Affinity‐Labeling‐Based Introduction of a Reactive Handle for Natural Protein Modification

A new chemical method to site‐specifically modify natural proteins without the need for genetic manipulation is described. Our strategy involves the affinity‐labeling‐based attachment of a unique reactive handle at the surface of the target protein, and the subsequent selective transformation of the reactive handle by a bioorthogonal reaction to introduce a variety of functional probes into the protein. To demonstrate this approach, we synthesized labeling reagents that contain: 1) a benzenesulfonamide ligand that directs specifically to bovine carbonic anhydrase II (bCA), 2) an electrophilic epoxide group for protein labeling, 3) an exchangeable hydrazone bond linking the ligand and the epoxide group, and 4) an iodophenyl or acetylene handle. By incubating the labeling reagent with bCA, the reactive handle was covalently attached at the surface of bCA through epoxide ring opening. Either after or before removing the ligand by a hydrazone/oxime‐exhange reaction, which restores the enzymatic activity, the reactive handle incorporated could be derivatized by Suzuki coupling or Huisgen cycloaddition reactions. This method is also applicable to the target‐specific multiple modification in a protein mixture. The availability of various (photo)affinity‐labeling reagents and bioorthogonal reactions should extend the flexibility of this strategy for the site‐selective incorporation of many functional molecules into proteins.     

One-pot and sequential organic chemistry on an enzyme surface to tether a fluorescent probe at the proximity of the active site with restoring enzyme activity

A new and simple method to tether a functional molecule at the proximity of the active site of an enzyme has been successfully developed without any activity loss. The one-pot sequential reaction was conducted on a surface of human carbonic anhydrase II (hCAII) based on the affinity labeling and the subsequent hydrazone/oxime exchange reaction. The reaction proceeds in a greater than 90% yield in the overall steps under mild conditions. The enzymatic activity assay demonstrated that the release of the affinity ligand from the active site of hCAII concurrently occurred with the replacement by the aminooxy derivatives, so that it restored the enzymatic activity from the completely suppressed state of the labeled hCAII. Such restoring of the activity upon the sequential modification is quite unique compared to conventional affinity labeling methods. The peptide mapping experiment revealed that the labeling reaction was selectively directed to His-3 or His-4, located on a protein surface proximal to the active site. When the fluorescent probe was tethered using the present sequential chemistry, the engineered hCAII can act as a fluorescent biosensor toward the hCAII inhibitors. This clearly indicates the two advantages of this method, that is (i) the modification is directed to the proximity of the active site and (ii) the sequential reaction re-opens the active site cavity of the target enzyme.     

Development of small molecule inhibitors and probes of human SUMO deconjugating proteases

Sentrin specific proteases (SENPs) are responsible for activating and deconjugating SUMO (Small Ubiquitin like MOdifier) from target proteins. It remains difficult to study this posttranslational modification due to the lack of reagents that can be used to block the removal of SUMO from substrates. Here, we describe the identification of small molecule SENP inhibitors and active site probes containing aza-epoxide and acyloxymethyl ketone (AOMK) reactive groups. Both classes of compounds are effective inhibitors of hSENPs 1, 2, 5, and 7 while only the AOMKs efficiently inhibit hSENP6. Unlike previous reported peptide vinyl sulfones, these compounds covalently labeled the active site cysteine of multiple recombinantly expressed SENP proteases and the AOMK probe showed selective labeling of these SENPs when added to complex protein mixtures. The AOMK compound therefore represents promising new reagents to study the process of SUMO deconjugation.     

Identification of Histone deacetylase (HDAC)‐Associated Proteins with DNA‐Programmed Affinity Labeling

Histone deacetylase (HDAC) is a major class of deacetylation enzymes. Many HDACs exist in large protein complexes in cells and their functions strongly depend on the complex composition. The identification of HDAC‐associated proteins is highly important in understanding their molecular mechanisms. Although affinity probes have been developed to study HDACs, they were mostly targeting the direct binder HDAC, while other proteins in the complex remain underexplored. We report a DNA‐based affinity labeling method capable of presenting different probe configurations without the need for preparing multiple probes. Using one binding probe, 9 probe configurations were created to profile HDAC complexes. Notably, this method identified indirect HDAC binders that may be inaccessible to traditional affinity probes, and it also revealed new biological implications for HDAC‐associated proteins. This study provided a simple and broadly applicable method for characterizing protein‐protein interactions.     

