GFP

From OpenWetWare

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  • Inherited parent variant mutations (GFPmut3b*): S2R, S65G, S72A (S65G mutation is superseded by S65T mutation, hence the alternative mutation designation of G65T)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

1. Cormack BP, Valdivia RH, and Falkow S. . pmid:8707053. PubMed HubMed [Cormack]
2. Andersen JB, Sternberg C, Poulsen LK, Bjorn SP, Givskov M, and Molin S. . pmid:9603842. PubMed HubMed [Andersen]
3. Li X, Zhang G, Ngo N, Zhao X, Kain SR, and Huang CC. . pmid:9353317. PubMed HubMed [li97]
4. Stricker J, Cookson S, Bennett MR, Mather WH, Tsimring LS, and Hasty J. . pmid:18971928. PubMed HubMed [Stricker08]
5. Pédelacq JD, Cabantous S, Tran T, Terwilliger TC, and Waldo GS. . pmid:16369541. PubMed HubMed [Pedelacq06]
6. Shaner NC, Steinbach PA, and Tsien RY. . pmid:16299475. PubMed HubMed [shaner05]
7. Crameri A, Whitehorn EA, Tate E, and Stemmer WP. . pmid:9630892. PubMed HubMed [Crameri96]
8. Prasher DC, Eckenrode VK, Ward WW, Prendergast FG, and Cormier MJ. . pmid:1347277. PubMed HubMed [prasher]

   original cloning of GFP

9. Chalfie M, Tu Y, Euskirchen G, Ward WW, and Prasher DC. . pmid:8303295. PubMed HubMed [chalfie]

   original use of GFP as a reporter

10. Tsien RY. . pmid:9759496. PubMed HubMed [tsien98]

    good review of GFP

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

    [zacharias]

GFP as a measure of gene expression

12. Leveau JH and Lindow SE. . pmid:11698362. PubMed HubMed [Lindow]
13. Iafolla MA, Mazumder M, Sardana V, Velauthapillai T, Pannu K, and McMillen DR. . pmid:18350571. PubMed HubMed [Iafolla]
14. Bagh S, Mazumder M, Velauthapillai T, Sardana V, Dong GQ, Movva AB, Lim LH, and McMillen DR. . pmid:18352063. PubMed HubMed [Bagh]
15. Dong GQ and McMillen DR. . pmid:18352052. PubMed HubMed [Dong]