GFP

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General Info

• From jellyfish Aeqorea victoria
• wtGFP sequence on NCBI
• 238 amino acids
• The fluorophore is tyrosine Y66 and the surrounding amino acids of Ser(65)-Tyr(66)-Gly(67) are also critical [1].
• Oxygen is required for maturation of the fluorophore

GFP Variants

• GFPmut3b - S65G, S72A
• GFPmut3* - mutations from wt: S2R,S65G,S72A
  • This is GFPmut3b[2] with an accidental mutation at position 2 that doesn't affect function according to the authors.[3]
• Emerald - F64L, S65T, S72A, N149K, M153T, I167T
• EGFP - F64L, S65T + optimized for human codons with 35-fold increase in fluorescence over GFP [4]
• Other variants and their mutations can be found in Shaner supplementary table 2 [5]

Mutations

• R96A: slows cyclization reaction from minutes to months
• S65T: 5-6x increase in amplitude and red shift
• Y203I: eliminate excitation peak at 475nm, leaving lower peak of 399nm. emission remains at 511nm, producing large Stokes shift
• S65(G|T) and T203(Y|F|W|H) -> YFP
• Y66W: GFP -> CFP
• Y66H: GFP -> BFP (more blue than CFP), dim, easily photobleached
• Y66F: excitation 360nm, emission 442nm
• A206K: make monomeric

References

1. Molecular Biology and Muation of Green Fluorescent Protein (Book Chapter, Zacharias & Tsien) [zacharias]
2. Cormack BP, Valdivia RH, and Falkow S. . pmid:8707053. PubMed HubMed [Cormack]
3. Andersen JB, Sternberg C, Poulsen LK, Bjorn SP, Givskov M, and Molin S. . pmid:9603842. PubMed HubMed [Andersen]
4. Li X, Zhang G, Ngo N, Zhao X, Kain SR, and Huang CC. . pmid:9353317. PubMed HubMed [li97]
5. Shaner NC, Steinbach PA, and Tsien RY. . pmid:16299475. PubMed HubMed [shaner05]