Identification of Histone deacetylase (HDAC)-Associated Proteins with DNA-Programmed Affinity Labeling

Histone deacetylase (HDAC) is a major class of deacetylation enzymes. Many HDACs exist in large protein complexes in cells and their functions strongly depend on the complex composition. The identification of HDAC-associated proteins is highly important in understanding their molecular mechanisms. Although affinity probes have been developed to study HDACs, they were mostly targeting the direct binder HDAC, while other proteins in the complex remain underexplored. We report a DNA-based affinity labeling method capable of presenting different probe configurations without the need for preparing multiple probes. Using one binding probe, 9 probe configurations were created to profile HDAC complexes. Notably, this method identified indirect HDAC binders that may be inaccessible to traditional affinity probes, and it also revealed new biological implications for HDAC-associated proteins. This study provided a simple and broadly applicable method for characterizing protein-protein interactions.     

Oligo-Asp tag/Zn(II) complex probe as a new pair for labeling and fluorescence imaging of proteins

To accomplish the selective labeling of a specific protein in complicated biological systems, a peptide tag incorporated into the protein and a complementary small molecular probe are required. Although a variety of peptide tag/probe pairs have been developed as molecular tools for protein analyses, the availability of pairs suitable for real-time imaging of proteins is still limited. We now report a new peptide tag/artificial probe pair composed of a genetically encodable oligo-aspartate sequence (D4 tag, (D4)n, n = 1-3) and the corresponding multinuclear Zn(II) complexes (Zn(II)-DpaTyrs). The strong binding affinity of the Zn(II)-DpaTyr probes with the D4 tag was a result of the multiple coordination bonds and the multivalent effect. It was measured quantitatively by isothermal titration calorimetry. The high affinity between the tag and the probe, indispensable for the selective protein labeling, enabled the pair to be used for the labeling and fluorescence imaging of a membrane-bound receptor protein tethering a triply repeated D4 tag ((D4)3) in an intact cell configuration without significantly affecting the receptor signal transduction.     

Photo-affinity labeling strategies in identifying the protein ligands of bioactive small molecules: examples of targeted synthesis of drug analog photoprobes

The discovery of many new targets by chemical genetics has frequently exploited the fact that their biologically active chemical ligands were reactive and thus could covalently bind to their protein target(s). When experimental compounds or therapeutic agents with unidentified mechanisms of action do not contain reactive groups that can covalently label the putative site of molecular action, it may be possible to create a reactive photo-affinity probe if there is sufficient knowledge of the structure-activity relationship of the chemical series. Two specific examples are presented. These include the use of photo-affinity probes in the identification of the mechanism of action of synthetic oxazolidinones, a class of novel acting antibiotics and in the identification of a novel target for the insulin-sensitizing thiazolidinediones. Developments in photo-affinity labeling and combinatorial library design now imply that the parallel incorporation of photo-probes into screening library design could, at least in principle, greatly facilitate reverse pharmacological and chemical genetics approaches to protein target discovery.     

Cleavable biotin probes for labeling of biomolecules via azide-alkyne cycloaddition

The azide-alkyne cycloaddition provides a powerful tool for bio-orthogonal labeling of proteins, nucleic acids, glycans, and lipids. In some labeling experiments, e.g., in proteomic studies involving affinity purification and mass spectrometry, it is convenient to use cleavable probes that allow release of labeled biomolecules under mild conditions. Five cleavable biotin probes are described for use in labeling of proteins and other biomolecules via azide-alkyne cycloaddition. Subsequent to conjugation with metabolically labeled protein, these probes are subject to cleavage with either 50 mM Na(2)S(2)O(4), 2% HOCH(2)CH(2)SH, 10% HCO(2)H, 95% CF(3)CO(2)H, or irradiation at 365 nm. Most strikingly, a probe constructed around a dialkoxydiphenylsilane (DADPS) linker was found to be cleaved efficiently when treated with 10% HCO(2)H for 0.5 h. A model green fluorescent protein was used to demonstrate that the DADPS probe undergoes highly selective conjugation and leaves a small (143 Da) mass tag on the labeled protein after cleavage. These features make the DADPS probe especially attractive for use in biomolecular labeling and proteomic studies.     

