Posttranslational modifications (PTMs) of lysine are crucial histone marks that regulate diverse biological processes. The functional roles and regulation mechanism of many newly identified lysine PTMs, however, remain yet to be understood. Here we report a photoaffinity crotonyl lysine (Kcr) analogue that can be genetically and site-specifically incorporated into histone proteins. This, in conjunction with the genetically encoded photo-lysine as a “control probe”, enables the capture and identification of enzymatic machinery and/or effector proteins for histone lysine crotonylation.

ConspectusThe cell envelope is an integral and essential component of Gram-negative bacteria. As the front line during host–pathogen interactions, it is directly challenged by host immune responses as well as other harsh extracellular stimuli. The high permeability of the outer-membrane and the lack of ATP energy system render it difficult to maintain important biological activities within the periplasmic space under stress conditions. The HdeA/B chaperone machinery is the only known acid resistant system found in bacterial periplasm, enabling enteric pathogens to survive through the highly acidic human stomach and establish infections in the intestine. These two homologous chaperones belong to a fast growing family of conditionally disordered chaperones that conditionally lose their well-defined three-dimensional structures to exert biological activities. Upon losing ordered structures, these proteins commit promiscuous binding of diverse clients in response to environmental stimulation. For example, HdeA and HdeB are well-folded inactive dimers at neutral pH but become partially unfolded to protect a wide array of acid-denatured proteins upon acid stress. Whether these conditionally disordered chaperones possess client specificities remains unclear. This is in part due to the lack of efficient tools to investigate such versatile and heterogeneous protein–protein interactions under living conditions.Genetically encoded protein photo-cross-linkers have offered a powerful strategy to capture protein–protein interactions, showing great potential in profiling protein interaction networks, mapping binding interfaces, and probing dynamic changes in both physiological and pathological settings. Despite great success, photo-cross-linkers that can simultaneously capture the promiscuous binding partners and directly identify the interaction interfaces remain technically challenging. Furthermore, methods for side-by-side profiling and comparing the condition-dependent client pools from two homologous chaperones are lacking.Herein, we introduce our recent efforts in developing a panel of versatile genetically encoded photo-cross-linkers to study the disorder-mediated chaperone–client interactions in living cells. In particular, we have developed a series of proteomic-based strategies relying on these new photo-cross-linkers to systematically compare the client profiles of HdeA and HdeB, as well as to map their interaction interfaces. These studies revealed the mode-of-action, particularly the client specificity, of these two conditionally disordered chaperones. In the end, some recent elegant work from other groups that applied the genetically encoded photo-cross-linking strategy to illuminate important protein–protein interactions within bacterial cell envelope is also discussed.

Multiple antibiotic resistance regulator (MarR) family proteins are widely conserved transcription factors that are important for regulating bacterial resistance to various stresses. Escherichia coli MarR, the prototype member of this family, has recently been shown to undergo interdimer disulfide bond formation via a unique cysteine residue (Cys80) that ultimately triggered MarR's dissociation from its promoter DNA. However, these structural studies were performed with cysteine mutants while the structure of wild type MarR remains uncharacterized. Here we report the crystal structure of wild type MarR in the absence of DNA, which further revealed the roles of cysteine residues in MarR's transcriptional regulation. In addition, we developed a circularly permuted yellow fluorescent protein (cpYFP)-based environmental-sensitive fluorescent reporter for in situ detection of the DNA-binding induced conformational change on MarR, which verified the induction mechanism of this prototypical MarR family protein. This strategy might potentially be applicable for monitoring local conformational change within diverse protein structures.Download high-res image (150KB)Download full-size image

AbstractA genetically encoded, multifunctional photocrosslinker was developed for quantitative and comparative proteomics. By bearing a bioorthogonal handle and a releasable linker in addition to its photoaffinity warhead, this probe enables the enrichment of transient and low-abundance prey proteins after intracellular photocrosslinking and prey–bait separation, which can be subject to stable isotope dimethyl labeling and mass spectrometry analysis. This quantitative strategy (termed isoCAPP) allowed a comparative proteomic approach to be adopted to identify the proteolytic substrates of an E. coli protease–chaperone dual machinery DegP. Two newly identified substrates were subsequently confirmed by proteolysis experiments.

