GFP

General Info

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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  • Inherited parent variant mutations (GFPmut3*): S2R, S65G, S72A (S65G mutation is superseded by S65T mutation, hence the alternative mutation designation of G65T)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]

Mutations

• 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]

References

General

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4. Stricker J, Cookson S, Bennett MR, Mather WH, Tsimring LS, and Hasty J. A fast, robust and tunable synthetic gene oscillator. Nature. 2008 Nov 27;456(7221):516-9. DOI:10.1038/nature07389 |
6. Shaner NC, Steinbach PA, and Tsien RY. A guide to choosing fluorescent proteins. Nat Methods. 2005 Dec;2(12):905-9. DOI:10.1038/nmeth819 |
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 |

   original cloning of GFP

9. Chalfie M, Tu Y, Euskirchen G, Ward WW, and Prasher DC. Green fluorescent protein as a marker for gene expression. Science. 1994 Feb 11;263(5148):802-5. DOI:10.1126/science.8303295 |

   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 |

    good review of GFP

11. Molecular Biology and Muation of Green Fluorescent Protein (Book Chapter, Zacharias & Tsien)

GFP as a measure of gene expression

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