Selective Covalent Labeling Research Articles

Self-Immobilizing Quinone Methides for the Fluorescent Sensing of Enzyme Activity

Gaining insight into biological processes relies on sensitive analytical techniques. These often require labeling of biomolecules that help visualize them. Selective covalent labeling without preliminary modification of the biomolecules is an advantageous method. For example, this can be achieved by using probes that are capable of in situ quinone methide (QM) formation. The QM can be masked to give a stable precursor, and the highly reactive form is only generated upon activation by a specific trigger. The in situ formed QM then binds covalently to the nucleophilic side chains of either the target protein or a protein in close proximity. Using fluorogenic probes further improves this method by reducing non-specific background signals, thus improving signal-to-noise ratios. In this review we summarize the development of quinone methide-based probes from mechanism-based inactivation to red-emitting, fluorogenic activity probes, focusing on enzyme-triggered activation.

Electron Microscopic Detection of Single Membrane Proteins by a Specific Chemical Labeling.

SummaryElectron microscopy (EM) is a technology that enables visualization of single proteins at a nanometer resolution. However, current protein analysis by EM mainly relies on immunolabeling with gold-particle-conjugated antibodies, which is compromised by large size of antibody, precluding precise detection of protein location in biological samples. Here, we develop a specific chemical labeling method for EM detection of proteins at single-molecular level. Rational design of α-helical peptide tag and probe structure provided a complementary reaction pair that enabled specific cysteine conjugation of the tag. The developed chemical labeling with gold-nanoparticle-conjugated probe showed significantly higher labeling efficiency and detectability of high-density clusters of tag-fused G protein-coupled receptors in freeze-fracture replicas compared with immunogold labeling. Furthermore, in ultrathin sections, the spatial resolution of the chemical labeling was significantly higher than that of antibody-mediated labeling. These results demonstrate substantial advantages of the chemical labeling approach for single protein visualization by EM.

Selective covalent labeling of miRNA and siRNA duplexes using HEN1 methyltransferase.

MicroRNAs regulate gene expression in numerous biological pathways and are typically methylated at their 3'-termini in plants but not in animals. Here we show that the HEN1 RNA 2'-O-methyltransferase from Arabidopsis thaliana catalyzes the transfer of extended propargylic moieties from synthetic AdoMet cofactor analogs to duplex miRNAs or siRNAs. The presented approach permits selective and efficient covalent labeling of small RNA duplexes with a variety of functional or reporter groups for their enrichment and analysis.

Chemoproteomic profiling of lysine acetyltransferases highlights an expanded landscape of catalytic acetylation.

Lysine acetyltransferases (KATs) play a critical role in the regulation of gene expression, metabolism, and other key cellular functions. One shortcoming of traditional KAT assays is their inability to study KAT activity in complex settings, a limitation that hinders efforts at KAT discovery, characterization, and inhibitor development. To address this challenge, here we describe a suite of cofactor-based affinity probes capable of profiling KAT activity in biological contexts. Conversion of KAT bisubstrate inhibitors to clickable photoaffinity probes enables the selective covalent labeling of three phylogenetically distinct families of KAT enzymes. Cofactor-based affinity probes report on KAT activity in cell lysates, where KATs exist as multiprotein complexes. Chemical affinity purification and unbiased LC–MS/MS profiling highlights an expanded landscape of orphan lysine acetyltransferases present in the human genome and provides insight into the global selectivity and sensitivity of CoA-based proteomic probes that will guide future applications. Chemoproteomic profiling provides a powerful method to study the molecular interactions of KATs in native contexts and will aid investigations into the role of KATs in cell state and disease.

Structural Insights into the Pre-Amyloid Tetramer of β-2-Microglobulin from Covalent Labeling and Mass Spectrometry

The main pathogenic process underlying dialysis-related amyloidosis is the accumulation of β-2-microglobulin (β2m) as amyloid fibrils in the musculoskeletal system, and some evidence suggests that Cu(II) may play a role in β2m amyloid formation. Cu(II)-induced β2m fibril formation is preceded by the formation of discrete, oligomeric intermediates, including dimers, tetramers, and hexamers. In this work, we use selective covalent labeling reactions combined with mass spectrometry to investigate the amino acids responsible for mediating tetramer formation in wild-type β2m. By comparing the labeling patterns of the monomer, dimer, and tetramer, we find evidence that the tetramer interface is formed by the interaction of D strands from one dimer unit and G strands from another dimer unit. These covalent labeling data along with molecular dynamics calculations allow the construction of a tetramer model that indicates how the protein might proceed to form even higher-order oligomers.

