publication . Article . 2015

Directed evolution of GFP with non-natural amino acids identifies residues for augmenting and photoswitching fluorescence†

Reddington, Samuel C.; Baldwin, Amy J.; Thompson, Rebecca; Brancale, Andrea; Tippmann, Eric M.; Jones, D. Dafydd;
Open Access
  • Published: 01 Feb 2015 Journal: Chemical Science, volume 6, pages 1,159-1,166 (issn: 2041-6520, eissn: 2041-6539, Copyright policy)
  • Publisher: Royal Society of Chemistry (RSC)
  • Country: United Kingdom
Abstract
Genetic code reprogramming allows proteins to sample new chemistry through the defined and targeted introduction of non-natural amino acids (nAAs). Many useful nAAs are derivatives of the natural aromatic amino acid tyrosine, with the para OH group replaced with useful but often bulkier substituents. Extending residue sampling by directed evolution identified positions in Green Fluorescent Protein tolerant to aromatic nAAs, including identification of novel sites that modulate fluorescence. Replacement of the buried L44 residue by photosensitive p-azidophenylalanine (azF) conferred environmentally sensitive photoswitching. In silico modelling of the L44azF dark ...
Subjects
free text keywords: Stereochemistry, Mutagenesis, Genetic code, Organic chemistry, Green fluorescent protein, Aromatic amino acids, chemistry.chemical_compound, chemistry, Chromophore, In silico, Amino acid, chemistry.chemical_classification, Directed evolution, QD, RM
Related Organizations
25 references, page 1 of 2

1 C. H. Kim, J. Y. Axup and P. G. Schultz, Protein conjugation with genetically encoded unnatural amino acids, Curr. Opin. Chem. Biol., 2013, 17, 412-419.

2 W. H. Zhang, G. Otting and C. J. Jackson, Protein engineering with unnatural amino acids, Curr. Opin. Struct. Biol., 2013, 23, 581-587.

3 A. K. Antonczak, J. Morris and E. M. Tippmann, Advances in the mechanism and understanding of site-selective noncanonical amino acid incorporation, Curr. Opin. Struct. Biol., 2011, 21, 481-487.

4 S. Reddington, P. Watson, P. Rizkallah, E. Tippmann and D. D. Jones, Genetically encoding phenyl azide chemistry: new uses and ideas for classical biochemistry, Biochem. Soc. Trans., 2013, 41, 1177-1182. [OpenAIRE]

5 L. Wang, T. Magliery, D. Liu and P. Schultz, A New Functional Suppressor tRNA/Aminoacyl-tRNA Synthetase Pair for the In Vivo Incorporation of Unnatural Amino Acids into Proteins, J. Am. Chem. Soc., 2000, 122, 5010-5011. [OpenAIRE]

6 M. J. Lajoie, A. J. Rovner, D. B. Goodman, H. R. Aerni, A. D. Haimovich, G. Kuznetsov, J. A. Mercer, H. H. Wang, P. A. Carr, J. A. Mosberg, N. Rohland, P. G. Schultz, J. M. Jacobson, J. Rinehart, G. M. Church and F. J. Isaacs, Genomically Recoded Organisms Expand Biological Functions, Science, 2013, 342, 357-360.

7 C. C. Liu and P. G. Schultz, Adding new chemistries to the genetic code, Annu. Rev. Biochem., 2010, 79, 413-444.

8 K. Lang and J. W. Chin, Cellular Incorporation of Unnatural Amino Acids and Bioorthogonal Labeling of Proteins, Chem. Rev., 2014, 114, 4764-4806.

9 S. C. Reddington, P. J. Rizkallah, P. D. Watson, R. Pearson, E. M. Tippmann and D. D. Jones, Different Photochemical Events of a Genetically Encoded Phenyl Azide Dene and Modulate GFP Fluorescence, Angew. Chem., Int. Ed., 2013, 52, 5974-5977. [OpenAIRE]

10 S. C. Reddington, E. M. Tippmann and D. D. Jones, Residue choice denes efficiency and inuence of bioorthogonal protein modication via genetically encoded strain promoted click chemistry, Chem. Commun., 2012, 48, 8419- 8421.

11 J. H. Bae, M. Rubini, G. Jung, G. Wiegand, M. H. J. Seifert, M. K. Azim, J. S. Kim, A. Zumbusch, T. A. Holak, L. Moroder, R. Huber and N. Budisa, Expansion of the genetic code enables design of a novel “gold” class of green uorescent proteins, J. Mol. Biol., 2003, 328, 1071- 1081.

12 F. Wang, W. Niu, J. T. Guo and P. G. Schultz, Unnatural excitation and photoisomerization of the Aequorea victoria Amino Acid Mutagenesis of Fluorescent Proteins, Angew. green uorescent protein, Proc. Natl. Acad. Sci. U. S. A., Chem., Int. Ed., 2012, 51, 10132-10135. 1997, 94, 2306-2311.

13 F. H. Arnold, Combinatorial and computational challenges 26 M. Ormo, A. B. Cubitt, K. Kallio, L. A. Gross, R. Y. Tsien and for biocatalyst design, Nature, 2001, 409, 253-257. S. J. Remington, Crystal structure of the Aequorea victoria

14 P. A. Dalby, Optimising enzyme function by directed green uorescent protein, Science, 1996, 273, 1392-1395. evolution, Curr. Opin. Struct. Biol., 2003, 13, 500-505. 27 J. A. J. Arpino, P. J. Rizkallah and D. D. Jones, Crystal

15 C. M. Yuen and D. R. Liu, Dissecting protein structure and Structure of Enhanced Green Fluorescent Protein to 1.35 function using directed evolution, Nat. Methods, 2007, 4, angstrom Resolution Reveals Alternative Conformations 995-997. for Glu222, PLoS One, 2012, 7, e47132.

