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 encode 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 cross-linking, 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.

Engineering of enzymes using non-natural amino acids

“…Furthermore, dual labelling has become feasible due to the emergence of multiple nnAA incorporation systems, which is especially useful in Förster resonance energy transfer (FRET) experiments. The reported coupled nnAAs include p -azidophenylalanine (pAzF)/N6-((2-propynyloxy) carbonyl)-L-lysine [ 102 ], p -acetylphenylalanine/alkynyllysine [ 103 ], p AzF/Tet3.0 [ 104 ], 4-biphenyl-L-phenylalanine/L-(7-hydroxycoumarin-4-yl)-ethylglycine [ 105 ], and have been successfully employed to illustrate the dynamic changes in a wide range of proteins. An elegant example of this is the dual site-specific fluorescent labelling toolbox developed by Meineke et al By combining trans -cyclooct-2-ene-L-lysine (TCO*K) and N -Propargyl-L-lysine (ProK) incorporation with CuAAC/SPIEDAC, the labelling of Notch receptors and a G protein-coupled receptor (GPCR) has been achieved for the first time in living cells ( Figure 6 ).…”

Dual Noncanonical Amino Acid Incorporation Enabling Chemoselective Protein Modification at Two Distinct Sites in Yeast

“…Covalent attachment of two distinct chemical probes to dual ncAA-substituted proteins has enabled diverse biological applications, including FRET for studying protein conformation and dynamics (Bednar et al, 2021;Meineke et al, 2020;Wang et al, 2014). Here, we investigate, for what we believe is the first time, the combination of SPAAC and CuAAC reactions to install two different chemical probes within yeast-displayed proteins.…”

“…The broad set of machineries now available for encoding ncAAs in proteins has led to the establishment of systems for encoding multiple, chemically distinct ncAAs in both bacterial and mammalian systems (Dunkelmann et al, 2020(Dunkelmann et al, , 2021Tharp et al, 2021;Zheng et al, 2018a). Dual ncAA incorporation systems have facilitated important biological applications, such as i) identifying mutually compatible bioconjugation chemistries for protein labeling or for monitoring protein dynamics; ii) aiding intra-and intermolecular protein crosslinking; and iii) mimicking epigenetic modifications for unravelling new protein-protein interactions (Bednar et al, 2021;Meineke et al, 2020;Sachdeva et al, 2014;Venkat et al, 2018;Wang et al, 2014;Xiao et al, 2013;Zheng et al, 2017Zheng et al, , 2018b. However, to the best of our knowledge, such dual ncAA incorporation systems have not yet been implemented in S. cerevisiae or any other yeast.…”

“…Orthogonal codons can be repurposed sense codons, nonsense ("stop") codons, or engineered frameshift codons. For both bacteria and mammalian cells, orthogonal codons have been discovered and utilized in various combinations (Bednar et al, 2021;Chen et al, 2022;Hankore et al, 2019;Meineke et al, 2020;Niu et al, 2013;de la Torre and Chin, 2021;Wang et al, 2014;Willis and Chin, 2018;Zheng et al, 2017Zheng et al, , 2018bZheng et al, , 2018a. However, to the best of our knowledge, in yeast only the amber (TAG) codon has been repurposed and employed for ncAA incorporation (Wiltschi, 2016).…”

Dual fluorescent labeling of GLP-1R in live cells via enzymatic tagging and bioorthogonal chemistry

“… 7 More recently, a strategy involving dual genetic encoding of two unnatural amino acids followed by a pair of mutually exclusive bioorthogonal reactions was reported to insert FRET pairs to a yeast protein site-specifically. 8 However, none of these strategies have been employed to construct biosensors to monitor class B GPCR dynamics.…”