Selective Covalent Labeling of Tag-Fused GPCR Proteins on Live Cell Surface with a Synthetic Probe for Their Functional Analysis

Selective protein labeling with a small molecular probe is a versatile method for elucidating protein functions in living cells. In this paper, we report a covalent labeling method of tag-fused G-protein coupled receptor (GPCR) proteins expressing on cell surfaces utilizing small functional molecules. This method employs the selective and rapid reaction of a peptide tag and a molecular probe, which comprises the cysteine-containing short CA6D4x2 tag (CAAAAAADDDDGDDDD) and a tetranuclear Zn(II)-DpaTyr probe containing a reactive alpha-chloroacetyl moiety. The covalent labeling of tag-fused GPCRs such as bradykinin receptor (B2R) and acetylcholine receptor (m1AchR) selectively proceeded under physiological conditions during short incubation (10-30 min) with Zn(II)-DpaTyr probes bearing various functional groups. Labeling with fluorophore-appended Zn(II)-DpaTyr probes enabled visualization of the GPCRs on the surface of HEK293 cells by fluorescence. Labeling with the biotin-appended probe allowed introduction of a biotin unit into the GPCRs. This biotin label was utilized for fluorescence bioimaging studies and postlabeling blotting analysis of the labeled GPCRs by use of the specific biotin-streptavidin interaction. The utility of this labeling method was demonstrated in several function analyses of GPCRs, such as fluorescence visualization of the stimuli-responsive internalization of GPCRs and pH change in endosomes containing the internalized GPCRs.

Structure of the preamyloid dimer of beta-2-microglobulin from covalent labeling and mass spectrometry.

Beta-2-microglobulin (beta2m) self-associates into fibrillar amyloid deposits in the musculoskeletal system of patients undergoing hemodialysis treatment. Previous studies have shown that stoichiometric amounts of Cu(II) at near physiological conditions can cause beta2m to organize into native-like dimers prior to forming amyloid fibrils. Here, we report the results from selective covalent labeling reactions combined with mass spectrometry that provide insight into the amino acid residues that mediate dimer formation in the wild-type protein. Using three complementary covalent labeling reagents, we find that the dimer interface is formed by the antiparallel stacking of ABED beta-sheets from two beta2m monomers. In addition, our data clearly indicate that a dimer interface involving the interactions of D-D strands from separate protein units as seen in the recent crystal structures of two mutant beta2m oligomers is unlikely.

Selective covalent labeling with radiolabeled suicide substrates for isolating P450s

This chapter describes the use of covalent tagging technique to isolate a target P450 protein, the obtainment of specific peptide sequences, and the subsequent cloning of the corresponding complementary deoxyribonucleic acid (cDNA). Mechanism-based enzyme inactivators are irreversible inactivators of specific target enzymes that catalyze their own destruction by unmasking a latent functional group during the catalytic cycle of the enzyme. The reactive intermediate thus formed may either evolve to a normal reaction product, which is then released from the enzyme, or become covalently attached. It is the ratio between these two competing reactions that decides primarily the rate of enzyme inactivation. A promising route is offered by introducing a substrate-specific step into the separation procedure. This may be achieved by using so-called “suicide substrates” really mechanism-based inhibitors) to introduce a covalent tag into the target enzyme and to allow tracking it during separation. As an example of the power of this approach and its shortcomings, this chapter describes the isolation of the first plant fatty acid ω-hydroxylase and its subsequent cloning.

