Engineering 'Golden' Fluorescence by Selective Pressure Incorporation of Non-canonical Amino Acids and Protein Analysis by Mass Spectrometry and Fluorescence

Abstract

Fluorescent proteins are fundamental tools for the life sciences, in particular for fluorescence microscopy of living cells. While wild-type and engineered variants of the green fluorescent protein from Aequorea victoria (avGFP) as well as homologs from other species already cover large parts of the optical spectrum, a spectral gap remains in the near-infrared region, for which avGFP-based fluorophores are not available. Red-shifted fluorescent protein (FP) variants would substantially expand the toolkit for spectral unmixing of multiple molecular species, but the naturally occurring red-shifted FPs derived from corals or sea anemones have lower fluorescence quantum yield and inferior photo-stability compared to the avGFP variants. Further manipulation and possible expansion of the chromophore's conjugated system towards the far-red spectral region is also limited by the repertoire of 20 canonical amino acids prescribed by the genetic code. To overcome these limitations, synthetic biology can achieve further spectral red-shifting via insertion of non-canonical amino acids into the chromophore triad. We describe the application of SPI to engineer avGFP variants with novel spectral properties. Protein expression is performed in a tryptophan-auxotrophic E. coli strain and by supplementing growth media with suitable indole precursors. Inside the cells, these precursors are converted to the corresponding tryptophan analogs and incorporated into proteins by the ribosomal machinery in response to UGG codons. The replacement of Trp-66 in the enhanced "cyan" variant of avGFP (ECFP) by an electron-donating 4-aminotryptophan results in GdFP featuring a 108 nm Stokes shift and a strongly red-shifted emission maximum (574 nm), while being thermodynamically more stable than its predecessor ECFP. Residue-specific incorporation of the non-canonical amino acid is analyzed by mass spectrometry. The spectroscopic properties of GdFP are characterized by time-resolved fluorescence spectroscopy as one of the valuable applications of genetically encoded FPs in life sciences.

Advances in Biosynthesis of Non-Canonical Amino Acids (ncAAs) and the Methods of ncAAs Incorporation into Proteins

“…This approach is based on the substrate tolerance of cellular systems [ 81 ]. Endogenous aaRS play a crucial role in replacing the cAAs in protein with one or more amino acid analogs that have similar structures to those of the cAAs [ 82 ]. The auxotrophic hosts are cultured in a medium that initially contains a limited amount of nAAs.…”

Unnatural Amino Acids for Biological Spectroscopy and Microscopy

“…For example, when the pH is increased from 3 to 7, the λ em of 4NH 2 -Trp-containing barstar is decreased from 358 to 338 nm, whereas the λ ab of the 5NH 2 -Trp variant exhibits shifts from 363 to 375 nm when the solution pH is increased from 7 to 9. In addition, Budisa and co-workers have demonstrated that aminotryptophans could be used to change the color of green fluorescence protein (GFP) as well as other protein systems. − …”

Combating Antimicrobial Resistance With New-To-Nature Lanthipeptides Created by Genetic Code Expansion

“…This provides suitable bio-orthogonal reactive groups (‘handles’) amenable for various chemoselective ligand couplings. In addition, both site- and residue-specific incorporation techniques generally allow for fine chemical manipulations of the amino acid side chains, e.g., of Pro, Trp, Tyr, Met and to visualize them in spectroscopic recordings ( Baumann et al, 2018 ).…”