Photochemical fishing approaches for identifying target proteins and elucidating the structure of a ligand-binding region using carbene-generating photoreactive probes.

Photoaffinity labeling enables the direct probing of a target protein through a covalent bond between a ligand and its binding protein, and even a complex formed by weak interactions can be isolated by the method. The photochemical fishing approach accelerates the throughput, isolating crosslinked complexes and analyzing the structure of the ligand binding site within the protein. We used carbene-generating phenyldiazirine for this approach because practical examinations had shown that the phenyldiazirine functioned as the powerful barb on the hook. Improving the synthetic pathways of the photoprobes and using chemoselective-integrated photoreactive units makes possible the easy and rapid preparation of carbene-generating photoreactive probes including the derivatives in peptides, proteins, DNAs, and carbohydrates. This review also shows several recent impacts of photoaffinity labeling, including the in vivo preparation of photoreactive proteins in living cells.     

Recent Applications of Diazirines in Chemical Proteomics

The elucidation of substrate–protein interactions is an important component of the drug development process. Due to the complexity of native cellular environments, elucidating these fundamental biochemical interactions remains challenging. Photoaffinity labeling (PAL) is a versatile technique that can provide insight into ligand-target interactions. By judicious modification of substrates with a photoreactive group, PAL creates a covalent crosslink between a substrate and its biological target following UV-irradiation. Among the commonly employed photoreactive groups, diazirines have emerged as the gold standard. In this Minireview, recent developments in the field of diazirine-based photoaffinity labeling will be discussed, with emphasis being placed on their applications in chemical proteomic studies.     

Secretory protein profiling reveals TNF-α inactivation by selective and promiscuous Sec61 modulators

Cotransins are cyclic heptadepsipeptides that bind the Sec61 translocon to inhibit cotranslational translocation of a subset of secreted and type I transmembrane proteins. The few known cotransin-sensitive substrates are all targeted to the translocon by?a cleavable signal sequence, previously shown to be a critical determinant of cotransin sensitivity. By profiling two cotransin variants against a panel of secreted and transmembrane proteins, we demonstrate that cotransin side-chain differences profoundly affect substrate selectivity. Among the most sensitive substrates we identified is the proinflammatory cytokine tumor necrosis factor alpha (TNF-α). Like all type II transmembrane proteins, TNF-α is targeted to the translocon by its membrane-spanning domain, indicating that a cleavable signal sequence is not strictly required for cotransin sensitivity. Our results thus reveal an unanticipated breadth of translocon substrates whose expression is inhibited by Sec61 modulators.     

Recent Trends in Photoaffinity Labeling

Investigation of receptor—ligand interactions remains an inexhaustible challenge for chemists and biologists. Structural exploration of biological receptors is the starting point for a better understanding of how they function. Photoaffinity labeling is a biochemical approach to identify and characterize receptors targeting further structural investigations. The primary structure of a receptor protein was typically obtained by reverse genetics after exhaustive purification and sequencing of the N-terminal peptide, which allowed the design of the corresponding oligonucleotide probes. Synthesis of these oligonucleotide probes then led to identification of cDNA clones by hybridization. Following this strategy, several membrane neurotransmitter receptors and constituent polypeptides, present in very small quantities in the central nervous system, were identified and their sequence deduced from the cDNA sequence. Since photoaffinity labeling implies the formation of a covalent bond between a radiolabeled ligand analogue and a receptor binding site, it becomes theoretically possible to isolate and sequence radiolabeled peptides and then synthesize the corresponding oligonucleotide probes. Photoaffinity labeling might avoid the critical solubilization and purification steps of the classical approach. To our knowledge, no such example of primary structure determination based on photoaffinity labeling experiments has been reported. However, the extraordinary developments in gene cloning technologies, in particular homology cloning and expression cloning, have made this approach obsolete and raised the question of new perspectives for photoaffinity labeling technology. In this article we present an update on selected original developments, as well as new challenges for this method. Photoaffinity labeling not only gives access to structural elements but is also a potential tool for the investigation of functional aspects of biological receptors, for example their role in signal transduction mechanisms.     