125I]azido-DTLET as a tool for selective covalent labeling of δ-opioid receptors in rat brain sections

The binding kinetics and pharmacological selectivity of the photoaffinity δ-opioid ligand [ 125I]azido-DTLET (Tyr-D-Thr-Gly-Phe(pN 3)-Leu-Thr) were investigated in serial frozen sections from rat neostriatum prior to ultraviolet irradiation (i.e., in conditions of reversibility). Scatchard analysis of saturation binding experiments indicated that [ 125I]azido-DTLET binds to both a high (K D = 5.04 nM) and a low (K D = 38 nM) affinity site. Binding to the low-affinity site was no longer detectable in the presence of unlabeled [D-Ala 2,N-MePhe 4,Gly-Ol 5]enkephalin (DAGO), suggesting that this site corresponds to μ-opioid receptors. This interpretation was further supported by the dose-dependent inhibition of the binding of [ 3H]DAGO by non-radioactive azido-DTLET. Binding to the high-affinity site was totally inhibited, in a dose-dependent fashion, by a variety of opioid drugs among which δ-opioid ligands showed the highest order of potency. It is concluded that, in the nanomolar range, [ 125I]azido-DTLET constitutes a highly selective tool for covalent labeling of δ-opioid receptors in rat brain sections.

Fate of antidiuretic hormone water channel proteins after retrieval from apical membrane.

In toad bladder granular cells, antidiuretic hormone (ADH) stimulates insertion of vesicles containing water channels (WCV), markedly increasing apical membrane osmotic water permeability (Pf). After withdrawal of ADH stimulation, WCV are removed from the apical membrane and fluid-phase markers endocytosed from the apical solution appear predominantly in endosomes at 10-15 min and multivesicular bodies at 30-60 min. Although the luminal contents of this endocytic pathway have been well characterized, the fate of membrane proteins, including functional ADH water channels in these vesicles remains unclear. Using electron microscopic, flow cytometric, and stopped-flow fluorescence measurements and characterization of labeled vesicle proteins, we examined the fate of membrane proteins contained within WCV. The protein complements of endosomes harvested after 10, 30, and 60 min of ADH withdrawal were similar. Selective covalent labeling of apical proteins during ADH stimulation followed by ADH reversal for 30 or 60 min showed that apical proteins colocalize with fluid-phase marker-labeled endosomes at all times, and most apically labeled protein bands present in the 10-min fraction were also present in the 30- and 60-min endosome fractions. Endosomes at 10 and 30 min but not at 60 min contained functional water channels revealed by high Pf and proton permeability, low activation energy of Pf, and sensitivity of Pf to mercurial reagents. We conclude that a portion of apically exposed membrane proteins, including candidate water channel proteins, travel together with fluid-phase markers from 10-min endosomes into later endosomal compartments. Functional water channels may be inactivated or some essential protein component selectively sorted away between 30 and 60 min after ADH withdrawal.

24] Transition-metal complexes as spectroscopic probes for selective covalent labeling and cross-linking of proteins

Publisher Summary A good reagent for covalent labeling and cross-linking of amino acid side chains should be selective in reactions with proteins under mild conditions, easily detected and quantitated, stable yet removable, and noninvasive. Well-chosen transition-metal complexes can meet all of these requirements. Because much is known about substitution reactions of metal complexes, their selectivity toward side chains of certain types or in certain regions of the protein surface can be controlled according to principles of coordination chemistry and stereochemistry. The methods described in this chapter are developed in experiments with different species of cytochrome c , a prototypical electron carrier, and with papain, chymotrypsin, and a-lytic protease, prototypical proteinase enzymes. A metal complex with two open coordination sites or a precursor to such a complex may in principle serve as a cross-linking reagent. Procedures in this chapter are described in sufficient detail to enable relatively easy optimization for other proteins, especially metalloproteins.

