Notes

The protein GFP is a fluorophore that absorbs light at particular wavelengths and then emits light at different wavelengths. The fluorescent protein is now a basic tool of molecular biology, helping to answer the most basic of questions about proteins in living organisms - “where is it?” Fluorescent microscopy enables a scientist to locate and watch particular proteins inside a living cell.

This is a video taken during mitosis - cellular division. The cells are producing green fluorescent protein that remains bound to DNA (H2B-GFP) while the skeletal protein Tubulin is fused to a red-fluorescent protein (mCherry-Tubulin). As one cell becomes two we can watch how the shape and location of the DNA changes in response to the coordinated restructuring of the cytoskeleton. Watching this choreography in a live cell would be significantly more challenging without access to genetically encoded fluorophores like GFP.

Genetic Palette

The original GFP protein found in jellyfish has been significantly engineered to be experimentally useful. Scientists have found and developed many additional colors of fluorophores, and even proteins that can switch between colors.

• Ultraviolet: Sirius
• Cyan: MiCy
• Green: Clover
• Yellow: mVenus
• Orange: mOrange
• Red: mScarlet
• Far-red: mKate2

Creative Uses

Florophores as one of the most basic tools in molecular biology act as a ‘hello world’ for genetic coding. Looking for a green cell is a basic positive control in understanding an experimental setup. By engineering the protein scientists have developed a number of different sensors that can be used to read out additional states of a live cell.

• gTemp is a cellular thermometer based on the ratiometric difference in fluoresence of two different fluorophores.
• FRET using fluorophore pairs like CFP and YFP is a technique used measure nanometer distances between objects in a live cell.
• Split fluorophores composed of an N-terminal component and a C-terminal component, become whole and fluorescent only when the two halves are brought together in a live cell.
• By attaching localization signals to fluorophores scientists can effectively ‘paint’ organelles like the Nucleus, golgi, ER, membrane, etc.

Nobel Prize

• 2008 Nobel Prize in Chemistry was awarded to Osamu Shimomura, Martin Chalfie, and Riger Tsien for the discovery and development of the green fluorescent protein, GFP.