Maiyun Yang

Palladium-triggered deprotection chemistry for protein activation in living cells

Employing small molecules or chemical reagents to modulate the function of an intracellular protein, particularly in a gain-of-function fashion, remains a challenge. In contrast to inhibitor-based loss-of-function approaches, methods based on a gain of function enable specific signalling pathways to be activated inside a cell. Here we report a chemical rescue strategy that uses a palladium-mediated deprotection reaction to activate a protein within living cells. We identify biocompatible and efficient palladium catalysts that cleave the propargyl carbamate group of a protected lysine analogue to generate a free lysine. The lysine analogue can be genetically and site-specifically incorporated into a protein, which enables control over the reaction site. This deprotection strategy is shown to work with a range of different cell lines and proteins. We further applied this biocompatible protection group/catalyst pair for caging and subsequent release of a crucial lysine residue in a bacterial Type III effector protein within host cells, which reveals details of its virulence mechanism.

Converting a solvatochromic fluorophore into a protein-based pH indicator for extreme acidity.

Live-cell pH measurements: An environment-sensitive fluorophore (green) was site-specifically introduced on HdeA, an acid-resistant chaperone showing pH-mediated conformational changes under low pH conditions. A survey of the attachment sites led to the discovery of one position on HdeA at which the attached fluorophore showed a strong fluorescence increase upon acidification.

Ligand-Free Palladium-Mediated Site-Specific Protein Labeling Inside Gram-Negative Bacterial Pathogens

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.

Biocompatible click chemistry enabled compartment-specific pH measurement inside E. coli

Bioorthogonal reactions, especially the Cu(I)-catalyzed azide-alkyne cycloaddition, have revolutionized our ability to label and manipulate biomolecules under living conditions. The cytotoxicity of Cu(I) ions, however, has hindered the application of this reaction in the internal space of living cells. By systematically surveying a panel of Cu(I)-stabilizing ligands in promoting protein labeling within the cytoplasm of E. coli, here we identify a highly efficient and biocompatible catalyst for intracellular modification of proteins by azide-alkyne cycloaddition. This reaction permits us to conjugate an environment-sensitive fluorophore site-specifically onto HdeA, an acid-stress chaperone that adopts pH-dependent conformational changes, in both the periplasm and cytoplasm of E. coli. The resulting protein-fluorophore hybrid pH indicators enable compartment-specific pH measurement to determine the pH gradient across the E. coli cytoplasmic membrane. This construct also allows the measurement of E. coli transmembrane potential, and the determination of the proton motive force across its inner membrane under normal and acid-stress conditions.

Ligation of Expressed Protein α-Hydrazides via Genetic Incorporation of an α-Hydroxy Acid

Expressed protein ligation bridges the gap between synthetic peptides and recombinant proteins and thereby significantly increases the size and complexity of chemically synthesized proteins. Although the intein-based expressed protein ligation method has been extensively used in this regard, the development of new expressed protein ligation methods may improve the flexibility and power of protein semisynthesis. In this study a new alternative version of expressed protein ligation is developed by combining the recently developed technologies of hydrazide-based peptide ligation and genetic code expansion. Compared to the previous intein-based expressed protein ligation method, the new method does not require the use of protein splicing technology and generates recombinant protein α-hydrazides as ligation intermediates that are more chemically stable than protein α-thioesters. Furthermore, the use of an evolved mutant pyrrolysyl-tRNA synthetase(PylRS), ACPK-RS, from M. barkeri shows an improved performance for the expression of recombinant protein backbone oxoesters. By using HdeA as a model protein we demonstrate that the hydrazide-based method can be used to synthesize proteins with correctly folded structures and full biological activity. Because the PylRS-tRNACUAPyl system is compatible with both prokaryotic and eukaryotic cells,the strategy presented here may be readily expanded to manipulate proteins produced in mammalian cells. The new hydrazide-based method may also supplement the intein-based expressed protein ligation method by allowing for a more flexible selection of ligation site.

Site‐Specific Engineering of Chemical Functionalities on the Surface of Live Hepatitis D Virus