A biosynthetic route to photoclick chemistry on proteins

Light-induced chemical reactions exist in nature, regulating many important cellular and organismal functions, e.g., photosensing in prokaryotes and vision formation in mammals. Here, we report the genetic incorporation of a photoreactive unnatural amino acid, p-(2-tetrazole)phenylalanine (p-Tpa), into myoglobin site-specifically in E. coli by evolving an orthogonal tRNA/aminoacyl-tRNA synthetase pair and the use of p-Tpa as a bioorthogonal chemical "handle" for fluorescent labeling of p-Tpa-encoded myoglobin via the photoclick reaction. Moreover, we elucidated the structural basis for the biosynthetic incorporation of p-Tpa into proteins by solving the X-ray structure of p-Tpa-specific aminoacyl-tRNA synthetase in complex with p-Tpa. The genetic encoding of this photoreactive amino acid should make it possible in the future to photoregulate protein function in living systems.     

A Designed Inhibitor of a CLC Antiporter Blocks Function through a Unique Binding Mode

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Highlights? OADS is a known small-molecule inhibitor of a CLC antiporter ? OADS specifically inhibits the ClC-ec1 antiporter but not the ClC-1 channel ? Photoaffinity labeling and mass spectrometry have localized OADS binding to two discrete sites ? The unique binding mode and lipid dependence of OADS suggest potential mechanisms of action     

In Vitro Synthesis of the E. coli Sec Translocon from DNA

Difficulties in constructing complex lipid/protein membranes have severely limited the development of functional artificial cells endowed with vital membrane‐related functions. The Sec translocon membrane channel, which mediates the insertion of membrane proteins into the plasma membrane, was constructed in the membrane of lipid vesicles through in vitro expression of its component proteins. The components of the Sec translocon were synthesized from their respective genes in the presence of liposomes, thereby bringing about a functional complex. The synthesized E. coli Sec translocon mediated the membrane translocation of single‐ and multi‐span membrane proteins. The successful translocation of a functional peptidase into the liposome lumen further confirmed the proper insertion of the translocon complex. Our results demonstrate the feasible construction of artificial cells, the membranes of which can be functionalized by directly decoding genetic information into membrane functions.     

Native FKBP12 engineering by ligand-directed tosyl chemistry: labeling properties and application to photo-cross-linking of protein complexes in vitro and in living cells

The ability to modify target "native" (endogenous) proteins selectively in living cells with synthetic molecules should provide powerful tools for chemical biology. To this end, we recently developed a novel protein labeling technique termed ligand-directed tosyl (LDT) chemistry. This method uses labeling reagents in which a protein ligand and a synthetic probe are connected by a tosylate ester group. We previously demonstrated its applicability to the selective chemical labeling of several native proteins in living cells and mice. However, many fundamental features of this chemistry remain to be studied. In this work, we investigated the relationship between the LDT reagent structure and labeling properties by using native FK506-binding protein 12 (FKBP12) as a target protein. In vitro experiments revealed that the length and rigidity of the spacer structure linking the protein ligand and the tosylate group have significant effects on the overall labeling yield and labeling site. In addition to histidine, which we reported previously, tyrosine and glutamate residues were identified as amino acids that are modified by LDT-mediated labeling. Through the screening of various spacer structures, piperazine was found to be optimal for FKBP12 labeling in terms of labeling efficiency and site specificity. Using a piperazine-based LDT reagent containing a photoreactive probe, we successfully demonstrated the labeling and UV-induced covalent cross-linking of FKBP12 and its interacting proteins in vitro and in living cells. This study not only furthers our understanding of the basic reaction properties of LDT chemistry but also extends the applicability of this method to the investigation of biological processes in mammalian cells.     