Multiple antibiotic resistance regulator (MarR) family proteins are widely conserved transcription factors that control bacterial resistance to antibiotics, environmental stresses, as well as the regulation of virulence determinants. Escherichia coli MarR, the prototype member of this family, has recently been shown to undergo copper(II)-catalyzed inter-dimer disulfide bond formation via a unique cysteine residue (Cys80) residing in its DNA-binding domain. However, despite extensive structural characterization of the MarR family proteins, the structural mechanism for DNA binding of this copper(II)-sensing MarR factor remains elusive. Here, we report the crystal structures of DNA-bound forms of MarR, which revealed a unique, concerted generation of two new helix–loop–helix motifs that facilitated MarR’s DNA binding. Structural analysis and electrophoretic mobility shift assays (EMSA) show that the flexibility of Gly116 in the center of helix α5 and the extensive hydrogen-bonding interactions at the N-terminus of helix α1 together assist the reorientation of the wHTH domains and stabilize MarR’s DNA-bound conformation.

Chemical de-caging has emerged as an attractive strategy for gain-of-function study of proteins via small-molecule reagents. The previously reported chemical de-caging reactions have been largely centered on liberating the side chain of lysine on a given protein. Herein, we developed an allene-based caging moiety and the corresponding palladium de-caging reagents for chemical rescue of tyrosine (Tyr) activity on intracellular proteins. This bioorthogonal de-caging pair has been successfully applied to unmask enzymatic Tyr sites (e.g., Y671 on Taq polymerase and Y728 on Anthrax lethal factor) as well as the post-translational Tyr modification site (Y416 on Src kinase) in vitro and in living cells. Our strategy provides a general platform for chemical rescue of Tyr-dependent protein activity inside cells.

Selective manipulation of protein kinases under living conditions is highly desirable yet extremely challenging, particularly in a gain-of-function fashion. Here we employ our recently developed bioorthogonal cleavage reaction as a general strategy for intracellular activation of individual kinases. Site-specific incorporation of trans-cyclooctene-caged lysine in place of the conserved catalytic lysine, in conjunction with the cleavage partner dimethyl-tetrazine, allowed efficient lysine decaging with the kinase activity chemically rescued in living systems.

HdeA and HdeB constitute the essential chaperone system that functions in the unique periplasmic space of Gram-negative enteric bacteria to confer acid resistance. How this two-chaperone machinery cooperates to protect a broad range of client proteins from acid denaturation while avoiding nonspecific binding during bacterial passage through the highly acidic human stomach remains unclear. We have developed a comparative proteomic strategy that combines the genetically encoded releasable protein photocross-linker with 2D difference gel electrophoresis, which allows an unbiased side-by-side comparison of the entire client pools from these two acid-activated chaperones in Escherichia coli. Our results reveal distinct client specificities between HdeA and HdeB in vivo that are determined mainly by their different responses to pH stimulus. The intracellular acidity serves as an environmental cue to determine the folding status of both chaperones and their clients, enabling specific chaperone–client binding and release under defined pH conditions. This cooperative and synergistic mode of action provides an efficient, economical, flexible, and finely tuned protein quality control strategy for coping with acid stress.

Coupling genetically encoded peptide tags or unnatural amino acids (UAAs) with bioorthogonal reactions allows for precise control over the protein-labeling sites as well as the wide choice of labeling dyes. However, the value of these site-specific protein labeling strategies in a real biology setting, particularly their advantages over conventional labeling methods including fluorescent proteins (FPs), remains to be fully demonstrated. In this tutorial review, we first introduce various strategies for site-specific protein labeling that utilize artificial peptide sequences or genetically encoded UAAs as the labeling handle. Emphasis will be placed on introducing the advantages of protein site-specific labeling techniques as well as their applications in solving biological problems, particularly as to why a site-specific protein labeling approach is needed. Finally, beyond the widely used single site-specific labeling methods, the recently emerged dual site-specific protein labeling strategies will be introduced together with their fast-growing potential in illustrating biological processes.

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.