The genetic code expansion strategy, the recently emerged pyrrolysine (Pyl)-based system in particular, has become a generally applicable method for site-specific incorporation of unnatural amino acids (UAAs) into a protein of interest in bacteria, yeast, mammalian cells, and even in animals. However, this technique has yet to be applied to intact and live viruses, which is largely due to the fragile nature as well as the complicated assembly process of many human viruses. To address this challenge, we here coupled the genetic-code expansion strategy with an engineered virus assembly process in human hepatocytes to site-specifically introduce unnatural chemical groups onto virus surface proteins by using hepatitis D virus (HDV) as a model system. HDV has infectedmore than 15 million people worldwide, and currently there are no drugs clinically available against this virus. HDV is a satellite virus of human hepatitis B virus (HBV), which has infected two billion people and among them about 240 million are currently chronically infected. Both HBV and HDV share the same envelope proteins for infection of hepatocytes. Study of HBV and HDV infection has long been hampered by the lack of efficient and easily accessible in vitro infection system. Recently, a bile acid transporter predominantly expressed in liver, sodium taurocholate cotransporting polypeptide (NTCP) was identified as a functional receptor for HDV and HBV. The NTCP complemented human hepatoma cell line HepG2 provided a feasible in vitro infection system for studying HBV and HDV infection. However, the lack of methods to selectively label, monitor, and/or manipulate an intact virus under living conditions still restricts investigations into molecular details of the infection. Many problems are due to the distinct topological features of the critical viral proteins, as well as complex virus assembly processes. For example, HDV has developed a tightly regulated assembly process to produce infectious viral particles in human hepatocytes: the HDV RNAs were first encapsulated with delta antigens and then packaged with three HBV envelope proteins, namely large (L), middle (M), and small (S) proteins, to produce the intact viral particle before being secreted to the extracellular space (Supporting Information, Figure S1). The resulting HDV, with a diameter of 36 nm, is one of the smallest animal viruses known to date. It is therefore exceedingly difficult to chemically label this tiny virus with delicate structures under living conditions. Furthermore, the virus surface envelope proteins contain many chemically active amino acids (for example, cysteine and lysine) that are essential for virus entry in host cells. Conjugation or modification of these natural residues will severely compromise viral infectivity. A noninvasive strategy for manipulation of living viral particles without impairment of their viability and infectivity is thus highly desired. Bioorthogonal reactions have revolutionized our ability to label and manipulate various biomolecules and even whole cells and organisms under living conditions. As a critical step for applying such chemistry for virus labeling, several approaches have been reported for installation of bioorthogonal handles, typically in the form of UAAs into proteins from sub-viral-like particle (SVP) or intact virus. For instance, site-specific or residue-specific incorporation of UAAs bearing an azide or an alkyne moiety into SVP has been demonstrated in bacterial cells by several laboratories. These methods allow the conjugation of SVPs with various fluorescent dyes or therapeutic reagents for biomedical or biomaterial applications. However, SVPs are non-infectious and not suitable for investigating virus infection mechanisms. Indeed, SVPs produced from prokaryotic cells lack posttranslational modifications, particularly on their surface envelope proteins, and therefore differ from the native SVPs. Although attempts have been made to extend some of these methods for virus production in mammalian cells, such strategies typically require the metabolic replacement of a specific type of amino acid permissive only to simple groups (for example, azide and ketone) from the entire virus proteome, which may disrupt the virion assembly process or permute the vulnerable virion structure, resulting in compromised viral infectivity. Taken together, a general approach for precise labeling and manipulation of intact [*] S.-X. Lin, M.-Y. Yang, Prof. Dr. P. R. Chen Synthetic and Functional Biomolecules Center, Beijing National Laboratory for Molecular Sciences, Department of Chemical Biology, College of Chemistry and Molecular Engineering, Synthetic and Functional Biomolecules Center and Peking-Tsinghua Center for Life Sciences Peking University, Beijing 100871 (China) E-mail: pengchen@pku.edu.cn H. Yan Graduate program in School of Life Sciences Peking University, Beijing (China) H. Yan, L. Li, B. Peng, S. Chen, Prof. Dr. W.-H. Li National Institute of Biological Sciences, Beijing (China) E-mail: liwenhui@nibs.ac.cn [] These authors contributed equally to this work.

Genetically encoded alkenyl–pyrrolysine analogues for thiol–ene reaction mediated site-specific protein labeling

A series of alkene-bearing pyrrolysine analogues were synthesized and subsequently incorporated into proteins at two sites by a mutant PylRS–tRNA pair with excellent efficiency. This strategy allowed the site-specific labeling of proteins carrying single or double genetically encoded alkene handles via bioorthogonal thiol–ene ligation reactions.

A Genetically Encoded FRET Sensor for Intracellular Heme

Heme plays pivotal roles in various cellular processes as well as in iron homeostasis in living systems. Here, we report a genetically encoded fluorescence resonance energy transfer (FRET) sensor for selective heme imaging by employing a pair of bacterial heme transfer chaperones as the sensory components. This heme-specific probe allows spatial-temporal visualization of intracellular heme distribution within living cells.

Transition-Metal-Catalyzed Bioorthogonal Cycloaddition Reactions

AbstractIn recent years, bioorthogonal reactions have emerged as a powerful toolbox for specific labeling and visualization of biomolecules, even within the highly complex and fragile living systems. Among them, copper(I)-catalyzed azide–alkyne cycloaddition (CuAAC) reaction is one of the most widely studied and used biocompatible reactions. The cytotoxicity of Cu(I) ions has been greatly reduced due to the use of Cu(I) ligands, which enabled the CuAAC reaction to proceed on the cell surface, as well as within an intracellular environment. Meanwhile, other transition metals such as ruthenium, rhodium and silver are now under development as alternative sources for catalyzing bioorthogonal cycloadditions. In this review, we summarize the development of CuAAC reaction as a prominent bioorthogonal reaction, discuss various ligands used in reducing Cu(I) toxicity while promoting the reaction rate, and illustrate some of its important biological applications. The development of additional transition metals in catalyzing cycloaddition reactions will also be briefly introduced.