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.

Fig. 1. Tolerance of sfGFP to aromatic nAA incorporation. (a) Structure of the aromatic non-natural amino acids used in this study including the 3 aromatic nAAs used in library screening (ioF, azF and acF). (b) Linear map of sfGFP sequence showing sites tolerant to aromatic nAA incorporation as arrows. Each colour represents variants found from screening with three different aromatic nAAs (ioF, black; azF, orange; acF, blue). The position of the sfGFP chromophore (“Cro”) is shown as a green arrow. A more detailed description is provided in ESI Table 1. (c) 3D structure of sfGFP showing the positions of tolerated aromatic nAA incorporation sites as red spheres.

Fig. 2. Spectral properties of sfGFP^nAA variants. Fluorescence excitation spectra of (a) sfGFP^ioF and (b) sfGFP^azF variants. Spectra were recorded on cell lysates (soluble fraction) by monitoring emission at 511 nm. Spectra were normalised to a value of 1 at 460 nm.

Fig. 3. The influence of L44 mutations on sfGFP fluorescence. (a) Position of L44 (magenta) in sfGFP in relation to the chromophore (Cro; green) with neighbouring residues shown as sticks. PDB accession 2B3P. (b) Fluorescence excitation spectra of sfGFP with the indicated amino acid at position 44. Spectra were measured by monitoring emission at 511 nm on the soluble fraction of cell lysates from protein production cultures that were diluted to equivalent OD₆₀₀ of 0.5.

Fig. 4. Photoswitching properties of sfGFP^L44azF. Fluorescence photoswitching of sfGFP^L44azF was monitored on (a) whole cell, (b) cell lysate and (c) 1 μM pure protein samples. Samples in (a) and (b) were standardised to an OD₆₀₀ of 0.5. Relative excitations was calculated by normalisation of fluorescence excitation intensity to 1 for the major 484 nm peak at time point 0 (no irradiation). Fluorescence excitation spectra were recorded following UV irradiation (302 nm, 6 W) for the indicated amounts of time by monitoring emission at 511 nm. Arrows indicate the change in fluorescence intensity over irradiation time. (d) Photoswitching behaviour of sfGFP^L44azF monitored by UV-vis absorbance. Spectra were recorded on 10 μM pure protein in phosphate buffer (100 mM, pH 8, 300 mM NaCl). The corresponding emission spectra are shown in ESI Fig. 5.

Fig. 5. Redox and molecular crowding sensitive photoswitching of sfGFP^L44azF. (a) Photoswitching of pure sfGFP^L44azF in the presence of different redox agents. The initial (‘dark’ reading) and final time points of irradiation for each redox agent are shown. (b) Photoswitching of sfGFP^L44azF in the presence of 200 mg mL^–1 BSA showing the initial (‘dark’) and final time point of irradiation (60 min). Fluorescence excitation spectra are shown in (a) and (b) and were recorded on 1 μM sfGFP^L44azF by monitoring emission at 511 nm.

Fig. 6. Molecular models of the sfGFP^L44 aromatic nAA mutants. (a) X-ray crystal structure of the parent protein sfGFP (PDB accession ; 2B3P). Representative structural models of (b) sfGFP^L44Y and (c) sfGFP^L44azF obtained by molecular dynamics (50 ns). Residue 44, E222 and the chromophore (Cro) are shown as sticks and coloured by element with green carbon atoms in each case. Nearby residues are shown as lines and coloured by element with grey carbon atoms. Suggested hydrogen bonds are shown as black dashed lines and structural water molecules as red spheres with distance shown in Å. (d) Overlay of sfGFP (green), sfGFP^L44Y (red) and sfGFP^L44azF (pink) showing residues 44, E222 and the chromophore. The inset shows E222 from a different orientation (∼90° rotation).

Fig. 7. Photoswitching properties of sfGFP^T203azF. (a) Structure of sfGFP showing the position of residue 203 in relation to the chromophore (Cro; green). Residue 203 is shown as the native Thr (grey) and Tyr as (yellow) as found in YFP (PDB accession ; 1YFP) demonstrating the π–π stacking interaction with the chromophore. (b) Ambient light photoswitching of sfGFP^T203azF. Images of a cell lysate sample left in ambient room light for the indicated amount of time (in min). Photoswitching of sfGFP^T203azF monitored by (c) absorbance and (d) fluorescence emission. Photolysis was performed with a handheld UV lamp (302 nm, 6 W) for the indicated amount of time. Emission spectra were recorded on 1 μM protein after excitation at 485 nm and absorbance spectra with 10 μM protein.