Considerable attention has been focused on improving the biocompatibility of Cu(I)-catalyzed azide–alkyne cycloaddition (CuAAC), a hallmark of bioorthogonal reaction, in living cells. Besides creating copper-free versions of click chemistry such as strain promoted azide–alkyne cycloaddition (SPAAC), a central effort has also been made to develop various Cu(I) ligands that can prevent the cytotoxicity of Cu(I) ions while accelerating the CuAAC reaction. Meanwhile, additional transition metals such as palladium have been explored as alternative sources to promote a bioorthogonal conjugation reaction on cell surface, as well as within an intracellular environment. Furthermore, transition metal mediated chemical conversions beyond conjugation have also been utilized to manipulate protein activity within living systems. We highlight these emerging examples that significantly enriched our protein chemistry toolkit, which will likely expand our view on the definition and applications of bioorthogonal chemistry.

We have developed a genetically encoded, selenium-based cleavable photo-cross-linker that allows for the separation of bait and prey proteins after protein photo-cross-linking. We have further demonstrated the efficient capture of the in situ generated selenenic acid on the cleaved prey proteins. Our strategy involves tagging the selenenic acid with an alkyne-containing dimethoxyaniline molecule and subsequently labeling with an azide-bearing fluorophore or biotin probe. This cleavage-and-capture after protein photo-cross-linking strategy allows for the efficient capture of prey proteins that are readily accessible by two-dimensional gel-based proteomics and mass spectrometry analysis.

We achieved single-molecule imaging and tracking of the transforming growth factor type II receptor (TβRII) that was labeled by an organic dye via a genetically encoded unnatural amino acid (UAA) and the copper-free click chemistry. The stoichiometry, mobility and dimerization kinetics of individual TβRII molecules were determined.

Two water-soluble triscyclometalated organoiridium complexes, 1 and 2, with polar side chains that form nanoparticles emitting bright-red phosphorescence in water were synthesized. The optimal emitting properties are related to both the triscyclometalated structure and nanoparticle-forming ability in aqueous solution. Nonlinear optical properties are also observed with the nanoparticles. Because of their proper cellular uptake in addition to high emission brightness and effective two-photon absorbing ability, cell imaging can be achieved with nanoparticles of 2 bearing quaternary ammonium side chains at ultra-low effective concentrations using NIR incident light via the multiphoton excitation phosphorescence process.Keywords: multiphoton excitation phosphorescence; phosphorescence cell imaging; phosphorescent nanoparticles; triscyclometalated iridium; water solubility;

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.

We developed a photoactivatable firefly luciferase (pfLuc) whose activation can be controlled by light. A photocaged Lys analogue was site-specifically incorporated into fLuc to replace its key catalytic Lys residue, Lys529, rendering fLuc inactive until light-triggered removal of the caging group. This photoinduced gain of luminescence provides a facile approach for assessing the photolysis efficiency of this valuable photosensitive Lys analogue within the context of its carrier protein in vitro and in living cells. We further took advantage of the spatial and temporal activation feature of pfLuc for intracellular measurement of labile ATP levels without impairment of cellular physiology.

Copper represents one of the most important biological metal ions due to its role as a catalytic cofactor in a multitude of proteins. However, an excess of copper is highly toxic. Thus, copper is heavily regulated, and copper homeostasis is controlled by many metalloregulatory proteins in various organisms. Here we report a genetically encoded copper(I) probe capable of monitoring copper fluctuations inside living cells. We insert the copper regulatory protein Ace1 into a yellow fluorescent protein, which selectively binds copper(I) and generates improved copper(I) probes.

A ratiometric and reversible organic hydroperoxide (OHP) sensor, rOHSer, was developed with high sensitivity and selectivity for subcellular OHP visualization. Through targeting rOHSer to the nucleus, we demonstrated that high levels of D-glucose cause elevated OHP production in this compartment. Further utilization of rOHSer probe may allow more elucidation of unique roles of OHPs in diverse biological processes.

A gold-specific sensory protein GolS from Samonella gol regulon was incorporated into E. coli, which in conjunction with an engineered downstream red fluorescence protein allowed the highly sensitive and selective whole-cell detection of gold(III) ions by naked eyes. The putative gold-chaperone, GolB, in the gol regulon was next verified to be specific to gold(I) ions over other metal ions including copper(I). The subsequent display of GolB on E. coli cell surface permitted selective enrichment of gold ions from media containing various thiophilic metal ions. The cell surface-enriched gold(I) was further shown to be easily recovered and the gold-deprived bacteria were capable for re-usage. E. coli bacteria harboring these gold-specific elements from the gol regulon could be a valuable tool for visual detection and facile recycling of gold ions from environmental resources.

