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General Info

GFP ribbon diagram. From PDB 1EMA.
  • Originally discovered and characterized in the jellyfish Aeqorea victoria
  • wtGFP sequence on NCBI
  • 238 amino acids
  • The tertiary structure is a fluorophore [Ser(65)-Tyr(66)-Gly(67)] nestled inside a protective beta barrel.
  • Oxygen is required for maturation of the fluorophore
  • Natural state is a dimer with a Kd of ~ 100 µM.

GFP Variants

  • GFPmut2 - S65A, V68L, S72A [1]
  • GFPmut3b - S65G, S72A [1]
  • GFPmut3* - mutations from wt: S2R,S65G,S72A
    • This is GFPmut3b with an accidental mutation at position 2 that doesn't affect function according to the authors.[2]
  • Emerald - F64L, S65T, S72A, N149K, M153T, I167T
  • EGFP - inserted GTG as second codon, F64L, S65T + optimized for human codons with 35-fold increase in fluorescence over GFP [3]
    • Additionally, "mutation of His231 to Leu, which was probably inadvertent and neutral". [3]
  • yemGFP - F64L, S65T, A206K [4]
  • superfolder GFP - S30R, Y39N, F64L, (S65T/G65T), F99S, N105T, Y145F, M153T, V163A, I171V, A206V [5]
    • Inherited parent variant mutations (GFPmut3*): S2R, S65G, S72A (S65G mutation is superseded by S65T mutation, hence the alternative mutation designation of G65T)
  • Other variants and their mutations can be found in Shaner supplementary table 2 [6]


  • F64L: improve folding @37C
  • S65T: 5-6x increase in amplitude and red shift
  • S65(G|T) and T203(Y|F|W|H): GFP -> YFP
  • Y66W: GFP -> CFP
  • Y66H: GFP -> BFP (more blue than CFP), dim, easily photobleached
  • Y66F: excitation 360nm, emission 442nm
  • R96A: slows cyclization reaction from minutes to months
  • F99S, M153T, V163A: "cycle 3" mutations (originally reported as F100S, M154T, V164A) [7]
  • Y203I: eliminate excitation peak at 475nm, leaving lower peak of 399nm. emission remains at 511nm, producing large Stokes shift
  • A206K: make monomeric
  • S30R, Y39N, N105T, Y145F, I171V, A206V: additional superfolder mutations [5]



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  1. Error fetching PMID 8707053: [Cormack]
  2. Error fetching PMID 9603842: [Andersen]
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  4. Error fetching PMID 18971928: [Stricker08]
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  8. Prasher DC, Eckenrode VK, Ward WW, Prendergast FG, and Cormier MJ. Primary structure of the Aequorea victoria green-fluorescent protein. Gene. 1992 Feb 15;111(2):229-33. DOI:10.1016/0378-1119(92)90691-h | PubMed ID:1347277 | HubMed [prasher]

    original cloning of GFP

  9. Error fetching PMID 8303295: [chalfie]

    original use of GFP as a reporter

  10. Tsien RY. The green fluorescent protein. Annu Rev Biochem. 1998;67:509-44. DOI:10.1146/annurev.biochem.67.1.509 | PubMed ID:9759496 | HubMed [tsien98]

    good review of GFP

  11. [zacharias]

All Medline abstracts: PubMed | HubMed

GFP as a measure of gene expression

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All Medline abstracts: PubMed | HubMed