Green fluorescent protein (GFP in short) was the first of a large (and still growing) family of proteins with the unique ability to fluoresce in different colors. But since GFP was the first, and it is still the most popularly used, and most known by the general public, I dedicate the first post to this protein.
But first, let’s briefly define the term fluorescence: it is the emission of light by a substance that absorbed light. The emitted light is at a longer wave-length than the exciting wave-length. However, under certain conditions where the fluorofore is simultaneously excited by two photos, the emission is shorter than the exciting wave-length. We will get to two- (and three-) photon excitation at a later post. Fluorescent is different than phosphorescent – which is emission of light independently of any excitation.
The common laboratory GFP is excited by blue light and emits green light.
But what is GFP?
GFP is a natural protein that was first isolated from the jellyfish Aequorea Victoria in 1962. Although 50 years has passed since its discovery, the biological function of GFP and GFP-like proteins remains controversial. I may dedicate a post about it at a later time.
GFP is a 238 amino-acid long protein, with a unique barrel-like structure. Unlike many proteins that utilize co-enzyme molecules to elicit their function (e.g hemoglobin that utilizes the heme molecule), the uniqueness of GFP is of creating its own chromofore by cyclization of amino acids number 65-67 (serine-tyrosine-glycine). This self-assembly is one of GFPs advantaged that made it so popular.
Another important feature of GFP is its monomeric, i.e. each protein functions alone, and does not associate with other GFP proteins (except when the concentration is high). There are other fluorescent proteins that act as dimers (i.e. two proteins) or even tetramers (4 proteins).
The wild-type GFP from the jellyfish is accustomed to low ambient temperatures (since the jellyfish is found in the cold Pacific Northwest). Therefore it has a low efficiency of folding at 37°C, which is required for studying many biological systems, from bacteria to mammalian. Another deficiency of the wild-type GFP is its low fluorescence intensity after excitation with blue light. To improve the quality of the protein for research, two mutations were implemented. The first is S65T (serine 65 changed to threonine). This made the protein fluoresce 35 times brighter than the wild-type GFP. The S65T version is often used in systems at low temperatures (20-30°C) such as insect of yeast. The second mutation, phenylalanine 64 changed to leucine (F64L), improve the folding of the protein at 37°C. GFP protein with the both mutations is called enhanced-GFP (EGFP in short). In the following posts, I may use the term GFP, instead of EGFP or GFP(S65T). In any case, few people still work with the wild-type GFP protein.
The above mutations also changed the spectral properties of the GFP protein. The wild-type GFP has two excitation peaks: a major one at 395nm and a minor at 475nm. If the GFP is excited at 395nm (UV light), it emits green light at a wavelength maximum of 508nm. Excitation at 475nm gives a maximum of 503nm. The S65T mutation leads to chromophore ionization. The excitation at 395nm is suppressed (due to the neutral phenol of the threonine) and the 475nm excitation peak is shifted to 488-490nm and enhanced 5-6 times.
Thus, you will find in most fluorescent microscopes a light source at 488nm, to suit the excitation peak of the commonly used EGFP.
There are several other classes of mutations that were introduced in GFP in order to create other spectral properties (e.g. red or blue-shifted, photoactivatable GFP).
So, how is GFP used as a fluorescent marker in biological research? Simply put, you can very easily fuse GFP to any protein or peptide of your choice. In some cases, this may disrupt the function of that protein. But in many cases the disruption is minimal if any. Once the GFP-fusion protein is expressed in the cell, you can answer, by using fluorescent microscopy, the following questions: WHERE in the cell does it reside? Does it MOVE? IF and/or WHEN is it expressed and what is the LEVEL of expression? What is its rate of SYNTHESIS or DEGRADATION? WHO does it associate with? And many more questions which we will explore in this blog.