LOGIN TO YOUR ACCOUNT

Username
Password
Remember Me
Or use your Academic/Social account:

CREATE AN ACCOUNT

Or use your Academic/Social account:

Congratulations!

You have just completed your registration at OpenAire.

Before you can login to the site, you will need to activate your account. An e-mail will be sent to you with the proper instructions.

Important!

Please note that this site is currently undergoing Beta testing.
Any new content you create is not guaranteed to be present to the final version of the site upon release.

Thank you for your patience,
OpenAire Dev Team.

Close This Message

CREATE AN ACCOUNT

Name:
Username:
Password:
Verify Password:
E-mail:
Verify E-mail:
*All Fields Are Required.
Please Verify You Are Human:
fbtwitterlinkedinvimeoflicker grey 14rssslideshare1
Reddington, Samuel C.; Baldwin, Amy Joy; Thompson, Rebecca; Brancale, Andrea; Tippmann, Eric M.; Jones, Darran Dafydd (2015)
Publisher: Royal Society of Chemistry
Journal: Chemical Science
Languages: English
Types: Article
Subjects: Chemistry, QD, RM
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 state provided an insight into the mechanism of action through modulation of the hydrogen bonding network surrounding the chromophore. Targeted mutagenesis of T203 with aromatic nAAs to introduce π-stacking with the chromophore successfully generated red shifted versions of GFP. Incorporation of azF at residue 203 conferred high photosensitivity on sfGFP with even ambient light mediating a functional switch. Thus, engineering proteins with non-natural aromatic amino acids by surveying a wide residue set can introduce new and beneficial properties into a protein through the sampling of non-intuitive mutations. Coupled with retrospective in silico modelling, this will facilitate both our understanding of the impact of nAAs on protein structure and function, and future design endeavours.
  • The results below are discovered through our pilot algorithms. Let us know how we are doing!

    • 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.
    • 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.
    • 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.
    • 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.
    • 16 A. Baldwin, J. Arpino, W. Edwards, E. Tippmann and 28 J. J. van Thor, Photoreactions and dynamics of the green D. Jones, Expanded chemical diversity sampling through uorescent protein, Chem. Soc. Rev., 2009, 38, 2935-2950. whole protein evolution, Mol. BioSyst., 2009, 5, 764-766. 29 Y. Chen, Y. Ebright and R. Ebright, Identication of the
    • 17 K. A. Daggett, M. Layer and T. A. Cropp, A General Method target of a transcription activator protein by proteinfor Scanning Unnatural Amino Acid Mutagenesis, ACS protein photocrosslinking, Science, 1994, 265, 90-92. Chem. Biol., 2009, 4, 109-113. 30 G. W. J. Fleet, R. R. Porter and J. R. Knowles, Affinity
    • 18 J. Lippincott-Schwartz and G. H. Patterson, Photoactivatable Labelling of Antibodies with Aryl Nitrene as Reactive uorescent proteins for diffraction-limited and super- Group, Nature, 1969, 224, 511-512. resolution imaging, Trends Cell Biol., 2009, 19, 555-565. 31 G. Schuster and M. Platz, Photochemistry of phenyl azide,
    • 19 G. H. Patterson and J. Lippincott-Schwartz, A Adv. Photochem., 1992, 17, 69-143. photoactivatable GFP for selective photolabeling of 32 J. H. Mills, S. D. Khare, J. M. Bolduc, F. Forouhar, proteins and cells, Science, 2002, 297, 1873-1877. V. K. Mulligan, S. Lew, J. Seetharaman, L. Tong,
    • 20 A. Baldwin, K. Busse, A. Simm and D. Jones, Expanded B. L. Stoddard and D. Baker, Computational Design of an molecular diversity generation during directed evolution by Unnatural Amino Acid Dependent Metalloprotein with trinucleotide exchange (TriNEx), Nucleic Acids Res., 2008, Atomic Level Accuracy, J. Am. Chem. Soc., 2013, 135, 13393- 36, 77-86. 13399.
    • 21 J. D. Pedelacq, S. Cabantous, T. Tran, T. C. Terwilliger and 33 A. B. Cubitt, L. A. Woollenweber and R. Heim, G. S. Waldo, Engineering and characterization of a Understanding structure-function relationships in the superfolder green uorescent protein, Nat. Biotechnol., Aequorea victoria green uorescent protein, Methods Cell 2006, 24, 79-88. Biol., 1999, 58, 19-30.
    • 22 N. C. Shaner, R. E. Campbell, P. A. Steinbach, 34 R. Wachter, M. Elsliger, K. Kallio, G. Hanson and B. N. Giepmans, A. E. Palmer and R. Y. Tsien, Improved S. Remington, Structural basis of spectral shis in the monomeric red, orange and yellow uorescent proteins yellow-emission variants of green uorescent protein, derived from Discosoma sp. red uorescent protein, Nat. Structure, 1998, 6, 1267-1277. Biotechnol., 2004, 22, 1567-1572. 35 K. Sivakumar, F. Xie, B. M. Cash, S. Long, H. N. Barnhill and
    • 23 M. Chattoraj, B. A. King, G. U. Bublitz and S. G. Boxer, Ultra- Q. Wang, A uorogenic 1,3-dipolar cycloaddition reaction of fast excited state dynamics in green uorescent protein: 3-azidocoumarins and acetylenes, Org. Lett., 2004, 6, 4603- Multiple states and proton transfer, Proc. Natl. Acad. Sci. U. 4606. S. A., 1996, 93, 8362-8367. 36 J. L. Morris, S. C. Reddington, D. M. Murphy, D. D. Jones,
    • 24 R. Tsien, The green uorescent protein, Annu. Rev. Biochem., J. A. Platts and E. M. Tippmann, Aryl Azide Photochemistry 1998, 67, 509-544. in Dened Protein Environments, Org. Lett., 2013, 15, 728-
    • 25 K. Brejc, T. K. Sixma, P. A. Kitts, S. R. Kain, R. Y. Tsien, 731. M. Ormo and S. J. Remington, Structural basis for dual
  • No related research data.
  • No similar publications.
  • BioEntity Site Name
    1yfpProtein Data Bank
    2b3pProtein Data Bank

Share - Bookmark

Funded by projects

  • RCUK | Electron transfer in engin...
  • RCUK | High-throughput engineerin...

Cite this article