Issue 2, 2015

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

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 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.

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

Supplementary files

Article information

Article type
Edge Article
Submitted
15 Sep 2014
Accepted
24 Oct 2014
First published
07 Nov 2014
This article is Open Access

All publication charges for this article have been paid for by the Royal Society of Chemistry
Creative Commons BY license

Chem. Sci., 2015,6, 1159-1166

Author version available

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

S. C. Reddington, A. J. Baldwin, R. Thompson, A. Brancale, E. M. Tippmann and D. D. Jones, Chem. Sci., 2015, 6, 1159 DOI: 10.1039/C4SC02827A

This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. You can use material from this article in other publications without requesting further permissions from the RSC, provided that the correct acknowledgement is given.

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