Improved Stabilities of Labeling Probes for the Selective Modification of Endogenous Proteins in Living Cells and In Vivo

To date, various affinity-based protein labeling probes have been developed and applied in biological research to modify endogenous proteins in cell lysates and on the cell surface. However, the reactive groups on the labeling probes are also the cause of probe instability and nonselective labeling in a more complex environment, e. g., intracellular and in vivo. Here, we show that labeling probes composed of a sterically stabilized difluorophenyl pivalate can achieve efficient and selective labeling of endogenous proteins on the cell surface, inside living cells and in vivo. As compared with the existing protein labeling probes, probes with the difluorophenyl pivalate exhibit several advantages, including long-term stability in stock solutions, resistance to enzymatic hydrolysis and can be customized easily with diverse fluorophores and protein ligands. With this probe design, endogenous hypoxia biomarker in living cells and nude mice were successfully labeled and validated by in vivo, ex vivo, and immunohistochemistry imaging.     

活性导向的化学探针在氨基酸反应活性表征中的应用进展

原创药物的研制得益于蛋白质新靶标的发现,而新靶标的发现依赖于高可信度、高通量的药物-蛋白质相互作用分析方法。蛋白质作为生命功能的执行者,其表达量、空间定位与结构差异直接影响药效的发挥。目前,超过85%的蛋白质尚被认为是无法成药的,主要原因是缺少药物分子靶向的空腔以及相应的反应活性位点。因此,基于蛋白质组学层次实现对氨基酸反应活性位点的表征成为原创共价靶向药物设计的关键,也是克服难以成药靶标蛋白问题的关键。近年来,质谱技术的飞速发展极大地推动了基于蛋白质组学技术的药物-靶蛋白相互作用研究。其中基于活性的蛋白质组分析(ABPP)策略是利用活性位点导向的化学探针分子在复杂样品中实现功能状态酶和药物靶标等蛋白质的检测。基于化学探针的开发和质谱定量技术的发展,ABPP技术在氨基酸反应活性表征研究中展现出重要的应用潜力,将助力于药物新靶标的发现和药物先导化合物的开发。ABPP策略主要基于蛋白质的活性特征进行富集,活性探针作为ABPP策略的核心,近年来取得了飞速进展。该文回顾了ABPP策略的发展历程,重点介绍基于广谱活性探针的ABPP技术在多种氨基酸反应活性筛选领域的研究进展,并对其在药物靶点发现中...     

Synthesis and characterization of a 'fluorous' (fluorinated alkyl) affinity reagent that labels primary amine groups in proteins/peptides

Strong non-covalent interactions such as biotin-avidin affinity play critical roles in protein/peptide purification. A new type of 'fluorous' (fluorinated alkyl) affinity approach has gained popularity due especially to its low level of non-specific binding to proteins/peptides. We have developed a novel water-soluble fluorous labeling reagent that is reactive (via an active sulfo-N-hydroxylsuccinimidyl ester group) to primary amine groups in proteins/peptides. After fluorous affinity purification, the bulky fluorous tag moiety and the long oligoethylene glycol (OEG) spacer of this labeling reagent can be trimmed via the cleavage of an acid labile linker. Upon collision-induced dissociation, the labeled peptide ion yields a characteristic fragment that can be retrieved from the residual portion of the fluorous affinity tag, and this fragment ion can serve as a marker to indicate that the relevant peptide has been successfully labeled. As a proof of principle, the newly synthesized fluorous labeling reagent was evaluated for peptide/protein labeling ability in phosphate-buffered saline (PBS). Results show that both the aqueous environment protein/peptide labeling and the affinity enrichment/separation process were highly efficient.     

