Tyrosine phosphorylation is a common protein posttranslational modification, which plays a critical role in signal transduction and the regulation of many cellular processes. Using a pro-peptide strategy to increase cellular uptake of O-phosphotyrosine (pTyr) and its nonhydrolyzable analog 4-phosphomethyl-L-phenylalanine (Pmp), we identified an orthogonal aminoacyl-tRNA synthetase/tRNA pair that allows the site-specific incorporation of both pTyr and Pmp into recombinant proteins in response to the amber stop codon in Escherichia coli in good yields. The X-ray crystal structure of the synthetase reveals a reconfigured substrate binding site formed by non-conservative mutations and substantial local structural perturbations. We demonstrate the utility of this method by introducing Pmp into a putative phosphorylation site whose corresponding kinase is unknown and determined the affinities of the individual variants for the substrate 3BP2. In summary, this work provides a useful recombinant tool to dissect the biological functions of tyrosine phosphorylation at specific sites in the proteome.

Detective work: Three highly sensitive and selective fluorescent probes for the detection of H2S have been developed. Two of the probes detect H2S by the reduction of an azide moiety to an amine group, which changes the probes into their fluorescent forms and generates a fluorescent signal. In the third method, the fluorescent signal is created by using H2S as a nucleophile in a reaction that unmasks fluorescein.

Inflammation is a prominent pathological feature in pulmonary arterial hypertension, as demonstrated by pulmonary vascular infiltration of inflammatory cells, including T and B lymphocytes. However, the contribution of the adaptive immune system is not well characterized in pulmonary hypertension caused by chronic hypoxia. CD4 T cells are required for initiating and maintaining inflammation, suggesting that these cells could play an important role in the pathogenesis of hypoxic pulmonary hypertension. Our objective was to test the hypothesis that CD4 T cells, specifically the T helper 17 subset, contribute to chronic hypoxia-induced pulmonary hypertension. We compared indices of pulmonary hypertension resulting from chronic hypoxia (3 wk) in wild-type mice and recombination-activating gene 1 knockout mice (RAG1, lacking mature T and B cells). Separate sets of mice were adoptively transferred with CD4, CD8, or T helper 17 cells before normoxic or chronic hypoxic exposure to evaluate the involvement of specific T cell subsets. RAG1 mice had diminished right ventricular systolic pressure and arterial remodeling compared with wild-type mice exposed to chronic hypoxia. Adoptive transfer of CD4 but not CD8 T cells restored the hypertensive phenotype in RAG1 mice. Interestingly, RAG1 mice receiving T helper 17 cells displayed evidence of pulmonary hypertension independent of chronic hypoxia. Supporting our hypothesis, depletion of CD4 cells or treatment with SR1001, an inhibitor of T helper 17 cell development, prevented increased pressure and remodeling responses to chronic hypoxia. We conclude that T helper 17 cells play a key role in the development of chronic hypoxia-induced pulmonary hypertension.

Thiopeptides are a subclass of ribosomally synthesized and posttranslationally modified peptides (RiPPs) with complex molecular architectures and an array of biological activities, including potent antimicrobial activity. Here we report the generation of thiopeptides containing noncanonical amino acids (ncAAs) by introducing orthogonal amber suppressor aminoacyl-tRNA synthetase/tRNA pairs into a thiocillin producer strain of Bacillus cereus. We demonstrate that thiopeptide variants containing ncAAs with bioorthogonal chemical reactivity can be further postbiosynthetically modified with biophysical probes, including fluorophores and photo-cross-linkers. This work allows the site-specific incorporation of ncAAs into thiopeptides to increase their structural diversity and probe their biological activity; similar approaches can likely be applied to other classes of RiPPs.noncanonical amino acid | biosynthesis | natural products | thiopeptides | antibiotic

A new internal charge transfer (ICT) fluorescent probe for highly selective and sensitive monitoring of H(2)S has been developed. The design takes advantage of the facile reduction of non-fluorescent hydroxyamine naphthalimide by H(2)S to highly fluorescent amine naphthalimide. It has been demonstrated that the probe is able to detect H(2)S at submicromolar concentrations in cells.

A FRET ratiometric fluorescent probe enabling a fast and highly sensitive response to OP nerve agent mimic DCP within 1 min and with as low as 0.17 ppm concentration detection limit has been developed. Moreover, the probe exhibits noticeable color changes under UV light and even with the naked eye. It is also demonstrated that it can detect both liquid and gas nerve agents.

Here we report the evolution of an orthogonal amber suppressor pyrrolysyl-tRNA synthetase (PylRS)/tRNACUAPyl pair that genetically encodes the post-translationally modified amino acid, ε-N-2-hydroxyisobutyryl-lysine (HibK), in bacteria and mammalian cells. HibK is a new type of histone mark that is widely distributed in histone proteins. The ability to site-specifically incorporate HibK into proteins provides a useful tool to probe the biological function of this newly identified post-translational modification.

The use of genetically encoded noncanonical amino acids (ncAAs) to construct crosslinks within or between proteins has emerged as a useful method to enhance protein stability, investigate protein-protein interactions and improve the pharmacological properties of proteins. Here we report ncAAs with aryl carbamate side chains (PheK and FPheK) that can react with proximal nucleophilic residues to form intra- or intermolecular protein crosslinks. We evolved a pyrrolysyl-tRNA synthetase that can site-specifically incorporate PheK and FPheK into proteins in both E. coli and mammalian cells. PheK and FPheK when incorporated into proteins showed good stability during protein expression and purification; crosslinking required incubation under weakly basic condition. We demonstrated that FPheK can react with adjacent Lys, Cys and Tyr residues in thioredoxin in high yields. In addition, crosslinks could be formed between FPheK and Lys residue of two interacting proteins, including the heavy chain and light chain of an antibody Fab. The high efficiency and specificity of FPheK crosslinking may make it a practical tool to engineer proteins with unnatural covalent linkages.