GFP

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(GFP Variants)
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* 238 amino acids
* 238 amino acids
* The fluorophore is tyrosine Y66 and the surrounding amino acids of Ser(65)-Tyr(66)-Gly(67) are also critical <cite>zacharias</cite>.
* The fluorophore is tyrosine Y66 and the surrounding amino acids of Ser(65)-Tyr(66)-Gly(67) are also critical <cite>zacharias</cite>.
 +
* Oxygen is required for maturation of the fluorophore
==GFP Variants==
==GFP Variants==
*GFPmut3b - S65G, S72A
*GFPmut3b - S65G, S72A
*[http://parts.mit.edu/registry/index.php/Part:BBa_E0040 ''GFP''mut3*] - mutations from wt: S2R,S65G,S72A
*[http://parts.mit.edu/registry/index.php/Part:BBa_E0040 ''GFP''mut3*] - mutations from wt: S2R,S65G,S72A
-
**This is GFPmut3b<cite>Cormack</cite> with a degredation tag attached + an accidental mutation at position 2 that doesn't affect function according to the authors.<cite>Anderson</cite>
+
**This is GFPmut3b<cite>Cormack</cite> with a degradation tag attached + an accidental mutation at position 2 that doesn't affect function according to the authors.<cite>Anderson</cite>
-
*[[Emerald]] - S65T, S72A, N149K, M153T, I167T
+
*[[Emerald]] - F64L, S65T, S72A, N149K, M153T, I167T
* EGFP - F64L, S65T + optimized for human codons with 35-fold increase in fluorescence over GFP <cite>li97</cite>
* EGFP - F64L, S65T + optimized for human codons with 35-fold increase in fluorescence over GFP <cite>li97</cite>
-
 
+
* Other variants and their mutations can be found in Shaner supplementary table 2 <cite>Shaner</cite>
==Mutations==
==Mutations==
* R96A: ''slows'' cyclization reaction from minutes to months
* R96A: ''slows'' cyclization reaction from minutes to months
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* 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
 +
* A206K: make monomeric
==References==
==References==
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#Andersen pmid=9603842
#Andersen pmid=9603842
#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
</biblio>
</biblio>

Revision as of 18:40, 30 December 2006

Contents

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 a degradation tag attached + 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. Li X, Zhang G, Ngo N, Zhao X, Kain SR, and Huang CC. . pmid:9353317. PubMed HubMed [li97]
  4. Andersen JB, Sternberg C, Poulsen LK, Bjorn SP, Givskov M, and Molin S. . pmid:9603842. PubMed HubMed [Andersen]
  5. Shaner NC, Steinbach PA, and Tsien RY. . pmid:16299475. PubMed HubMed [shaner05]
All Medline abstracts: PubMed HubMed
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