Optimization of activity-based probes for proteomic profiling of histone deacetylase complexes

Histone deacetylases (HDACs) are key enzymatic regulators of the epigenome and serve as promising targets for anticancer therapeutics. Recently, we developed a photoreactive "clickable" probe, SAHA-BPyne, to report on HDAC activity and complex formation in native biological systems. Here, we investigate the selectivity, sensitivity, and inhibitory properties of SAHA-BPyne and related potential activity-based probes for HDACs. While we identified several probes that are potent HDAC inhibitors and label HDAC complex components in native proteomic preparations, SAHA-BPyne was markedly superior for profiling HDAC activities in live cells. Interestingly, the enhanced performance of SAHA-BPyne as an in situ activity-based probe could not be solely ascribed to potency in HDAC binding, implying that other features of the molecule were key to efficient active site-directed labeling in living systems. Finally, we demonstrate the value of in situ profiling of HDACs by comparing the activity and expression of HDAC1 in cancer cells treated with the cytotoxic agent parthenolide. These results underscore the utility of activity-based protein profiling for studying HDAC function and may provide insight for the future development of click chemistry-based photoreactive probes for the in situ analysis of additional enzyme activities.     

Synthesis of AX7593, a quinazoline-derived photoaffinity probe for EGFR

[structure: see text] The synthesis of a photoaffinity probe for EGFR is described. O-Alkylation of 4-(meta-azidoanilino)-6-methoxy-7-hydroxy-quinazoline with a protected tetraethyleneglycol linker followed by the attachment of tetramethylrhodamine yielded the fluorescent probe AX7593. Photoaffinity labeling of EGFR by AX7593 (K(b) = 280 nM) was shown to have an efficiency of 34% and to be competitive with the EGFR inhibitors PP2 and AG1478.     

Photochemical Labeling: Can Photoaffinity Labeling be Differentiated from Site-Directed Photochemical Coupling?

Site-directed photochemical labeling is a methodology designed to irreversibly and specifically label, through the action of light, a ligand binding site of a biological mac-romolecule. Photoaffinity labeling, a widely used site-directed labeling methodology, uses photosensitive ligand analogs generally obtained after chemical modification of the ligand by introducing an appropriate photoactivata-ble moiety. This methodology can be applied to natural ligands showing inherent photosensitivity, without any additional modification, and which can be linked efficiently to their receptor binding site by direct photoac-tivation. The emergence of an alternative methodology that links nonphotosensitive ligands to their receptors has raised the question of their potential use and their mechanisms of photocoupling. This article presents a series of examples that are meant to compare the general characteristics of the different site-directed labeling reactions and proposes distinct photochemical activation processes between photoaffinity labeling and site-directed photochemical coupling reactions. We suggest in particular that the former is necessarily a ligand-mediated activation process while the latter might involve a receptor-mediated mechanism.     

一种含光亲和标记异戊烯侧链探针的合成与初步表征

经5步反应、以9%的总收率合成了一种含生物素修饰和光亲和标记的异戊烯侧链功能探针分子, 初步考察了反应条件下该探针分子与Saccharomyces cerevisiae AS 2.399粗蛋白的相互作用; 生物素印迹分析结果表明, 酵母中多种蛋白被探针分子修饰, 为进一步开展化学蛋白组学研究奠定了基础.     

Novel bifunctional probe for radioisotope-free photoaffinity labeling: compact structure comprised of photospecific ligand ligation and detectable tag anchoring units

A novel method for radioisotope-free photoaffinity labeling was developed, in which a bifunctional ligand is connected to a target protein by activation of a photoreactive group, such as an aromatic azido or 3-trifluoromethyl-3H-diazirin-3-yl group, and identification of the ligated product is achieved by anchoring of a detectable tag through the Staudinger-Bertozzi reaction with an alkyl azido moiety that survives photolysis. The chemical ground of this method was confirmed using model compounds with the bifunctional group under photoirradiation in the presence of trapping agents for reactive intermediates. The utility of the method has been demonstrated by specific labeling of the catalytic portion of human HMG-CoA reductase.     

