GFP

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==General Info==
==General Info==
[[Image:GFP structure.png|thumb|200px|GFP ribbon diagram. From [http://www.rcsb.org/pdb/explore/explore.do?structureId=1EMA PDB 1EMA].]]
[[Image:GFP structure.png|thumb|200px|GFP ribbon diagram. From [http://www.rcsb.org/pdb/explore/explore.do?structureId=1EMA PDB 1EMA].]]
-
* From jellyfish ''Aeqorea victoria''
+
* Originally discovered and characterized in the jellyfish ''Aeqorea victoria''
*[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 fluorophore is Ser(65)-Tyr(66)-Gly(67) <cite>zacharias</cite>.
+
* 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
 +
* Natural state is a dimer with a Kd of ~ 100 µM.
==GFP Variants==
==GFP Variants==
-
*GFPmut3b - S65G, S72A
+
* '''GFPmut2''' - S65A, V68L, S72A <cite>Cormack</cite>
-
*[http://parts.mit.edu/registry/index.php/Part:BBa_E0040 ''GFP''mut3*] - mutations from wt: S2R,S65G,S72A
+
* '''GFPmut3b''' - S65G, S72A <cite>Cormack</cite>
-
**This is GFPmut3b<cite>Cormack</cite> with an accidental mutation at position 2 that doesn't affect function according to the authors.<cite>Andersen</cite>
+
*[http://partsregistry.org/wiki/index.php/Part:BBa_E0040 '''''GFP''mut3*'''] - mutations from wt: S2R,S65G,S72A
-
*[[Emerald]] - F64L, S65T, S72A, N149K, M153T, I167T
+
**This is GFPmut3b with an accidental mutation at position 2 that doesn't affect function according to the authors.<cite>Andersen</cite>
-
* EGFP - added GTG as second codon, F64L, S65T + optimized for human codons with 35-fold increase in fluorescence over GFP <cite>li97</cite>
+
*'''[[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 <cite>li97</cite>
 +
** Additionally, "mutation of His231 to Leu, which was probably inadvertent and neutral". <cite>li97</cite>
 +
* '''yemGFP''' - F64L, S65T, A206K <cite>Stricker08</cite>
 +
* [http://partsregistry.org/Part:BBa_I746916 '''superfolder GFP'''] - S30R, Y39N, F64L, (S65T/G65T), F99S, N105T, Y145F, M153T, V163A, I171V, A206V <cite>Pedelacq06</cite>
 +
** Inherited parent variant mutations (GFPmut3*): S2R, <s>S65G</s>, 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 <cite>shaner05</cite>
* Other variants and their mutations can be found in Shaner supplementary table 2 <cite>shaner05</cite>
==Mutations==
==Mutations==
-
* F64L: improve folding @37C
+
* '''F64L''': improve folding @37C
-
* S65T: 5-6x increase in amplitude and red shift  
+
* '''S65T''': 5-6x increase in amplitude and red shift  
-
* S65(G|T) and T203(Y|F|W|H) -> YFP
+
* '''S65(G|T) and T203(Y|F|W|H)''': GFP -> YFP
-
* Y66W: GFP -> CFP
+
* '''Y66W''': GFP -> CFP
-
* Y66H: GFP -> BFP (more blue than CFP), dim, easily photobleached
+
* '''Y66H''': GFP -> BFP (more blue than CFP), dim, easily photobleached
-
* Y66F: excitation 360nm, emission 442nm
+
* '''Y66F''': excitation 360nm, emission 442nm
-
* R96A: ''slows'' cyclization reaction from minutes to months
+
* '''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
+
* '''F99S, M153T, V163A''': "cycle 3" mutations (originally reported as F100S, M154T, V164A) <cite>Crameri96</cite>
-
* A206K: make monomeric
+
* '''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 <cite>Pedelacq06</cite>
==References==
==References==
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#zacharias [http://www.tsienlab.ucsd.edu/Publications/Zacharias%202006%20-%20Molecular%20Biology%20and%20Mutation%20of%20GFP.pdf Molecular Biology and Muation of Green Fluorescent Protein (Book Chapter, Zacharias & Tsien)]
#zacharias [http://www.tsienlab.ucsd.edu/Publications/Zacharias%202006%20-%20Molecular%20Biology%20and%20Mutation%20of%20GFP.pdf Molecular Biology and Muation of Green Fluorescent Protein (Book Chapter, Zacharias & Tsien)]
#shaner05 pmid=16299475
#shaner05 pmid=16299475
 +
#Stricker08 pmid=18971928
 +
#Pedelacq06 pmid=16369541
 +
#Crameri96 pmid=9630892
</biblio>
</biblio>

Current revision

Contents

General Info

GFP ribbon diagram. From PDB 1EMA.
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

  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]
All Medline abstracts: PubMed HubMed

GFP as a measure of gene expression

  1. Leveau JH and Lindow SE. . pmid:11698362. PubMed HubMed [Lindow]
  2. Iafolla MA, Mazumder M, Sardana V, Velauthapillai T, Pannu K, and McMillen DR. . pmid:18350571. PubMed HubMed [Iafolla]
  3. Bagh S, Mazumder M, Velauthapillai T, Sardana V, Dong GQ, Movva AB, Lim LH, and McMillen DR. . pmid:18352063. PubMed HubMed [Bagh]
  4. Dong GQ and McMillen DR. . pmid:18352052. PubMed HubMed [Dong]
All Medline abstracts: PubMed HubMed
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