Difference between revisions of "GFP"

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(General Info)
(General Info)
Line 4: Line 4:
 
*[http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=protein&val=1169893 wtGFP sequence] on NCBI
 
*[http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=protein&val=1169893 wtGFP sequence] on NCBI
 
* 238 amino acids
 
* 238 amino acids
* The tertiary structure is a fluorophore [Ser(65)-Tyr(66)-Gly(67) <cite>zacharias</cite>] nestled inside a protective beta barrel.
+
* 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
 
* Oxygen is required for maturation of the fluorophore
  

Revision as of 12:42, 20 July 2011

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

GFP Variants

  • GFPmut3b - S65G, S72A
  • GFPmut3* - mutations from wt: S2R,S65G,S72A
    • This is GFPmut3b[1] 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 - added GTG as second codon, F64L, S65T + optimized for human codons with 35-fold increase in fluorescence over GFP [3]
  • Other variants and their mutations can be found in Shaner supplementary table 2 [4]

Mutations

  • F64L: improve folding @37C
  • S65T: 5-6x increase in amplitude and red 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
  • R96A: slows cyclization reaction from minutes to months
  • Y203I: eliminate excitation peak at 475nm, leaving lower peak of 399nm. emission remains at 511nm, producing large Stokes shift
  • A206K: make monomeric

References

General

  1. Cormack BP, Valdivia RH, and Falkow S. FACS-optimized mutants of the green fluorescent protein (GFP). Gene. 1996;173(1 Spec No):33-8. PubMed ID:8707053 | HubMed [Cormack]
  2. Andersen JB, Sternberg C, Poulsen LK, Bjorn SP, Givskov M, and Molin S. New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria. Appl Environ Microbiol. 1998 Jun;64(6):2240-6. PubMed ID:9603842 | HubMed [Andersen]
  3. Li X, Zhang G, Ngo N, Zhao X, Kain SR, and Huang CC. Deletions of the Aequorea victoria green fluorescent protein define the minimal domain required for fluorescence. J Biol Chem. 1997 Nov 7;272(45):28545-9. PubMed ID:9353317 | HubMed [li97]
  4. 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 | PubMed ID:16299475 | HubMed [shaner05]
  5. 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. PubMed ID:1347277 | HubMed [prasher]
    original cloning of GFP
  6. 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. PubMed ID:8303295 | HubMed [chalfie]
    original use of GFP as a reporter
  7. 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
  8. Molecular Biology and Muation of Green Fluorescent Protein (Book Chapter, Zacharias & Tsien) [zacharias]
All Medline abstracts: PubMed | HubMed

GFP as a measure of gene expression

  1. Leveau JH and Lindow SE. Predictive and interpretive simulation of green fluorescent protein expression in reporter bacteria. J Bacteriol. 2001 Dec;183(23):6752-62. DOI:10.1128/JB.183.23.6752-6762.2001 | PubMed ID:11698362 | HubMed [Lindow]
  2. Iafolla MA, Mazumder M, Sardana V, Velauthapillai T, Pannu K, and McMillen DR. Dark proteins: effect of inclusion body formation on quantification of protein expression. Proteins. 2008 Sep;72(4):1233-42. DOI:10.1002/prot.22024 | PubMed ID:18350571 | HubMed [Iafolla]
  3. Bagh S, Mazumder M, Velauthapillai T, Sardana V, Dong GQ, Movva AB, Lim LH, and McMillen DR. Plasmid-borne prokaryotic gene expression: sources of variability and quantitative system characterization. Phys Rev E Stat Nonlin Soft Matter Phys. 2008 Feb;77(2 Pt 1):021919. DOI:10.1103/PhysRevE.77.021919 | PubMed ID:18352063 | HubMed [Bagh]
  4. Dong GQ and McMillen DR. Effects of protein maturation on the noise in gene expression. Phys Rev E Stat Nonlin Soft Matter Phys. 2008 Feb;77(2 Pt 1):021908. DOI:10.1103/PhysRevE.77.021908 | PubMed ID:18352052 | HubMed [Dong]
All Medline abstracts: PubMed | HubMed