Fluorosulfate as a Latent Sulfate in Peptides and Proteins.

Sulfation widely exists in the eukaryotic proteome. However, understanding the biological functions of sulfation in peptides and proteins has been hampered by the lack of methods to control its spatial or temporal distribution in the proteome. Herein, we report that fluorosulfate can serve as a latent precursor of sulfate in peptides and proteins, which can be efficiently converted to sulfate by hydroxamic acid reagents under physiologically relevant conditions. Photocaging the hydroxamic acid reagents further allowed for the light-controlled activation of functional sulfopeptides. This work provides a valuable tool for probing the functional roles of sulfation in peptides and proteins.

A Translation-Independent Directed Evolution Strategy to Engineer Aminoacyl-tRNA Synthetases.

Using directed evolution, aminoacyl-tRNA synthetases (aaRSs) have been engineered to incorporate numerous noncanonical amino acids (ncAAs). Until now, the selection of such novel aaRS mutants has relied on the expression of a selectable reporter protein. However, such translation-dependent selections are incompatible with exotic monomers that are suboptimal substrates for the ribosome. A two-step solution is needed to overcome this limitation: (A) engineering an aaRS to charge the exotic monomer, without ribosomal translation; (B) subsequent engineering of the ribosome to accept the resulting acyl-tRNA for translation. Here, we report a platform for aaRS engineering that directly selects tRNA-acylation without ribosomal translation (START). In START, each distinct aaRS mutant is correlated to a cognate tRNA containing a unique sequence barcode. Acylation by an active aaRS mutant protects the corresponding barcode-containing tRNAs from oxidative treatment designed to damage the 3'-terminus of the uncharged tRNAs. Sequencing of these surviving barcode-containing tRNAs is then used to reveal the identity of the aaRS mutants that acylated the correlated tRNA sequences. The efficacy of START was demonstrated by identifying novel mutants of the Methanomethylophilus alvus pyrrolysyl-tRNA synthetase from a naïve library that enables incorporation of ncAAs into proteins in living cells.

A Facile Platform to Engineer Escherichia coli Tyrosyl-tRNA Synthetase Adds New Chemistries to the Eukaryotic Genetic Code, Including a Phosphotyrosine Mimic.

The Escherichia coli tyrosyl-tRNA synthetase (EcTyrRS)/tRNAEcTyr pair offers an attractive platform for genetically encoding new noncanonical amino acids (ncAA) in eukaryotes. However, challenges associated with a eukaryotic selection system, which is needed to engineer the platform, have impeded its success in the past. Recently, using a facile E. coli-based selection system, we showed that EcTyrRS could be engineered in a strain where the endogenous tyrosyl pair was substituted with an archaeal counterpart. However, significant cross-reactivity between the UAG-suppressing tRNACUA EcTyr and the bacterial glutaminyl-tRNA synthetase limited the scope of this strategy, preventing the selection of moderately active EcTyrRS mutants. Here we report an engineered tRNACUA EcTyr that overcomes this cross-reactivity. Optimized selection systems based on this tRNA enabled the efficient enrichment of both strongly and weakly active ncAA-selective EcTyrRS mutants. We also developed a wide dynamic range (WiDR) antibiotic selection to further enhance the activities of the weaker first-generation EcTyrRS mutants. We demonstrated the utility of our platform by developing several new EcTyrRS mutants that efficiently incorporated useful ncAAs in mammalian cells, including photoaffinity probes, bioconjugation handles, and a nonhydrolyzable mimic of phosphotyrosine.