Ribosomal Synthesis of Natural‐Product‐Like Bicyclic Peptides in Escherichia coli

Methods to access natural‐product‐like macrocyclic peptides can disclose new opportunities for the exploration of this important structural class for chemical biology and drug discovery applications. Here, the scope and mechanism of a novel strategy for directing the biosynthesis of thioether‐bridged bicyclic peptides in bacterial cells was investigated. This method entails split intein‐catalyzed head‐to‐tail cyclization of a ribosomally produced precursor peptide, combined with inter‐side‐chain crosslinking through a genetically encoded cysteine‐reactive amino acid. This strategy could be successfully applied to achieve formation of structurally diverse bicyclic peptides with high efficiency and selectivity in Escherichia coli. Insights into the sequence of reactions underlying the peptide bicyclization process were gained from time‐course experiments. Finally, the potential utility of this methodology toward the discovery of macrocyclic peptides with enhanced functional properties was demonstrated through the isolation of a bicyclic peptide with sub‐micromolar affinity for streptavidin.     

Linkage-Specific Synthesis of Di-ubiquitin Probes Enabled by the Incorporation of Unnatural Amino Acid ThzK

Di-ubiquitin (diUB) conjugates of defined linkages are useful tools for probing the functions of UB ligases, UB-binding proteins and deubiquitinating enzymes (DUBs) in coding, decoding and editing the signals carried by the UB chains. Here we developed an efficient method for linkage-specific synthesis of diUB probes based on the incorporation of the unnatural amino acid (UAA) N^ϵ-L-thiaprolyl-L-Lys (L-ThzK) into UB for ligation with another UB at a defined Lys position. The diUB formed by the UAA-mediated ligation reaction has a G76C mutation on the side of donor UB for conjugation with E2 and E3 enzymes or undergoing dethiolation to generate a covalent trap for DUBs. The development of UAA mutagenesis for diUB synthesis provides an easy route for preparing linkage-specific UB-based probes to decipher the biological signals mediated by protein ubiquitination.     

Genetic Encoding of a Photocaged Histidine for Light-Control of Protein Activity

The use of light to control protein function is a critical tool in chemical biology. Here we describe the addition of a photocaged histidine to the genetic code. This unnatural amino acid becomes histidine upon exposure to light and allows for the optical control of enzymes that utilize active-site histidine residues. We demonstrate light-induced activation of a blue fluorescent protein and a chloramphenicol transferase. Further, we genetically encoded photocaged histidine in mammalian cells. We then used this approach in live cells for optical control of firefly luciferase and, Renilla luciferase. This tool should have utility in manipulating and controlling a wide range of biological processes.     

Olefin Metathesis for Site‐Selective Protein Modification

Protein compatible . Olefin metathesis has emerged as a viable strategy for site‐selective protein modification. This minireview traces its development from early peptide models and metathesis in water to its ultimate application to protein substrates. Prospects in chemistry and biology are also discussed.

     

Efficient Generation of Hydrazides in Proteins by RadA Split Intein

Protein C-terminal hydrazides are useful for bioconjugation and construction of proteins from multiple fragments through native chemical ligation. To generate C-terminal hydrazides in proteins, an efficient intein-based preparation method has been developed by using thiols and hydrazine to accelerate the formation of the transient thioester intermediate and subsequent hydrazinolysis. This approach not only increases the yield, but also improves biocompatibility. The scope of the method has been expanded by employing Pyrococcus horikoshii RadA split intein, which can accommodate a broad range of extein residues before the site of cleavage. The use of split RadA minimizes premature intein N cleavage in vivo and offers control over the initiation of the intein N cleavage reaction. It is expected that this versatile preparation method will expand the utilization of protein C-terminal hydrazides in protein preparation and modification.     

Expanding the Genetic Code for Neuronal Studies

Genetic code expansion is one of the most powerful technologies in protein engineering. In addition to the 20 canonical amino acids, the expanded genetic code is supplemented by unnatural amino acids, which have artificial side chains that can be introduced into target proteins in vitro and in vivo. A wide range of chemical groups have been incorporated co-translationally into proteins in single cells and multicellular organisms by using genetic code expansion. Incorporated unnatural amino acids have been used for novel structure-function relationship studies, bioorthogonal labelling of proteins in cellulo for microscopy and in vivo for tissue-specific proteomics, the introduction of post-translational modifications and optical control of protein function, to name a few examples. In this Minireview, the development of genetic code expansion technology is briefly introduced, then its applications in neurobiology are discussed, with a focus on studies using mammalian cells and mice as model organisms.     

