An orthogonal aminoacyl-tRNA synthetase/tRNA pair is a crucial prerequisite for site-specific incorporation of unnatural amino acids. Due to its high codon suppression efficiency and full ...orthogonality, the pyrrolysyl-tRNA synthetase/pyrrolysyl-tRNA pair is currently the ideal system for genetic code expansion in both eukaryotes and prokaryotes. There is a pressing need to discover or engineer other fully orthogonal translation systems. Here, through rational chimera design by transplanting the key orthogonal components from the pyrrolysine system, we create multiple chimeric tRNA synthetase/chimeric tRNA pairs, including chimera histidine, phenylalanine, and alanine systems. We further show that these engineered chimeric systems are orthogonal and highly efficient with comparable flexibility to the pyrrolysine system. Besides, the chimera phenylalanine system can incorporate a group of phenylalanine, tyrosine, and tryptophan analogues efficiently in both E. coli and mammalian cells. These aromatic amino acids analogous exhibit unique properties and characteristics, including fluorescence, post-translation modification.
Methionine is one of the most hydrophobic, redox-sensitive, and one of the only two sulfur-containing amino acids on protein. Because of these biochemical properties, the methionine residue plays a ...central role in a variety of biological processes, such as metal coordination, antioxidant stress, and aging. However, studies on the molecular functions of methionine are much less common than the other primary sulfur-containing amino acid, cysteine. The limited number of publications on methionine-related studies is partially due to the lack of tools for methionine modification. Methionine bioconjugation offers a new strategy to decipher the biological function of methionine and expands the toolbox for protein functionalization in the context of the application, such as synthesizing proteins with novel properties and producing new biomaterials. The purpose of this Perspective is to highlight the biochemical properties and functions of methionine, list recent progress in the development of methionine bioconjugation reagents, and briefly demonstrate the application of these reagents on polypeptides, proteins, and proteomes.
Site-specific incorporation of unnatural amino acids (UAAs) with similar incorporation efficiency to that of natural amino acids (NAAs) and low background activity is extremely valuable for efficient ...synthesis of proteins with diverse new chemical functions and design of various synthetic auxotrophs. However, such efficient translation systems remain largely unknown in the literature. Here, we describe engineered chimeric phenylalanine systems that dramatically increase the yield of proteins bearing UAAs, through systematic engineering of the aminoacyl-tRNA synthetase and its respective cognate tRNA. These engineered synthetase/tRNA pairs allow single-site and multi-site incorporation of UAAs with efficiencies similar to those of NAAs and high fidelity. In addition, using the evolved chimeric phenylalanine system, we construct a series of E. coli strains whose growth is strictly dependent on exogenously supplied of UAAs. We further show that synthetic auxotrophic cells can grow robustly in living mice when UAAs are supplemented.
Cysteine can be specifically functionalized by a myriad of acid-base conjugation strategies for applications ranging from probing protein function to antibody-drug conjugates and proteomics. In ...contrast, selective ligation to the other sulfur-containing amino acid, methionine, has been precluded by its intrinsically weaker nucleophilicity. Here, we report a strategy for chemoselective methionine bioconjugation through redox reactivity, using oxaziridine-based reagents to achieve highly selective, rapid, and robust methionine labeling under a range of biocompatible reaction conditions. We highlight the broad utility of this conjugation method to enable precise addition of payloads to proteins, synthesis of antibody-drug conjugates, and identification of hyperreactive methionine residues in whole proteomes.
Palladium, a key transition metal in advancing modern organic synthesis, mediates diverse chemical conversions including many carbon–carbon bond formation reactions between organic compounds. ...However, expanding palladium chemistry for conjugation of biomolecules such as proteins, particularly within their native cellular context, is still in its infancy. Here we report the site-specific protein labeling inside pathogenic Gram-negative bacterial cells via a ligand-free palladium-mediated cross-coupling reaction. Two rationally designed pyrrolysine analogues bearing an aliphatic alkyne or an iodophenyl handle were first encoded in different enteric bacteria, which offered two facial handles for palladium-mediated Sonogashira coupling reaction on proteins within these pathogens. A GFP-based bioorthogonal reaction screening system was then developed, allowing evaluation of both the efficiency and the biocompatibilty of various palladium reagents in promoting protein–small molecule conjugation. The identified simple compound–Pd(NO3)2 exhibited high efficiency and biocompatibility for site-specific labeling of proteins in vitro and inside living E. coli cells. This Pd-mediated protein coupling method was further utilized to label and visualize a Type-III Secretion (T3S) toxin-OspF in Shigella cells. Our strategy may be generally applicable for imaging and tracking various virulence proteins within Gram-negative bacterial pathogens.
