is stable across a wide range of temperatures and pH levels.
• Versatility: Applicable to diverse organisms and experimental systems, from bacteria to human cells.

Limitations

Despite its versatility, GFP also has some limitations:

• Photobleaching: Prolonged exposure to light can diminish fluorescence over time.
• Maturation Time: Newly synthesised GFP requires time (tens of minutes) to fold and form the functional chromophore.
• Size: The relatively large size (27 kDa) may occasionally interfere with the function of fusion partner proteins.
• pH Sensitivity: Fluorescence intensity can vary under highly acidic or basic conditions.

Researchers often address these limitations by using improved variants such as superfolder GFP or alternative fluorescent proteins with faster folding and greater resistance to photobleaching.

Role in Modern Research

GFP has transformed the field of cell biology and molecular genetics, allowing visualisation of processes that were previously invisible. Some key areas of its application include:

• Tracking cell migration, neuronal development, and embryonic growth.
• Observing signal transduction pathways and protein–protein interactions.
• Monitoring disease progression, such as tumour formation and metastasis in cancer studies.
• Developing biosensors that change fluorescence in response to environmental signals or molecular binding events.

In biotechnology and medicine, GFP serves as a diagnostic and research tool, facilitating discoveries in gene therapy, developmental biology, and synthetic biology.

Nobel Prize and Scientific Recognition

The discovery and application of GFP earned the 2008 Nobel Prize in Chemistry for:

• Osamu Shimomura, who first isolated and characterised GFP from Aequorea victoria.
• Martin Chalfie, who demonstrated its utility as a genetic marker in living cells.
• Roger Y. Tsien, who expanded its palette and improved its optical properties.