Green Fluorescent Protein: Research Tool

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Use in the Laboratory

Green fluorescent protein has had great success as a marker protein in a variety of biological systems due to its inherent stability, which only adds to its many other desirable characteristics as a marker proteins.[1] GFP is rather resistant to denaturation,[2] sustaining structure and function up to 65°C, pH 11, 1% sodium dodecyl sulphate (SDS), or 6 M guanidinium chloride. GFP can also withstand the presence of most proteases for many hours.[3] Due to this stability, GFP could be applied to numerous other applications such as cell lineage tracing, gene expression reporting, or protein-protein interactions.[2]

While GFP can be incorporated into most prokaryotic systems, expression in eukaryotic systems may be limited to the cytoplasm and the nucleus, as GFP does not penetrate the nucleolus or vesicular organelles. However, highly specific intracellular localization can still be achieved in eukaryotes,[2] which can help to avoid the difficulties associated with adding extrinsic dyes.[4] However, this ability to generate fluorescence within live tissues in the absence of cofactors gives GFP the key for use in biological research. After an in-frame fusion to the protein of interest, the resulting chimeric protein can be expressed in a cellular environment to monitor function or activity[5].

Use of GFP as a Fusion Tag

One of the most common applications of GFP in research utilizes GFP as a fusion tag to a protein of interest in order to observe the activity of the protein. GFP is fused in frame with the gene encoding the protein of interest, resulting in a chimera that is both functional (hopefully) and fluorescent to be expressed in the organism. This technique has been used successfully in nearly every cell organelle, including the plasma membrane, nucleus, endoplasmic reticulum, Golgi apparatus, secretory vesicles, mitochondria, peroxisomes, vacuoles, and phagosomes. GFP is most commonly fused to either terminal end of the protein gene, although it may be possible to insert it onto a noncritical exterior loop or domain depending on the structure of the protein in question. For example, residues 2-233 of GFP were inserted between the last transmembrane segment and long cytoplasmic tail of a Shaker potassium channel in experiments done by Siegel and Isacoff (1997).[6]

Use of GFP as an Active Indicator

Because the β-can structure protects the chromophore from the surrounding environment, it is difficult to use wild-type GFP as an active indicator of changing conditions. However, environmental indicators have been created by combining various GFP mutants created from random and directed mutagenesis. It is also possible to incorporate phosphorylation sites into the GFP structure such that phosphorylation or dephosphorylation will induce major changes in fluorescence. For example, the Shaker fusion protein mentioned in the section above was the first genetically encoded optical sensor of membrane potential that induced at most a 5% decrease in fluorescence in phosphorylated conditions. However, the most general way to make biochemically sensitive GFPs is to exploit FRET as described in the following sections.[6]

Use of GFP in FRET

Förster resonance energy transfer (FRET) is a laboratory technique used to detect the proximity of two biological molecules by the fusion of fluorescent proteins with each of the proteins of interest. When the two fluorophores come within a certain proximity (typcially <100Å) in the proper orientation, the excited fluorophore (donor) emits energy that excites the second longer-wavelength fluorophore (acceptor) suc that it also fluoresces. Results can then be seen by the ratio of the donor and acceptor emission intensities.[7] FRET is noninvasive and is therefore safe for using within live cells[5].

While the wild-type GFP protein has not been particularly useful as a sensor in FRET, several mutants of GFP have been manufactured to create proteins with distinct fluorescence spectra. FRET has been done between two GFP molecules or a single GFP molecule and a secondary fluorophore [5].

GFP Mutants Used In FRET

(Green links depict the chromophore of each GFP variant. The applet is currently showing the chromophore of .)

  • GFP (eGFP): contains a mutation at Ser65 in which the residue is most commonly replaced by Thr, Ala, or Gly. Lacks the 395 nm excitation peak, but still emits light at 508 nm like wild-type GFP.
  • (1bfp): blue-shifted GFP that has replaced Tyr66 with His (Y66H mutation).
  • (1oxd): contains Y66H mutation with an emission spectra between BFP and eGFP.
  • Sapphire: excitation spectra at 495 nm has been suppressed while the 395 nm spectra is retained; still emits light at 508 nm.
  • (1yfp): red-shifted GFP that has replaced with an aromatic amino acid.