Photo-crosslinking: An Emerging Chemical Tool for Investigating Molecular Networks in Live Cells

Studying protein–protein interactions (PPIs) is useful for understanding cellular functions and mechanisms. Evaluating these PPIs under conditions as similar as possible to native conditions can be achieved using photo-crosslinking methods because of their on-demand ability to generate reactive species in situ by irradiation with UV light. Various fusion tag, metabolic incorporation, and amber codon suppression approaches using various crosslinkers containing aryl azide, benzophenone, and diazirines have been applied in live cells. Mass spectrometry and immunological techniques are used to identify crosslinked proteins based on their capture transient and context-dependent interactions. Herein we discuss various incorporation methods and crosslinkers that have been used for interactome mapping in live cells.     

Exploring the Functional Consequences of Protein Backbone Alteration in Ubiquitin through Native Chemical Ligation

Ubiquitin (Ub) plays critical roles in myriad protein degradation and signaling networks in the cell. We report herein Ub mimetics based on backbones that blend natural and artificial amino acid units. The variants were prepared by a modular route based on native chemical ligation. Biological assays show that some are enzymatically polymerized onto protein substrates, and that the resulting Ub tags are recognized for downstream pathways. These results advance the size and complexity of folded proteins mimicked by artificial backbones and expand the functional scope of such agents.     

Enhancing the Efficiency and Regioselectivity of P450 Oxidation Catalysts by Unnatural Amino Acid Mutagenesis

The development of effective strategies for modulating the reactivity and selectivity of cytochrome P450 enzymes represents a key step toward expediting the use of these biocatalysts for synthetic applications. We have investigated the potential of unnatural amino acid mutagenesis to aid efforts in this direction. Four unnatural amino acids with diverse aromatic side chains were incorporated at 11 active‐site positions of a substrate‐promiscuous CYP102A1 variant. The resulting “uP450s” were then tested for their catalytic activity and regioselectivity in the oxidation of two representative substrates: a small‐molecule drug and a natural product. Large shifts in regioselectivity resulted from these single mutations, and in particular, for para‐acetyl‐Phe substitutions at positions close to the heme cofactor. Screening this mini library of uP450s enabled us to identify P450 catalysts for the selective hydroxylation of four aliphatic positions in the target substrates, including a C(sp³)?H site not oxidized by the parent enzyme. Furthermore, we discovered a general activity‐enhancing effect of active‐site substitutions involving the unnatural amino acid para‐amino‐Phe, which resulted in P450 catalysts capable of supporting the highest total turnover number reported to date on a complex molecule (34 650). The functional changes induced by the unnatural amino acids could not be reproduced by any of the 20 natural amino acids. This study thus demonstrates that unnatural amino acid mutagenesis constitutes a promising new strategy for improving the catalytic activity and regioselectivity of P450 oxidation catalysts.     

Structure Prediction of Partial-Length Protein Sequences

Protein structure information is essential to understand protein function. Computational methods to accurately predict protein structure from the sequence have primarily been evaluated on protein sequences representing full-length native proteins. Here, we demonstrate that top-performing structure prediction methods can accurately predict the partial structures of proteins encoded by sequences that contain approximately 50% or more of the full-length protein sequence. We hypothesize that structure prediction may be useful for predicting functions of proteins whose corresponding genes are mapped expressed sequence tags (ESTs) that encode partial-length amino acid sequences. Additionally, we identify a confidence score representing the quality of a predicted structure as a useful means of predicting the likelihood that an arbitrary polypeptide sequence represents a portion of a foldable protein sequence (“foldability”). This work has ramifications for the prediction of protein structure with limited or noisy sequence information, as well as genome annotation.     

Genetic Encoding of a Bioconjugation Handle for [2+2+2] Cycloaddition Reactions

Protein bioconjugates have many critical applications, especially in the development of therapeutics. Consequently, the design of novel methodologies to prepare protein bioconjugates is of great importance. Herein we present the development and optimization of a novel strategy to prepare bioconjugates through a genetically encoded [2+2+2] cycloaddition reaction. To do this, a novel unnatural amino acid (UAA) containing a dipropargyl amine functionality was synthesized and incorporated site specifically. This UAA-containing protein was reacted with an alkyne-containing fluorophore to afford a covalently linked, well-defined protein bioconjugate. This reaction is convenient with an optimized reaction time of just two hours at room temperature and yields a stable, polysubstituted benzene ring. Overall, this work contributes a new bioconjugation strategy to the growing toolbox of reactions to develop protein bioconjugates, which have a myriad of applications.     