25 references, page 1 of 2
Abstract
Genetic code reprogramming allows proteins to sample new chemistry through the defined and targeted introduction of non-natural amino acids (nAAs). Many useful nAAs are derivatives of the natural aromatic amino acid tyrosine, with the para OH group replaced with useful but often bulkier substituents. Extending residue sampling by directed evolution identified positions in Green Fluorescent Protein tolerant to aromatic nAAs, including identification of novel sites that modulate fluorescence. Replacement of the buried L44 residue by photosensitive p-azidophenylalanine (azF) conferred environmentally sensitive photoswitching. In silico modelling of the L44azF dark ...
Subjects
free text keywords: Stereochemistry, Mutagenesis, Genetic code, Organic chemistry, Green fluorescent protein, Aromatic amino acids, chemistry.chemical_compound, chemistry, Chromophore, In silico, Amino acid, chemistry.chemical_classification, Directed evolution, QD, RM
Related Organizations
25 references, page 1 of 2

1 C. H. Kim, J. Y. Axup and P. G. Schultz, Protein conjugation with genetically encoded unnatural amino acids, Curr. Opin. Chem. Biol., 2013, 17, 412-419.

2 W. H. Zhang, G. Otting and C. J. Jackson, Protein engineering with unnatural amino acids, Curr. Opin. Struct. Biol., 2013, 23, 581-587.

3 A. K. Antonczak, J. Morris and E. M. Tippmann, Advances in the mechanism and understanding of site-selective noncanonical amino acid incorporation, Curr. Opin. Struct. Biol., 2011, 21, 481-487.

4 S. Reddington, P. Watson, P. Rizkallah, E. Tippmann and D. D. Jones, Genetically encoding phenyl azide chemistry: new uses and ideas for classical biochemistry, Biochem. Soc. Trans., 2013, 41, 1177-1182. [OpenAIRE]

5 L. Wang, T. Magliery, D. Liu and P. Schultz, A New Functional Suppressor tRNA/Aminoacyl-tRNA Synthetase Pair for the In Vivo Incorporation of Unnatural Amino Acids into Proteins, J. Am. Chem. Soc., 2000, 122, 5010-5011. [OpenAIRE]

6 M. J. Lajoie, A. J. Rovner, D. B. Goodman, H. R. Aerni, A. D. Haimovich, G. Kuznetsov, J. A. Mercer, H. H. Wang, P. A. Carr, J. A. Mosberg, N. Rohland, P. G. Schultz, J. M. Jacobson, J. Rinehart, G. M. Church and F. J. Isaacs, Genomically Recoded Organisms Expand Biological Functions, Science, 2013, 342, 357-360.

7 C. C. Liu and P. G. Schultz, Adding new chemistries to the genetic code, Annu. Rev. Biochem., 2010, 79, 413-444.

8 K. Lang and J. W. Chin, Cellular Incorporation of Unnatural Amino Acids and Bioorthogonal Labeling of Proteins, Chem. Rev., 2014, 114, 4764-4806.

9 S. C. Reddington, P. J. Rizkallah, P. D. Watson, R. Pearson, E. M. Tippmann and D. D. Jones, Different Photochemical Events of a Genetically Encoded Phenyl Azide Dene and Modulate GFP Fluorescence, Angew. Chem., Int. Ed., 2013, 52, 5974-5977. [OpenAIRE]

10 S. C. Reddington, E. M. Tippmann and D. D. Jones, Residue choice denes efficiency and inuence of bioorthogonal protein modication via genetically encoded strain promoted click chemistry, Chem. Commun., 2012, 48, 8419- 8421.

11 J. H. Bae, M. Rubini, G. Jung, G. Wiegand, M. H. J. Seifert, M. K. Azim, J. S. Kim, A. Zumbusch, T. A. Holak, L. Moroder, R. Huber and N. Budisa, Expansion of the genetic code enables design of a novel “gold” class of green uorescent proteins, J. Mol. Biol., 2003, 328, 1071- 1081.

12 F. Wang, W. Niu, J. T. Guo and P. G. Schultz, Unnatural excitation and photoisomerization of the Aequorea victoria Amino Acid Mutagenesis of Fluorescent Proteins, Angew. green uorescent protein, Proc. Natl. Acad. Sci. U. S. A., Chem., Int. Ed., 2012, 51, 10132-10135. 1997, 94, 2306-2311.

13 F. H. Arnold, Combinatorial and computational challenges 26 M. Ormo, A. B. Cubitt, K. Kallio, L. A. Gross, R. Y. Tsien and for biocatalyst design, Nature, 2001, 409, 253-257. S. J. Remington, Crystal structure of the Aequorea victoria

14 P. A. Dalby, Optimising enzyme function by directed green uorescent protein, Science, 1996, 273, 1392-1395. evolution, Curr. Opin. Struct. Biol., 2003, 13, 500-505. 27 J. A. J. Arpino, P. J. Rizkallah and D. D. Jones, Crystal

15 C. M. Yuen and D. R. Liu, Dissecting protein structure and Structure of Enhanced Green Fluorescent Protein to 1.35 function using directed evolution, Nat. Methods, 2007, 4, angstrom Resolution Reveals Alternative Conformations 995-997. for Glu222, PLoS One, 2012, 7, e47132.

25 references, page 1 of 2
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