Förster Distances (nm) for Energy Transfer

Acceptor (GFP variant or DsRed)
Donor Blue Cyan Green Yellow Red
Blue 2.61 ± 0.05 3.77 ± 0.08 4.14 ± 0.08 3.82 ± 0.08 3.17 ± 0.06
Cyan - 3.28 ± 0.07 4.82 ± 0.10 4.92 ± 0.10 4.17 ± 0.08
Green - 1.93 ± 0.04 4.65 ± 0.09 5.64 ± 0.11 4.73 ± 0.09
Yellow - 1.00 ± 0.02 3.25 ± 0.07 5.11 ± 0.10 4.94 ± 0.10
Red - 1.40 ± 0.03 2.84 ± 0.06 3.14 ± 0.06 3.54 ± 0.07
  • Data provided by [8]

Common Pairs of GFP Molecules Used in FRET

BFP-eGFP: The BFP-eGFP donor-acceptor pair is the most traditionally used pair of GFP molecules used in FRET, but BFP is only weakly fluorescent, creating a limitation for most applications aside from microscopy and flow cytometry.

CFP-YFP: This pair has been used more recently as an alternative to the BFP-eGFP pair because CFP is significantly brighter than BFP, allowing for more accurate ratiometric measurement of donor to acceptor emissions. However, filters must be used carefully while using CFP-YFP in FRET because of the bleeding of the CFP spectra into the YFP spectra. Therefore, proper filters must be set in order to distinguish the fluorescence from donor versus acceptor [5].

Related Structures

  • 1ema Aequorea victoria Green Fluorescent Protein, monomer
  • 1gfl Aequorea victoria Green Fluorescent Protein, dimer
  • 1w7s Aequorea victoria Green Fluorescent Protein, wild-type tetramer
  • 1emb Aequorea victoria Green Fluorescent Protein, Gln80 replaced with Arg
  • 1b9c Aequorea victoria Green Fluorescent Protein Mutant F99s, M153t And V163a
  • 1qxt Crystal structure of precyclized intermediate for the green fluorescent protein R96A variant (A)
  • 1qy3 Crystal structure of precyclized intermediate for the green fluorescent protein R96A variant (B)
  • 1qyo Anaerobic precylization intermediate crystal structure for S65 Y66 GFP variant
  • 1qyq Crystal Structure of the cyclized S65 Y66 GFP variant
  • 1jbz Crystal Structure analysis of a dual-wavelength emission green fluorescent protein variant at high pH
  • 1jby Crystal Structure analysis of a dual-wavelength emission green fluorescent protein variant at low pH
  • 1bfp Aequorea victoria Blue Variant of Green Fluorescent Protein
  • 1yfp Aequorea victoria Yellow Variant of Green Fluorescent Protein
  • 1huy Crystal structure of citrine, an improved yellow variant of green fluorescent protein
  • 1oxd Aequorea victoria Cyan Variant of Green Fluorescent Protein

List of 3D structures of green fluorescent protein

Green Fluorescent Protein

Reference for this Structure

Ormo M, Cubitt AB, Kallio K, Gross LA, Tsien RY, Remington SJ. 1996. Crystal structure of the Aequorea victoria green fluorescent protein. Science. 273(5280):1392-1395. DOI 10.1126/science.273.5280.1392.


  1. [1], Haldar S, Chattopadhyay A. 2009. The green journey. J Fluoresc. 19:1-2. DOI 10.1007/s10895-008-0455-6.
  2. 2.0 2.1 2.2 [2], Yang F, Moss LG, Phillips GN Jr. 1996. The molecular structure of green fluorescent protein. Biotechnology. 14: 1246-1251. DOI 10.1038/nbt1096-1246.
  3. [3],Cubitt AB, Heim R, Adams SR, Boyd AE, Gross LA, Tsien R. 1995. Understanding, improving, and using green fluorescent protein. Trends in Biochemical Sciences. 20(11): 448-455. DOI 0.1016/S0968-0004(00)89099-4.
  4. van Thor JJ, Sage, JT. 2006. Charge transfer in green fluorescent protein. Photochemical & Photobiological Sciences. 5:597-602. DOI 10.1039/b516525c.
  5. 5.0 5.1 5.2 5.3 [4], Pollock, BA, and Heim, R. 1999. Using GFP in FRET-based applications. Trends in Cell Biology. 9 (2): 57-60. DOI 10.1016/SO962-8924(98)01434-2.
  6. 6.0 6.1 Tsien, Roger Y. 1998. The Green Fluorescent Protein. Annual Review in Biochemistry. 67:509-544.
  7. For a more extensive description and development of this topic, go to Wikipedia
  8. [5], Patterson, GH, Piston, DW, and Barisas, BG. 2000. Förster Distances between Green Fluorescent Protein Pairs. Analytical Biochemistry. 284 (2): 438-440. DOI 10.1006/abio.2000.4708.

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