Genetic Incorporation of Two Mutually Orthogonal Bioorthogonal Amino Acids That Enable Efficient Protein Dual-Labeling in Cells

Abstract

The ability to site-specifically modify proteins at multiple sites in vivo will enable the study of protein function in its native environment with unprecedented levels of detail. Here, we present a versatile two-step strategy to meet this goal involving site-specific encoding of two distinct noncanonical amino acids bearing bioorthogonal handles into proteins in vivo followed by mutually orthogonal labeling. This general approach, that we call dual encoding and labeling (DEAL), allowed us to efficiently encoded tetrazine- and azide-bearing amino acids into a protein and demonstrate for the first time that the bioorthogonal labeling reactions with strained alkene and alkyne labels can function simultaneously and intracellularly with high yields when site-specifically encoded in a single protein. Using our DEAL system, we were able to perform topologically-defined protein-protein crosslinking, intramolecular stapling, and site-specific installation of fluorophores all inside living Escherichia coli cells, as well as study the DNA-binding properties of yeast Replication Protein A in vitro. By enabling the efficient dual modification of proteins in vivo, this DEAL approach provides a tool for the characterization and engineering of proteins in vivo.

Faster Surface Ligation Reactions Improve Immobilized Enzyme Structure and Activity

“…At these positions, AzF, Cys, or Tet3.0 was introduced such that each construct contained a combination of two of the three amino acids for labeling, where the third amino acid was employed for the site-specific immobilization reaction at position 126 (e.g., for CA AzF , Tet3.0 was introduced at residue 20 and Cys at residue 233, thus maintaining the ability for site-specific immobilization through the azido- l -phenylalanine residue at position 126). Critical to this approach was the simultaneous incorporation of AzF and Tet3.0 (i.e., two distinct unnatural amino acids) via suppression of two blank codons (TAG and TAA) in Escherichia coli using a set of mutually orthogonal aminoacyl-tRNA synthetase/tRNA pairs . To confirm that all three amino acids were present and reactive, the constructs were reacted with a Tet-reactive sTCO-bearing polyethylene glycol polymer ( M w 5000), an azide-reactive DBCO-bearing TAMRA, and thiol-reactive methanethiosulfonate-bearing BODIPY.…”

“…Critical to this approach was the simultaneous incorporation of AzF and Tet3.0 (i.e., two distinct unnatural amino acids) via suppression of two blank codons (TAG and TAA) in Escherichia coli using a set of mutually orthogonal aminoacyl-tRNA synthetase/tRNA pairs. 35 To confirm that all three amino acids were present and reactive, the constructs were reacted with a Tet-reactive sTCO-bearing polyethylene glycol polymer (M w 5000), an azide-reactive DBCO-bearing TAMRA, and thiol-reactive methanethiosulfonate-bearing BODIPY. As shown in Figure S6, the triple mutant constructs designed for FRET showed signatures of modification with all three reagents via in-gel SDS-PAGE imaging.…”

“…12 Following cloning, CA constructs were produced using the DEAL approach as previously described. 35 Briefly, CA constructs were paired with AzF-and Tet3.0-encoding machinery plasmids in Escherichia coli BL21(DE3) cells to generate the strains listed in Table S1. These strains were grown overnight in non-inducing media, 50 which was used to inoculate 50 mL of autoinduction media supplemented with 200 μM ZnSO 4 , 0.05% (w/v) arabinose, 0.02% (w/v) lactose, appropriate antibiotics at a 1% (v/v) inoculum, and 500 μM Tet3.0 and/or 1 mM AzF (when necessary).…”

“…CA constructs (Table S1) were cloned from a C205S thermal stable variant using methods that were previously described elsewhere . Following cloning, CA constructs were produced using the DEAL approach as previously described . Briefly, CA constructs were paired with AzF- and Tet3.0-encoding machinery plasmids in Escherichia coli BL21­(DE3) cells to generate the strains listed in Table S1.…”

Rational design for high bioorthogonal fluorogenicity of tetrazine‐encoded green fluorescent proteins

“…17,[21][22][23] However, exogenous organic fluorophores are intrinsically more invasive than endogenous reporters, such as the heterologously expressed green fluorescent protein (GFP), whose discovery and development have revolutionized bioimaging and biomedicine. 24,25 The genetic code expansion (GCE) technique 26 has further allowed the site-specific incorporation of noncanonical amino acid (ncAA) derivatives with tetrazine into FPs and other proteins, [27][28][29][30][31] We previously encoded a superfolder (sf)GFP 32 with a tetrazine-containing amino acid referred to as Tet-v2.0 (i.e., Tet-v2.0-Me, Me indicates a methyl group on Tet ring) 29 and showed that this genetically encoded handle can effectively react with a cyclopropane-fused sTCO (a strained TCO). 27 Interestingly, we observed that in addition to enabling ultrafast labeling, incorporation of Tet-v2.0-Me at TAG150 (ASN149Tet-v2.0 mutation) significantly quenches the fluorescence, the restoration of which can be achieved through reaction with sTCO.…”