Mapping enzyme active sites in complex proteomes

Genome sequencing projects have uncovered many novel enzymes and enzyme classes for which knowledge of active site structure and mechanism is limited. To facilitate mechanistic investigations of the numerous enzymes encoded by prokaryotic and eukaryotic genomes, new methods are needed to analyze enzyme function in samples of high biocomplexity. Here, we describe a general strategy for profiling enzyme active sites in whole proteomes that utilizes activity-based chemical probes coupled with a gel-free analysis platform. We apply this gel-free strategy to identify the sites of labeling on enzymes targeted by sulfonate ester probes. For each enzyme examined, probe labeling was found to occur on a conserved active site residue, including catalytic nucleophiles (e.g., C32 in glutathione S-transferase omega) and bases/acids (e.g., E269 in aldehyde dehydrogenase-1; D204 in enoyl CoA hydratase-1), as well as residues of unknown function (e.g., D127 in 3 beta-hydroxysteroid dehydrogenase/isomerase-1). These results reveal that sulfonate ester probes are remarkably versatile activity-based profiling reagents capable of labeling a diversity of catalytic residues in a range of mechanistically distinct enzymes. More generally, the gel-free strategy described herein, by consolidating into a single step the identification of both protein targets of activity-based probes and the specific residues labeled by these reagents, provides a novel platform in which the proteomic comparison of enzymes can be accomplished in unison with a mechanistic analysis of their active sites.     

Intracellular protein labeling with prodrug-like probes using a mutant β-lactamase tag

Intracellular protein labeling with small molecular probes that do not require a washing step for the removal of excess probe is greatly desired for real-time investigation of protein dynamics in living cells. Successful labeling of proteins on the cell membrane has been performed using mutant β-lactamase tag (BL-tag) technology. In the present study, intracellular protein labeling with novel cell membrane permeable probes based on β-lactam prodrugs is described. The prodrug-based probes quickly permeated the plasma membranes of living mammalian cells, and efficiently labeled intracellular proteins at low probe concentrations. Because these cell-permeable probes were activated only inside cells, simultaneous discriminative labeling of intracellular and cell surface BL-tag fusion proteins was attained by using cell-permeable and impermeable probes. Thus, this technology enables adequate discrimination of the location of proteins labeled with the same protein tag, in conjunction with different color probes, by dual-color fluorescence. Moreover, the combination of BL-tag technology and the prodrug-based probes enabled the labeling of target proteins without requiring a washing step, owing to the efficient entry of probes into cells and the fast covalent labeling achieved with BL-tag technology after bioactivation. This prodrug-based probe design strategy for BL-tags provides a simple experimental procedure with application to cellular studies with the additional advantage of reduced stress to living cells.     

Site-specific protein double labeling by expressed protein ligation: applications to repeat proteins

In the last few years, the use of labeled proteins has significantly expanded in the life sciences. Now, labeled proteins are indispensable tools for a wide spectrum of biophysical and chemical biology applications. In particular, the quest for more sophisticated experimental setups requires the development of new synthetic methodology, especially for multiple site-specific labeling. In this paper, we describe a synthetic strategy based on expressed protein ligation to prepare proteins in high purity and homogeneity, in which two different molecular probes are incorporated specifically at any desired position. Proteins are sequentially labeled in solution, with the advantage that a large excess of probes is not required and the labeled fragments are not restricted to peptide synthesis length limitations. This strategy was applied to selectively label a repeat protein with a fluorophores pair in different positions along the protein sequence. The doubly labeled proteins were prepared at high purity and homogeneity, as required for single molecule FRET studies. Remarkably, this approach can be adapted to the introduction of more than two molecular probes.     

Fast and efficient MCR-based synthesis of clickable rhodamine tags for protein profiling

Protein profiling probes are important tools for studying the composition of the proteome and as such have contributed greatly to the understanding of various complex biological processes in higher organisms. For this purpose the application of fluorescently labeled activity or affinity probes is highly desirable. Especially for in vivo detection of low abundant target proteins, otherwise difficult to analyse by standard blotting techniques, fluorescently labeled profiling probes are of high value. Here, a one-pot protocol for the synthesis of activated fluorescent labels (i.e. azide, alkynyl or NHS), based on the Ugi-4-component reaction (Ugi-4CR), is presented. As a result of the peptoidic structure formed, the fluorescent properties of the products are pH insensitive. Moreover, the applicability of these probes, as exemplified by the labeling of model protein BSA, will be discussed.     

