Green Fluorescent Protein (GFP) originated in the Pacific jellyfish, Aequorea victoria. Researchers widely use this protein in molecular biology as a reporter, allowing them to visualize cellular processes in real-time. By attaching the gene for GFP to the gene for a protein of interest, scientists can track where and when that protein is produced inside a living cell. When the GFP gene is inserted into bacteria, the cells begin to glow bright green under specific light, providing a simple, non-invasive method to monitor gene expression. Understanding the physical form GFP takes inside the bacterial cell is central to its utility as a scientific tag.
The GFP Molecule’s Unique Shape
The green fluorescence is the result of a highly protected and specific three-dimensional structure. The main body of the protein forms a rigid, cylindrical shape often described as a beta-barrel or a “beta-can.” This structure is composed of eleven strands of beta-sheet that wrap around a central alpha-helix. The overall dimensions of this cylinder are approximately 30 Angstroms wide by 40 Angstroms long.
The protective beta-barrel shields the source of the fluorescence, which is a small chemical structure called the chromophore. This chromophore is nestled deep within the center of the barrel, protected from the surrounding cellular environment. The surrounding protein matrix dictates the chromophore’s final structure and its light-emitting properties.
The formation of the chromophore is a fascinating chemical process that happens entirely on its own, without the help of external enzymes. It begins with the cyclization of a three-amino-acid sequence (Serine-Tyrosine-Glycine) within the central alpha-helix. This cyclization is followed by an oxidation step, which requires molecular oxygen to be present. The surrounding barrel structure is essential for holding the three amino acids in the precise alignment needed for this autocatalytic reaction.
The complete maturation of the chromophore, from the initial protein folding to the final fluorescent state, is a relatively slow process. It can take anywhere from 90 minutes to four hours for a newly synthesized GFP molecule to become fully fluorescent. Once formed, the chromophore-containing barrel is rigid and stable, enabling the protein to resist changes in temperature and pH.
Cellular Location and Folding State
When expressed in a bacterial cell, the majority of functional GFP is found freely dissolved in the cytoplasm. In this ideal configuration, the protein is properly folded into its beta-barrel shape and is actively fluorescent, moving throughout the cell’s interior. This soluble, functional state allows the bacteria to exhibit a diffuse, uniform green glow when viewed under a microscope.
However, the configuration is not always ideal, especially when GFP is produced rapidly. A common alternative configuration is the aggregated, non-functional state known as an inclusion body. Inclusion bodies are dense, insoluble clumps of protein that form when the rate of synthesis overwhelms the cell’s natural machinery for folding proteins correctly. These aggregates often appear as bright, concentrated spots or clusters within the cytoplasm, typically at the cell poles.
The visibility of fluorescence is the key distinction between the two configurations. Soluble GFP is fluorescent because its chromophore is fully mature and protected within the barrel. Conversely, aggregated GFP is generally non-fluorescent, or only weakly so, because it was trapped in an unfolded or partially folded state before the chromophore could properly mature. This dual configuration of soluble-functional versus aggregated-non-functional is the primary answer to how GFP exists inside the bacterial cell.
The Bacterial Expression Process
The configuration that GFP ultimately adopts within the bacteria is directly related to the speed and conditions of the protein production process. Bacteria like E. coli are designed for rapid growth and protein synthesis. When scientists induce the bacteria to produce large amounts of GFP using strong genetic promoters, the cell’s machinery often runs too fast for the GFP to keep up.
The conflict arises because the physical folding of the GFP polypeptide chain is fast, but the chemical maturation of the chromophore is inherently slow, requiring up to several hours. The cell quickly generates numerous newly synthesized GFP chains. If these chains cannot fold and mature fast enough, they begin to interact with each other in unproductive ways, leading to the formation of insoluble aggregates that constitute the inclusion bodies.
Researchers can influence this process to favor the soluble, fluorescent configuration by adjusting growth conditions. One common strategy is to lower the cultivation temperature significantly, often from the typical 37°C down to 18°C or 25°C. This decrease in temperature slows down the overall bacterial metabolism and the rate of protein synthesis. The slower production rate allows more time for the GFP to correctly fold and for the chromophore to mature, thereby increasing the final yield of functional, soluble protein. The concentration of the inducing agent can also be carefully managed to control the rate of GFP production and minimize aggregation.
Visualizing GFP Configuration
Scientists confirm the configuration and location of GFP inside living bacterial cells primarily through fluorescence microscopy. By exciting the cells with a specific wavelength of light, the green glow reveals the protein’s distribution. The soluble, functional state appears as a diffuse, uniform fluorescence that fills the entire cytoplasm of the bacterial cell.
In contrast, the aggregated, non-functional state is visible as one or more distinct, bright spots or clumps within the cell, which directly represent the inclusion bodies. This simple visual difference allows researchers to quickly distinguish between the soluble configuration and the aggregated configuration. Confocal microscopy provides high-resolution images to precisely map the location of these aggregates.
Advanced techniques are used to confirm the dynamic state of the protein. Fluorescence Recovery After Photobleaching (FRAP) is a method that assesses whether the protein is free-floating or aggregated. A strong laser pulse is used to irreversibly destroy the fluorescence in a small region of the cell. If the GFP is soluble, unbleached molecules quickly diffuse into the bleached spot, causing the fluorescence to rapidly recover. If the GFP is aggregated and immobile, the fluorescence recovery is slow or non-existent, confirming the protein is trapped in an inclusion body.