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.

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.

The multiple antibiotic resistance regulatory protein (MarR) binds to two promoter sites on the marO operator in Escherichia coli. Our study showed that more than one MarR dimer proteins bound to either of its two promoter sites (Site I and Site II), suggesting that MarR might form higher complexes than homodimers when bound to DNA inside E. coli cells. To further verify this hypothesis, we site-specifically incorporated a photocrosslinking probe at the interface between two MarR dimer proteins. Photolysis in living E. coli cells revealed a covalent linkage between the two interdimer subunits of MarR, suggesting that MarR forms dimer of dimers in vivo.

Proteins are the workhorses of the cell, playing crucial roles in virtually every biological process. The revolutionary ability to visualize and monitor proteins in living systems, which is largely the result of the development of green fluorescence protein (GFP) and its derivatives, has dramatically expanded our understanding of protein dynamics and function. Still, GFPs are ill suited in many circumstances; one major drawback is their relatively large size, which can significantly perturb the functions of the native proteins to which they are fused.To bridge this gap, scientists working at the chemistry–biology interface have developed methods to install bioorthogonal functional groups into proteins in living cells. The bioorthogonal group is, by definition, a non-native and nonperturbing chemical group. But more importantly, the installed bioorthogonal handle is able to react with a probe bearing a complementary functionality in a highly selective fashion and with the cell operating in its physiological state. Although extensive efforts have been directed toward the development of bioorthogonal chemical reactions, introducing chemical functionalities into proteins in living systems remains an ongoing challenge. In this Account, we survey recent progress in this area, focusing on a genetic code expansion approach.In nature, a cell uses posttranslational modifications to append the necessary functional groups into proteins that are beyond those contained in the canonical 20 amino acids. Taking lessons from nature, scientists have chosen or engineered certain enzymes to modify target proteins with chemical handles. Alternatively, one can use the cell’s translational machinery to genetically encode bioorthogonal functionalities, typically in the form of unnatural amino acids (UAAs), into proteins; this can be done in a residue-specific or a site-specific manner. For studying protein dynamics and function in living cells, site-specific modification by means of genetic code expansion is usually favored.A variety of UAAs bearing bioorthogonal groups as well as other functionalities have been genetically encoded into proteins of interest. Although this approach is well established in bacteria, tagging proteins in mammalian cells is challenging. A facile pyrrolysine-based system, which might potentially become the “one-stop shop” for protein modification in both prokaryotic and eukaryotic cells, has recently emerged. This technology can effectively introduce a series of bioorthogonal handles into proteins in mammalian cells for subsequent chemical conjugation with small-molecule probes. Moreover, the method may provide more precise protein labeling than GFP tagging. These advancements build the foundation for studying more complex cellular processes, such as the dynamics of important receptors on living mammalian cell surfaces.

Enteric bacterial pathogens are known to effectively pass through the extremely acidic mammalian stomachs and cause infections in the small and/or large intestine of human hosts. However, their acid-survival strategy and pathogenesis mechanisms remain elusive, largely due to the lack of tools to directly monitor and manipulate essential components (e.g., defense proteins or invasive toxins) participating in these processes. Herein, we have extended the pyrrolysine-based genetic code expansion strategy for encoding unnatural amino acids in enteric bacterial species, including enteropathogenic Escherichia coli, Shigella, and Salmonella. Using this system, a photo-cross-linking amino acid was incorporated into a Shigella acid chaperone HdeA (shHdeA), which allowed the identification of a comprehensive list of in vivo client proteins that are protected by shHdeA upon acid stress. To further demonstrate the application of our strategy, an azide-bearing amino acid was introduced into a Shigella type 3 secretion effector, OspF, without interruption of its secretion efficiency. This site-specifically installed azide handle allowed the facile detection of OspF’s secretion in bacterial extracellular space. Taken together, these bioorthogonal functionalities we incorporated into enteric pathogens were shown to facilitate the investigation of unique and important proteins involved in the pathogenesis and stress-defense mechanisms of pathogenic bacteria that remain exceedingly difficult to study using conventional methodologies.

