The Green Fluorescent Protein (GFP) was originally isolated from the jellyfish Aequorea victoria and is of interest because it fluoresces by molecular biological means. Its uses so far have been as reporters of gene expression, tracers of cell lineage and as fusion tags to monitor protein localization within living cells. GFP is a unique protein because it fluoresces through an intramolecular reaction between three amino acids: Ser 65, Tyr 66 and Gly 67. In other words, it is its own ligand.
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The GFP protein is a cylinder with a unique secondary structure to it called a beta-can which will be discussed later. The active site exists in the middle of the cylinder as we will see later. To get a clearer picture of the molecule try visualizing the protein backbone by right clicking on the molecule and going to display backbone. You can also visualize the main chain atoms by going under select protein side chains and then hiding the side chains. To visualize the hydrogens, select hydrogens on.
The space filling view shows the porportion of oxygens to nitrogens. You can visualize the Van der Waals radii by right clicking on the image and going to display spacefill Van der Waals radii.
By right clicking on the molecule and selecting select protein charged or hydrophobic and then changing it to a new color you can visualize the charged atoms and hydrophobicity present in the molecule. Neither seem to play a significant role in the active site.
The cartoon view gives a clear picture of the secondary structure. Beta-sheets form the outside of the cylinder with an alpha helix running down the middle of the cylinder. This is typical of a beta-can motif. Note that the beta sheets of each monomer run in the same direction. The alpha helix contains the active site, specifically the three residues mentioned in the introduction. The active site is protected from the external environment and it is speculated that certain amino acid side chains in the cylinder can be modified to change the range that GFP fluoresces at. A hydrophobic pocket is used to shield the active site from an aqueous environment. A polar environment surrounding the nonpolar side chains present in the active site would decrease fluorescence intensity and fluorescence life. You can see the beta sheets and the alpha helices by right clicking on the molecule and going to select protein sheets or helix and then coloring them a different color. When the beta sheets are colored, note the alpha helix running down the middle (characteristic of the beta-can structure). Might we suggest a purple beta sheet and a white helix for optimal visualization.
The ribbon view is another way to visualize the secondary structure.
Ramachandran plots show secondary structures of proteins. The Ramachandran plot above is for green fluorescent protein. It shows both beta sheets and alpah helices present in the molecule. The upper left corner of the plot shows both parallel and antiparallel beta sheets are present in the molecule. The groups in the middle are for right and left-handed alpha helices. The most prominent secondary structure is beta sheet followed by the right-handed alpha helix. This is consistent with beta can structure observed through molecular modeling.
You can visualize the solvent accessible areas of the molecule by right clicking on the molecule and going to options dot surfact Connolly/Richards Solvent (1.2A). This shows where solvent molecules associate with the GFP protein.
Now we can zoom in on the active site of the alpha helix.
Once again, the segment of interest includes Ser 65, Tyr 66 and Gly 67. An aromatic system is formed among these three amino acids to form the active site. A double bond in formed between the alpha and beta carbons of the tyrosine residue. The nitrogen on Gly 67 attacks the base of the carbonyl on Ser 65, water is released and the cyclic structure is formed. Photons can then be aborbed in the pi bonds of the new conjugated system. When the electrons that are excited by the photons relax, fluorescence occurs in the range of green light.
There are two hydrogen bonds present in the active site. However these particular hydrogen bonds do not appear to play a role in the formation of the active site but may help in stabilization.
Note the two nitrogens in the cyclic structure.
Note the missing oxygen in the serine residue.
These are mixed renderings of the protein with the alpha carbons in a ribbon cartoon and the active site (Ser 65, Tyr 66, Gly 67) as Van der Waals spheres. Note how the beta can encloses the active site and shielding it from an aqueous environment (important for long-lived fluorescence).The side view looks at the active site through the beta sheets. The front view looks at the active site down the axis of the alpha helix.
The energy of the GFP protein as given by Quanta Charmm is 9.48467 x 10^14 kJ/mol.