Rational design of the genetic code expansion toolkit for in vivo encoding of D-amino acids

Abstract

Once thought to be non-naturally occurring, D-amino acids (DAAs) have in recent years been revealed to play a wide range of physiological roles across the tree of life, including in human systems. Synthetic biologists have since exploited DAAs' unique biophysical properties to generate peptides and proteins with novel or enhanced functions. However, while peptides and small proteins containing DAAs can be efficiently prepared in vitro, producing large-sized heterochiral proteins poses as a major challenge mainly due to absence of pre-existing DAA translational machinery and presence of endogenous chiral discriminators. Based on our previous work demonstrating pyrrolysyl-tRNA synthetase's (PylRS') remarkable substrate polyspecificity, this work attempts to increase PylRS' ability in directly charging tRNA^Pyl with D-phenylalanine analogs (DFAs). We here report a novel, polyspecific Methanosarcina mazei PylRS mutant, DFRS2, capable of incorporating DFAs into proteins via ribosomal synthesis in vivo. To validate its utility, in vivo translational DAA substitution were performed in superfolder green fluorescent protein and human heavy chain ferritin, successfully altering both proteins' physiochemical properties. Furthermore, aminoacylation kinetic assays further demonstrated aminoacylation of DFAs by DFRS2 in vitro.

Keywords: D-phenylalanine analogs; amber suppression; genetic code expansion; noncanonical amino acids; pyrrolysyl-tRNA synthetase; synthetic biology.

FIGURE 1

Substrate selectivity of PylRS variants. (A) Structures of phenylalanine analogs employed in this study. (B) Suppression efficiencies of PylRS variants were examined using an sfGFP-27TAG reporter in BL21 (DE3) cells in GMML medium containing a dedicated ncAA. UAG translation efficiencies were obtained by normalizing fluorescence emission of sfGFP-27TAG according to that of wild-type sfGFP (with the signal of wild-type sfGFP considered to be 100%). Data are presented the mean ± SD for four replicates.

FIGURE 2

Crystal structures of DFRSc complexed with AMP-PNP and LFA/DFA. (A) Three overlapping crystal structures of LFA (2, blue stick; 4, white stick; 6, orange stick)-bound DFRSc. (B) The overlapping crystal structures of DFRSc complexed with D-3-chlorophenylalanine (3, blue stick) and L-3-chlorophenylalanine (4, white stick). Mutations N346G, C348Q and V401G are presented in magenta. The resolution of each crystal: 2: 2.2 Å; 3: 1.9 Å; 4: 1.9 Å; 6: 2.5 Å.

FIGURE 3

Biophysical analysis of DFA encoded proteins. (A) Excitation and emission spectra of sfGFP-Y66-3 (red) and sfGFP-Y66-4 (blue). (B) Intrinsic fluorescence spectra of sfGFP-Y66-3 (red) and sfGFP-Y66-4 (blue). (C) Temperature course of ellipticity for sfGFP-Y66-3 monitored by CD spectroscopy. (D) Temperature course of ellipticity at 200 nm for sfGFP-Y66-3 (red) and sfGFP-Y66-4 (blue) monitored by CD spectroscopy. (E) DLS analysis of FTH1-2x-1 (red) and FTH1-2x-2 (blue). (F) ICP-MS analysis of FTH1-H60 and FTH1-2x variants. Labeled numbers on the top of each bar indicate the chelated iron per FTH1 molecule.