The Green Fluorescent Protein (GFP) allows scientists to visualize previously invisible processes within living organisms. This protein has transformed how we study cellular functions and biological systems. Its ability to produce a bright glow under specific light conditions has made it a widely used and important tool. Understanding how GFP interacts with light is fundamental to its widespread utility in various scientific disciplines.
What is GFP?
GFP is a protein originally isolated from the Pacific Northwest jellyfish Aequorea victoria. This jellyfish naturally exhibits bioluminescence, a phenomenon where living organisms produce light. Within the jellyfish, GFP works in conjunction with another protein called aequorin, which emits blue light when calcium ions are present. GFP then absorbs this blue light and re-emits it as green light, contributing to the jellyfish’s characteristic green glow.
How GFP Interacts with Light
GFP’s ability to glow stems from fluorescence, a process involving specific light wavelengths. Wild-type GFP, from Aequorea victoria, has two main excitation peaks where it absorbs light most efficiently: a major peak at 395 nanometers (in the ultraviolet range) and a smaller peak at 475 nanometers (in the blue light spectrum). When GFP absorbs light at these wavelengths, its internal chromophore, a light-absorbing part of the protein, becomes energized. This energy is then re-emitted as light at a longer wavelength, around 509 nanometers, which falls within the green portion of the visible spectrum.
This shift from a shorter absorbed wavelength to a longer emitted wavelength defines fluorescence. For example, exciting GFP with blue light around 475 nm or 488 nm results in green light emission at approximately 509 nm. This property allows researchers to illuminate GFP with a specific color of light and observe it glowing green, making it a visible marker in biological systems.
Applications of GFP
GFP’s unique fluorescent properties have led to its widespread adoption across many scientific fields. It serves as a biological marker, allowing scientists to visualize processes. One common application involves tracking gene expression by fusing the GFP gene to a gene of interest. When the target gene is expressed, GFP is also produced, causing cells or tissues to glow, indicating gene activity.
GFP is also used to visualize cellular structures and observe protein movement within living cells. By attaching GFP to specific proteins, researchers can track their location and dynamic changes in real-time. This has provided insights into processes like mitochondrial movement, cytoskeleton remodeling, and transport within cellular compartments. Additionally, GFP has found utility in medical diagnostics, drug discovery, and studying host-pathogen interactions, acting as a biological tracer to analyze pathogen spread.
Variations of Fluorescent Proteins and Their Wavelengths
While GFP was the pioneer, scientists have developed or discovered a diverse palette of other fluorescent proteins that emit different colors. These include blue fluorescent proteins (BFPs), cyan fluorescent proteins (CFPs), yellow fluorescent proteins (YFPs), and red fluorescent proteins (RFPs). Each variation has distinct excitation and emission wavelengths, resulting in a spectrum of colors.
For instance, enhanced blue fluorescent protein (EBFP) has an excitation maximum at 380 nm and an emission maximum at 448 nm, producing blue light. Yellow fluorescent proteins (YFPs) have excitation and emission maxima shifted to longer wavelengths, such as 514 nm and 527 nm respectively, resulting in a yellow glow.
These different colored fluorescent proteins are useful for multi-color imaging, enabling researchers to label and observe multiple structures or processes simultaneously within a single living cell or organism. For example, a blue fluorescent protein could label one cellular component, while a red fluorescent protein labels another, providing a comprehensive view of complex biological interactions. This expanded palette allows for more detailed investigations of living systems and processes.