Photochemical patterning of a self-assembled monolayer of 7-diazomethylcarbonyl-2,4,9-trithiaadmantane on gold films via Wolff rearrangement

Photolithographic attachment of functional organic molecules via ester or amide linkages to self-assembled monolayers (SAMs) on gold thin films was achieved by employing a novel photoreactive surface anchor, 7-diazomethylcarbonyl-2,4,9-trithiaadmantane. The photoreactive SAM was prepared by the spontaneous physical adsorption of the photoreactive surface anchor onto gold surfaces. The alpha-diazo ketone moiety of the SAM was found to display the classical Wolff rearrangement reactivity to produce a ketene intermediate on the exposed area. Organic molecules such as alcohols and amines can thus be attached to the gold surfaces selectively by the facile in situ formation of ester or amide linkages. The structure and reactivity of the photoreactive surface anchor were characterized by real-time FT-IR, fluorescence, and polarization modulation infrared reflectance absorption spectroscopy (PM-IRRAS). The Wolff rearrangement reactivity of the SAM suggested that a "surface-isolated" carbonylcarbene may be generated when the SAM was exposed to 255-nm irradiation.     

Analysis of transmembrane dynamics of cholera toxin using photoreactive probes.

Using sodium dodecyl sulfate--polyacrylamide gel electrophoresis and autoradiography, we have shown that 125I-labeled cholera toxin binds to Newcastle disease virus. Pretreatment of Newcastle disease virus with "cold" cholera toxin (at 37 degrees C for 30 minutes) inhibits the binding of 125I-labeled toxin in a subsequent incubation (at 37 degrees C for 30 minutes). These results suggest that cholera toxin binds to Newcastle disease virus in a specific manner. The precise receptor for toxin is unknown in Newcastle disease virus but it is presumed to be the ganglioside GM1. We have previously shown that the photoreactive probe 12-(4-azido-2-nitrophenoxy)stearoylglucosamine[1-14C] labels the membrane proteins of Newcastle disease virus. Since the reactive group of the probe, ie, N3, resides within the membrane bilayer, studies were initiated to determined which, if any, of the subunits of cholera toxin cross the membrane of Newcastle disease virus and become radioactively labeled upon photoactivation of the probe at 360 nm. After a 15-minute incubation of cholera toxin with Newcastle disease virus containing the photoreactive probe, irradiation effected the 14C-labeling of the active A1 subunit of cholera toxin. Irradiation of cholera toxin in solution with an equivalent amount of probe but without virus resulted in no labeling of toxin subunits.     

Cross-linking chemistry and biology: development of multifunctional photoaffinity probes

An efficient method of photoaffinity labeling has been developed based on rationally designed multifunctional photoprobes. Photoaffinity techniques have been used to elucidate the protein structure at the interface of biomolecules by the photochemical labeling of interacting sites. However, the identification of labeled sites within target proteins is often difficult. Novel biotinyl bioprobes bearing a diazirine photophore have contributed significantly to the rapid elucidation of ligand binding sites within proteins, thereby extending conventional photoaffinity methods. This article discusses the synthesis and applications of various photoprobes bearing a biotin, including strategies using cleavable linkages between photophores. The combination of photoaffinity methods with chip technology is also described as a novel entry to rapid affinity-based screening of inhibitors. This review focuses on a rapid and reliable photoaffinity method utilizing diazirine-based multifunctional photoprobes with numerous potential applications in functional proteomics of biomolecular interactions.     

THE USE OF BENZOPHENONE AS A PHOTOAFFINITY LABEL, LABELING IN p-BENZOYLPHENYLACETYL CHYMOTRYPSIN AT UNIT EFFICIENCY

Abstract— p -Benzoylphenylacetyl chymotrypsin, an acyl enzyme derivative containing the benzophenone group in the hydrophobic binding pocket, was prepared and is indefinitely stable at low pH. Photolysis of this covalent derivative leads to loss of enzymic activity and incorporation of the labeling group via formation of a second covalent bond. The efficiency of the photochemical processes is exceptionally high, producing 100% incorporation and at least 92% inactivation. Analysis of active site titration data for the photolyzed enzyme show that at least two different photochemical processes must be involved. Elimination of phosphorescence emission and reduction of UV absorption upon photolysis are consistent with initial hydrogen abstraction by benzophenone triplet state, followed by radical coupling, much as has been observed for the photoreaction of benzophenone with model systems. Photoaffinity labeling of chymotrypsin is also efficiently accomplished using two benzophenone derivatives which bind noncovalently to the enzyme's active site, although the rates of labeling are somewhat less than in the covalent complex.     

