GFP making the invisible light visible: The physics behind!

Elodie Chardin, Engenheira Física formada pela École Central de Marseille

e-mail: elodieygtc@gmail.com

Everything starts from a jellyfish Aequorea aequorea (1) that lives in the cold waters of the north Pacific.

The jellyfish contains a Ca-sensitive photoprotein, aequorin, that emits blue light. The green fluorescent protein (GPF) converts then this light to green light, actually seen when the jellyfish lights up. For proof, purified yellowish GFP solutions once taken outdoors in sunlight, glow with a bright green color. The protein absorbs ultraviolet light from the sunlight, and then emits it as lower-energy green light. GFP is also naturally present in coral reef organisms, fungi (Figure 1 a, b) and now has been engineered to attach the gene to any living body like specific plant tissues (Figure 1 c, d).

Figure 1. a) Bioluminescent jellyfish Aequorea aequorea in nature; b) Zebrafish; c) Colored bacteria; d) Bioluminescence of Colletotrichum falcatum

Who cares about this obscure little green protein from a jellyfish?

Nobel Prize awarded in 2008, a sole gene, GPF is sufficient to let an organism phosphorescent, i.e. allows to look directly into the inner workings of cells without any toxicity and therefore turned out an invaluable tool in the scientific world. Shine ultraviolet light and if there is any GFP, it will glow bright green, as well as any object linked to of your interest. For instance, attach it to a virus, then, as the virus spreads through the host, you can watch the spread by following the green glow. Attached to a protein, you will see through the microscope as it moves around inside cells. Used in the development of optical sensors, it provides information on cellular properties and processes (2). Nowadays variants of GFP are available in all spectrum colors and the GFP chromophore studied and engineered to design new functional biomaterials with optimal photo-response.

But how does it really work, what is happening behind!

The GFP is a single peptide chain containing 238 amino acid residues. Due to a self-contain fluorescent chromophore (absorbs light at a specific frequency and imparts color to a molecule (3)) in their peptide chains and these proteins can be expressed in living bodies (1).

GFP absorbs energy in the form of light and stores it in its chromophore’s electrons, which leads to the so-called excited state.To return to the ground energy state, the protein within less than 10-9 sec releases an electron, i.e., loses its excess energy through two different deactivation processes, as shown Figure 2: the first one is fluorescence – re-emitting energy in the form of light, and the second one is called nonradiative relaxation, during which electronic energy, is transformed into vibrational energy of nuclei.

Figure 2. Jablonski Diagram. A fluorescent molecule absorbs light energy and rapidly re-emit it in the fluorescence form. To pass from the ground state to an excited state, a molecule must receive an amount of energy equivalent to the difference between these two levels.

At this point electrons released very quickly react with oxygen, forming highly toxic oxygen radicals, which damage cellular components, causing the cells to die: it is bleached! But GFP’s structure acts as a shield, protecting the cell. When the fluorophore releases an electron, the radicals that are formed react within GFP, so they do damage to GFP but not to the cell!

GFP can be today color-coded to follow different cell-types and is used to study different processes at different scales, tagging anything from groups of cells to individual molecules. Used in the development of optical sensors, it provides information on cellular properties and processes (2). By controlling the release process, exhibit intrinsic energy barriers on the non-radiative pathway (4) and exhibit a self-fluorescence of the chromophore. The radiative channel turned predominant let imaging new functional biomaterials with optimal photo-response. Seems GFP is still full of surprise!