A concise route was developed for the facile synthesis of a cyclic pyrrolysine analogue bearing an azide handle. Directed evolution enabled the encoding of this non-natural amino acid in both prokaryotic and eukaryotic cells, which offers a highly efficient approach for the site-specific protein labeling using click chemistry.

The transcriptional regulatory protein OhrR is converted into a fluorescent bioprobe capable of detecting organic hydroperoxides in living cells with high sensitivity and selectivity.

We achieved single-molecule imaging and tracking of the transforming growth factor type II receptor (TβRII) that was labeled by an organic dye via a genetically encoded unnatural amino acid (UAA) and the copper-free click chemistry. The stoichiometry, mobility and dimerization kinetics of individual TβRII molecules were determined.

A concise route was developed for the facile synthesis of a cyclic pyrrolysine analogue bearing an azide handle. Directed evolution enabled the encoding of this non-natural amino acid in both prokaryotic and eukaryotic cells, which offers a highly efficient approach for the site-specific protein labeling using click chemistry.

A gold-specific sensory protein GolS from Samonella gol regulon was incorporated into E. coli, which in conjunction with an engineered downstream red fluorescence protein allowed the highly sensitive and selective whole-cell detection of gold(III) ions by naked eyes. The putative gold-chaperone, GolB, in the gol regulon was next verified to be specific to gold(I) ions over other metal ions including copper(I). The subsequent display of GolB on E. coli cell surface permitted selective enrichment of gold ions from media containing various thiophilic metal ions. The cell surface-enriched gold(I) was further shown to be easily recovered and the gold-deprived bacteria were capable for re-usage. E. coli bacteria harboring these gold-specific elements from the gol regulon could be a valuable tool for visual detection and facile recycling of gold ions from environmental resources.

Coupling genetically encoded peptide tags or unnatural amino acids (UAAs) with bioorthogonal reactions allows for precise control over the protein-labeling sites as well as the wide choice of labeling dyes. However, the value of these site-specific protein labeling strategies in a real biology setting, particularly their advantages over conventional labeling methods including fluorescent proteins (FPs), remains to be fully demonstrated. In this tutorial review, we first introduce various strategies for site-specific protein labeling that utilize artificial peptide sequences or genetically encoded UAAs as the labeling handle. Emphasis will be placed on introducing the advantages of protein site-specific labeling techniques as well as their applications in solving biological problems, particularly as to why a site-specific protein labeling approach is needed. Finally, beyond the widely used single site-specific labeling methods, the recently emerged dual site-specific protein labeling strategies will be introduced together with their fast-growing potential in illustrating biological processes.

Considerable attention has been focused on improving the biocompatibility of Cu(I)-catalyzed azide–alkyne cycloaddition (CuAAC), a hallmark of bioorthogonal reaction, in living cells. Besides creating copper-free versions of click chemistry such as strain promoted azide–alkyne cycloaddition (SPAAC), a central effort has also been made to develop various Cu(I) ligands that can prevent the cytotoxicity of Cu(I) ions while accelerating the CuAAC reaction. Meanwhile, additional transition metals such as palladium have been explored as alternative sources to promote a bioorthogonal conjugation reaction on cell surface, as well as within an intracellular environment. Furthermore, transition metal mediated chemical conversions beyond conjugation have also been utilized to manipulate protein activity within living systems. We highlight these emerging examples that significantly enriched our protein chemistry toolkit, which will likely expand our view on the definition and applications of bioorthogonal chemistry.

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.

The genetic code expansion strategy allowed incorporation of unnatural amino acids (UAAs) bearing diverse functional groups into proteins, providing a powerful toolkit for protein manipulation in living cells. We report a multifunctional UAA, Nε-p-azidobenzyloxycarbonyl lysine (PABK), that possesses a panel of unique properties capable of fulfilling various protein manipulation purposes. In addition to being used as a bioorthogonal ligation handle, an infrared probe and a photo-affinity reagent, PABK was shown to be chemically decaged by trans-cyclooctenols via a strain-promoted 1,3-dipolar cycloaddition, which provides a new bioorthogonal cleavage strategy for intracellular protein activation. The biocompatibility and efficiency of this method were demonstrated by decaging of a PABK-caged firefly luciferase under living conditions. We further extended this method to chemically rescue a bacterial toxin OspF inside mammalian host cells.