Green Fluorescent Protein

3D Structure of GFP

CPK Color Scheme C O N P S

Green Fluorescent Protein is an important protein for use in experimental procedures due to its popularity as a fluorescent indicator for protein localization and gene expression in cell and molecular biology research.

To reset the molecule:

The protein is tubular with beta sheets enclosing the chromophore, which is what causes the protein to fluoresce.

The length of the cylinder is 4.2 nm, and the diameter is 2.4 nm.

The chromophore (green) is covalently attached within the polypeptide and is made of modified amino acids.

The sequence of the chromophore is Ser-dehydroTyr-Gly at amino acids 65-67 near the N-terminus.

Get a closer view of the chromophore here, and the structure of the protein around it keeping it in place.

The chromophore is kept rigidly within the center of the protein and always rotates with the protein tube around it, never rotating on its own.

The amino acids on either side of the chromophore are phenylalanine 64 (green-blue) and valine 68 (magenta), which are highlighted here:

An important molecule in the fluorescence of the GFP protein is Glutamic acid 222, highlighted here

in blue, which donates its charge to the fluorophore by proton abstraction through a hydrogen bond network, involving Ser-205 (green)

and water. UV irradiation shifts absorption by pumping a proton relay from the neutral chromophore's excited state to Glutamic acid 222. Zoom in:

Threonine 203 (red) is a stabilizer and the side chain oxygen stabilizes the ionic state of the fluorophore by donating a hydrogen bond to it. Here is a look into the middle of the protein with all three key amino acids highlighted in their proximity to the chromophore which is important in their stabilization of its fluroescence

When Thr 203 is mutated to either Tyrosine or Histidine, the fluorescence of the protein becomes red-shifted.

There are two key amino acids in the flourescence of GFP that do not interact with the chromophore, and those are Glutamic acid 6 (dark purple) and Isoleucine 229 (light purple)- when either of these are changed, the protein no longer functions properly even if the rest of the protein remains intact.

Research has shown that the amino acids necessary for fluorescence are 7-229

, starting at the middle of the first small alpha helix at the N terminus, and ending immediately following the last beta sheet. The white section highlighted here is the only part of the protein that can be deleted without seriously affecting the fluorescence of the protein. As you can see, almost all of the protein is needed for properly functioning GFP (although there are several amino acids (230-238) excluded in this Chime image that could also be deleted from the C-terminus with no effect). There are places within the brown area, however, where point mutations can be made without adversly affecting the fluorescence as long as the mutation results in an amino acid with similar properties.

For example, this particular chime image has replaced Glutamine 80 with Arginine and the overall structure and function of the protein remained the same.

References:

Brejc, Katjusa; Sixma, Titia; Kitts, Paul; Kain, Steven; Tsien, Roger; Ormo, Mats; Remington, S. James. "Structural basis for dual excitation and photoisomerization of Aequorea victoria green fluorescent protein".Proceedings of the National Academy of Sciences of the USA; 1997, 94:2306-2311.

Cody, Chris; Prasher, Douglas; Westler, William; Prendergast, Franklyn; Ward, William. "Chemical Structure of the Hexapeptide Chromophore of the Aequorea Green-Fluorescent Protein". Biochemistry; 1993, 32:1212-1218.

Kogoy, John. (2003). CPK Color Scheme. Permission to use granted.

Li, Xianqiang; Zhang, Guohong; Ngo, Nhatanh; Zhao, Xiaoning; Kain, Steven; Huang, Chiao-Chain. "Deletions of the Aeqourea victoria Green Fluorescent Protein Define The Minimal Domain Required for Fluorescence". The Journal of Biological Chemistry; 1997, 272(45):28545-28549.