Acid chaperones are essential factors in preserving the protein homeostasis for enteric pathogens to survive in the extremely acidic mammalian stomach (pH 1-3). The client proteins of these ...chaperones remain largely unknown, primarily because of the exceeding difficulty of determining protein-protein interactions under low-pH conditions. We developed a genetically encoded, highly efficient protein photocrosslinking probe, which enabled us to profile the in vivo substrates of a major acid-protection chaperone, HdeA, in Escherichia coli periplasm. Among the identified HdeA client proteins, the periplasmic chaperones DegP and SurA were initially found to be protected by HdeA at a low pH, but they subsequently facilitated the HdeA-mediated acid recovery of other client proteins. This unique, ATP-independent chaperone cooperation in the ATP-deprived E. coli periplasm may support the acid resistance of enteric bacteria. The crosslinker would be valuable in unveiling the physiological interaction partners of any given protein and thus their functions under normal and stress conditions.
Coupling photocrosslinking reagents with mass spectrometry has become a powerful tool for studying protein-protein interactions in living systems, but it still suffers from high rates of ...false-positive identifications as well as the lack of information on interaction interface due to the challenges in deciphering crosslinking peptides. Here we develop a genetically encoded photo-affinity unnatural amino acid that introduces a mass spectrometry-identifiable label (MS-label) to the captured prey proteins after photocrosslinking and prey-bait separation. This strategy, termed IMAPP (In-situ cleavage and MS-label transfer After Protein Photocrosslinking), enables direct identification of photo-captured substrate peptides that are difficult to uncover by conventional genetically encoded photocrosslinkers. Taking advantage of the MS-label, the IMAPP strategy significantly enhances the confidence for identifying protein-protein interactions and enables simultaneous mapping of the binding interface under living conditions.
A 1 × 2 low-profile filtering antenna array is proposed, using an H-shape resonator to feed two strip dense dielectric patch (DDP) resonators. The even–odd mode of the H-shape resonator and the TMδ1 ...mode of the strip DDP resonator form the radiation band. Additionally, the odd–odd mode of the H-shape resonator excites the TMδ2 mode of the strip DDP resonator, thus achieving an upper-edge radiation null for the filtering response. The H-shape resonator not only participates in the antenna array radiation, but also excites two strip media patches at the same time, avoiding the traditional power distribution network and effectively reducing the complexity of the antenna array. In addition, compared with the reported dielectric filtering antenna designs, this design has the advantageous features of a low profile, a compact structure, wide bandwidth and a simplified structure. For example, the antenna prototype operating at 4.9 GHz achieves 10 dB impedance, a matching bandwidth of 7.1%, a maximum gain of 8.6 dBi and the compact size of 1.29 × 0.18 × 0.038 λ03, without requiring a traditional power division network.
A low-profile dielectric resonator (DR) filter is proposed to achieve the feature of high integration and wide stopband. The high integration is due to the structure of printed circuit board (PCB) ...substrate instead of metal cavity, which can be easily integrated with other planar circuits. Thus, the proposed design can improve the integration level and reduce installation errors. Moreover, the out-of-band harmonics can be well suppressed by the structure combined with introducing rectangular hollowing in the center of the dielectric block, coupling the feed and loading 1/4λ wavelength branch. For demonstration, it is fabricated and measured. The simulated and experimental results with good agreement are presented, the insertion loss is as low as 1.1 dB, the profile height is only 0.77λg, and the stopband reaches 2.61f0.
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