Probing enzymatic activity – a radical approach

Deubiquitinating enzymes (DUBs) are known to have numerous important interactions with the ubiquitin cascade and their dysregulation is associated with several diseases, including cancer and neurodegeneration. They are an important class of enzyme, and activity-based probes have been developed as an effective strategy to study them. Existing activity-based probes that target the active site of these enzymes work via nucleophilic mechanisms. We present the development of latent ubiquitin-based probes that target DUBs via a site selective, photoinitiated radical mechanism. This approach differs from existing photocrosslinking probes as it requires a free active site cysteine. In contrast to existing cysteine reactive probes, control over the timing of the enzyme–probe reaction is possible as the alkene warhead is completely inert under ambient conditions, even upon probe binding. The probe''s reactivity has been demonstrated against recombinant DUBs and to capture endogenous DUB activity in cell lysate. This allows more finely resolved investigations of DUBs.

Latent activity-based probes have been developed for deubiquitinating enzymes using a thiol–ene strategy, labelling following a specific binding interaction.     

Chemoselective and Site‐Selective Lysine‐Directed Lysine Modification Enables Single‐Site Labeling of Native Proteins

The necessity for precision labeling of proteins emerged during the efforts to understand and regulate their structure and function. It demands selective attachment of tags such as affinity probes, fluorophores, and potent cytotoxins. Here, we report a method that enables single‐site labeling of a high‐frequency Lys residue in the native proteins. At first, the enabling reagent forms stabilized imines with multiple solvent‐accessible Lys residues chemoselectively. These linchpins create the opportunity to regulate the position of a second Lys‐selective electrophile connected by a spacer. Consequently, it enables the irreversible single‐site labeling of a Lys residue independent of its place in the reactivity order. The user‐friendly protocol involves a series of steps to deconvolute and address chemoselectivity, site‐selectivity, and modularity. Also, it delivers ordered immobilization and analytically pure probe‐tagged proteins. Besides, the methodology provides access to antibody‐drug conjugate (ADC), which exhibits highly selective anti‐proliferative activity towards HER‐2 expressing SKBR‐3 breast cancer cells.     

In‐Situ Spin Labeling of His‐Tagged Proteins: Distance Measurements under In‐Cell Conditions

New spin labeling strategies have immense potential in studying protein structure and dynamics under physiological conditions with electron paramagnetic resonance (EPR) spectroscopy. Here, a new spin‐labeled chemical recognition unit for switchable and concomitantly high affinity binding to His‐tagged proteins was synthesized. In combination with an orthogonal site‐directed spin label, this novel spin probe, Proxyl‐trisNTA (P‐trisNTA) allows the extraction of structural constraints within proteins and macromolecular complexes by EPR. By using the multisubunit maltose import system of E. coli: 1) the topology of the substrate‐binding protein, 2) its substrate‐dependent conformational change, and 3) the formation of the membrane multiprotein complex can be extracted. Notably, the same distance information was retrieved both in vitro and in situ allowing for site‐specific spin labeling in cell lysates under in‐cell conditions. This approach will open new avenues towards in‐cell EPR.     

Design and synthesis of an affinity probe that targets caspases in proteomic experiments

The field of proteomics aims to study all proteins in the human proteome. This huge task may be accelerated by using active-site directed probes which profile proteins in an activity-dependent manner. Herein, we have developed a fluorescently-labeled affinity probe containing chemical reactivity specific towards caspases. Preliminary assays and proof-of-concept experiments demonstrated that this probe exhibits strong chemical reactivity towards caspase-1 over other enzymes, capable of covalently labeling caspapse-1 over other non-caspase enzymes. This thus demonstrates its selectivity and potential in high-throughput screenings of other unknown caspases in a large-scale proteomics experiment.     

蛋白质定点标记的近红外荧光探针的合成研究

生物大分子定点标记的荧光探针可以用来研究蛋白质的结构和功能,荧光探针良好的刚性和高连接特异性对于使用荧光共振能量转移(FRET)实验来解析生物大分子动态学特征来说有着重要的意义。本文报道两种花菁素类荧光探针IAM-Cyanine3和IAM-Cyanine5的合成方法,该探针通过碘乙酰胺基团特异性地标记在生物大分子的巯基上,相对于商业化的产品,其连接蛋白后的探针分布更加紧密,更有利于对生物大分子的结构和动态学进行更加精确的描述。