Phosphine-Activated Lysine Analogues for Fast Chemical Control of Protein Subcellular Localization and Protein SUMOylation

The Staudinger reduction and its variants have exceptional compatibility with live cells but can be limited by slow kinetics. Herein we report new small-molecule triggers that turn on proteins through a Staudinger reduction/self-immolation cascade with substantially improved kinetics and yields. We achieved this through site-specific incorporation of a new set of azidobenzyloxycarbonyl lysine derivatives in mammalian cells. This approach allowed us to activate proteins by adding a nontoxic, bioorthogonal phosphine trigger. We applied this methodology to control a post-translational modification (SUMOylation) in live cells, using native modification machinery. This work significantly improves the rate, yield, and tunability of the Staudinger reduction-based activation, paving the way for its application in other proteins and organisms.     

Distinct Mechanisms for Processing Autophagy Protein LC3-PE by RavZ and ATG4B

Autophagy is a conserved catabolic process involved in the elimination of proteins, organelles and pathogens in eukaryotic cells. Lipidated LC3 proteins that are conjugated to phosphatidylethanolamine (PE) play a key role in autophagosome biogenesis. Endogenous ATG4-mediated deconjugation of LC3-PE is required for LC3 recycling. However, the Legionella effector RavZ irreversibly deconjugates LC3-PE to inhibit autophagy. It is not clear how ATG4 and RavZ process LC3-PE with distinct modes. Herein, a series of semisynthetic LC3-PE proteins containing C-terminal mutations or insertions were used to investigate the relationship of the C-terminal structure of LC3-PE with ATG4/RavZ-mediated deconjugation. Using a combination of molecular docking and biochemical assays, we found that Gln116, Phe119 and Gly120 of LC3-PE are required for cleavage by both RavZ and ATG4B, whereas Glu117(LC3) is specific to cleavage by RavZ. The molecular ruler mechanism exists in the active site of ATG4B, but not in RavZ. Met63 and Gln64 at the active site of RavZ are involved in accommodating LC3 C-terminal motif. Our findings show that the distinct binding modes of the LC3 C-terminal motif (116–120) with ATG4 and RavZ might determine the specificity of cleavage site.     

Generation of a mono-ubiquitinated PCNA mimic by click chemistry

Genotoxic stress results in more than 50 000 damaged DNA sites per cell per day. During DNA replication, processive high‐fidelity DNA polymerases generally stall at DNA lesions and have to be displaced by translesion synthesis DNA polymerases, which are able to bypass the lesion. This switch is mediated by mono‐ubiquitination of the processivity factor proliferating cell nuclear antigen (PCNA). To further investigate the regulation of the DNA polymerase exchange, we developed an easy and efficient method to synthesize site‐specifically mono‐ubiquitinated PCNA by click chemistry. By incorporating artificial amino acids that carry an azide (Aha) or an alkyne (Plk) in their side chains, into ubiquitin (Ub) and PCNA, respectively, we were able to link the two proteins site‐specifically by the Cu^I‐catalyzed azide–alkyne cycloaddition. Finally, we show that the synthetic PCNA–Ub is able to stimulate DNA synthesis by DNA polymerase δ, and that DNA polymerase η has a higher affinity for PCNA–Ub than to PCNA.     

Dimerization determines substrate specificity of a bacterial prenyltransferase

We have identified the native dimer interface of heptaprenylglyceryl phosphate synthase PcrB from the bacterium Bacillus subtilis and analyzed the significance of oligomer formation for stability and catalytic activity. Computational methods predicted two different surface regions of the PcrB protomer that could be responsible for dimer formation. These bona fide interfaces were assessed both in silico and experimentally by the introduction of amino acid substitutions that led to monomerization, and by incorporation of an unnatural amino acid to allow cross-linking of the two protomers. The results showed that, in contrast to previous assumptions, PcrB uses the same interface for dimerization as the homologous geranylgeranylglyceryl phosphate synthase from Archaea. Thermal unfolding demonstrated that the monomeric proteins are only slightly less stable than wild-type PcrB. However, activity assays showed that monomerization limits the length of accepted polyprenyl pyrophosphates to three isoprene units, whereas the native PcrB substrate contains seven isoprene entities. We provide a plausible hypothesis as to how dimerization determines substrate specificity of PcrB.