Fluorogenic Photo-Crosslinking of Glycan-Binding Protein Recognition Using a Fluorinated Azido-Coumarin Fucoside

Glycan recognition by glycan-binding proteins is central to the biology of all living organisms. The efficient capture and characterization of relatively weak non-covalent interactions remains an important challenge in various fields of research. Photoaffinity labeling strategies can create covalent bonds between interacting partners, and photoactive scaffolds such as benzophenone, diazirines and aryl azides have proved widely useful. Since their first introduction, relatively few improvements have been advanced and products of photoaffinity labeling remain difficult to detect. We report a fluorinated azido-coumarin scaffold which enables photolabeling under fast and mild activation, and which can leave a fluorescent tag on crosslinked species. Coupling this scaffold to an α-fucoside, we demonstrate fluorogenic photolabeling of glycan-protein interactions over a wide range of affinities. We expect this strategy to be broadly applicable to other chromophores and we envision that such “fluoro-crosslinkers” could become important tools for the traceable capture of non-covalent binding events.     

Facile Recoding of Selenocysteine in Nature

Selenocysteine (Sec or U) is encoded by UGA, a stop codon reassigned by a Sec‐specific elongation factor and a distinctive RNA structure. To discover possible code variations in extant organisms we analyzed 6.4 trillion base pairs of metagenomic sequences and 24 903 microbial genomes for tRNA^Sec species. As expected, UGA is the predominant Sec codon in use. We also found tRNA^Sec species that recognize the stop codons UAG and UAA, and ten sense codons. Selenoprotein synthesis programmed by UAG in Geodermatophilus and Blastococcus, and by the Cys codon UGU in Aeromonas salmonicida was confirmed by metabolic labeling with ⁷⁵Se or mass spectrometry. Other tRNA^Sec species with different anticodons enabled E. coli to synthesize active formate dehydrogenase H, a selenoenzyme. This illustrates the ease by which the genetic code may evolve new coding schemes, possibly aiding organisms to adapt to changing environments, and show the genetic code is much more flexible than previously thought.     

Recent bioorganic studies on rhodopsin and visual transduction

Rhodopsin, the pigment responsible for vision in animals, insect and fish is a typical G protein (guanyl-nucleotide binding protein) consisting of seven transmembrane alpha helices and their interconnecting extramembrane loops. In the case of bovine rhodopsin, the best studied of the visual pigments, the chromophore is 11-cis retinal attached to the terminal amino group of Lys296 through a protonated Schiff base linkage. Photoaffinity labeling with a 3-diazo-4-oxo-retinoid shows that C-3 of the ionone ring moiety is close to Trp265 in helix F (VI) in dark inactivated rhodopsin. Irradiation causes a cis to trans isomerization of the 11-cis double bond giving rise to the highly strained intermediate bathorhodopsin. This undergoes a series of thermal relaxation through lumi-, meta-I and meta-II intermediates after which the retinal chromophore is expelled from the opsin binding pocket. Photoaffinity labeling performed with 3-diazo-4-oxoretinal at -196 degrees C for batho-, -80 degrees C for lumi-, -40 degrees C for meta-I, and 0 degrees C for meta-II rhodopsin showed that in bathorhodopsin the ring is still close to Trp265. However, in lumi-, meta-I and meta-II intermediates crosslinking occurs unexpectedly at A169 in helix D (IV). This shows that large movements in the helical arrangements and a flip over of the ring moiety accompanies the transduction (or bleaching) process. These changes in retinal/opsin interactions are necessarily accompanied by movements of the extramembrane loops, which in turn lead to activation of the G protein residing in the cytoplasmic side. Of the numerous G protein coupled receptors, this is the first time that the outline of transduction pathway has been clarified.