Fluorescent modification and orientation of myosin sulfhydryl 2 in skeletal muscle fibers

We describe a protocol for the selective covalent labeling of the sulfhydryl 2 (SH2) on the myosin cross-bridge in glycerinated muscle fibers using the sulfhydryl-selective label 4-[N-[(iodoacetoxy)ethyl]-N-methylamino]-7-nitrobenz-2-oxa-1,3-diazole (IANBD). The protocol promotes the specificity of IANBD by using the ability to protect sulfhydryl 1 (SH1) from modification by binding the cross-bridge to the actin filament and using cross-bridge-bound MgADP to promote the accessibility of SH2. We determined the specificity of the probe using fluorescence gel scanning of fiber-extracted proteins to isolate the probe on myosin subfragment 1 (S1), limited proteolysis of the purified S1 to isolate the probe on the 20-kilodalton fragment of S1, and titration of the free SH1's on purified S1 using the radiolabeled SH1-specific reagent [14C]iodoacetamide or enzymatic activity measurements. We estimated the distribution of the IANBD on the fiber proteins to be approximately 77% on SH2, approximately 5% on SH1, and approximately 18% on troponin I. We characterized the angular distribution of the IANBD on cross-bridges in fibers when the fibers are in rigor, in relaxation, in the presence of MgADP, and in isometric contraction using wavelength-dependent fluorescence polarization [Ajtai, K., & Burghardt, T. P. (1987) Biochemistry 26, 4517-4523]. With wavelength-dependent fluorescence polarization we use the ability to rotate the transition dipole in the molecular frame using excitation wavelength variation to investigate the three angular degrees of freedom of the cross-bridge.(ABSTRACT TRUNCATED AT 250 WORDS)

Selective covalent labeling of cysteines in bovine serum albumin and in hepatoma tissue culture cell glucocorticoid receptors by dexamethasone 21-mesylate.

The specificity of protein labeling by an affinity label of glucocorticoid receptors, dexamethasone 21-mesylate (Dex-Mes), was investigated using bovine serum albumin (BSA) as a model. During the early stages of [3H]Dex-Mes labeling at pH 8.8, approximately 90% of the covalent bond formation occurred at the one non-oxidized cysteine (Cys-34) of BSA. The nonspecific labeling was equally distributed over the rest of the BSA molecule. [3H]Dex-Mes labeling of Cys-34 was totally, and specifically inhibited by nearly stoichiometric amounts of the thiol-specific reagent methyl methanethiolsulfonate (MMTS). Thus both Dex-Mes and MMTS appear to react very selectively with thiols under our conditions. In reactions with hepatoma tissue culture (HTC) cell glucocorticoid receptors, MMTS was equally efficient in preventing [3H]dexamethasone binding to receptors and [3H]Dex-Mes labeling of the 98-kDa receptor protein. These results indicate that Dex-Mes labeling of the glucocorticoid receptor involves covalent reaction with at least one cysteine in the steroid binding site of the receptor. Small (approximately 1600-dalton) fragments of the [3H]Dex-Mes-labeled 98-kDa receptor were generated by limit proteolysis with trypsin, chymotrypsin, and Staphylococcus aureus V8 protease under denaturing conditions. Data from these fragments on 15% sodium dodecyl sulfate-polyacrylamide gels were consistent with all of the covalent [3H] Dex-Mes being located on one or a few cysteines in one approximately 15-residue stretch of the receptor. Further studies revealed no differences in the limit protease digestion patterns of activated and unactivated [3H]Dex-Mes-labeled receptors with trypsin, chymotrypsin, or V8 protease under denaturing conditions. These data suggest that activation does not cause any major covalent modifications of the amino acids immediately surrounding the affinity-labeled cysteine(s) of the steroid binding site.

A transition-metal chromophore as a new, sensitive spectroscopic tag for proteins. Selective covalent labeling of histidine residues in cytochromes c with chloro(2,2':6'2"-terpyridine)platinum(II) chloride

Reactivity and selectivity of Pt(trpy)Cl/sup +/ toward proteins are studied with cytochromes c from horse and tuna as examples. The new transition-metal reagent is specific for histidine residues at pH 5. The reaction, facile one-step displacement of the Cl/sup -/ ligand by imidazole, produces good yield. The binding sites, His 26 and His 33 in the horse protein and His 26 in the tuna protein, are identified by UV-vis spectrophotometry and by peptide-mapping experiments. Model complexes with imidazole, histidine, histidine derivatives, and histidine-containing peptides are prepared and characterized. The covalently attached Pt(trpy)/sup 2 +/ labels allow easy separation of the protein derivatives by cation-exchange chromatography. The labels do not perturb the conformation and reduction potential of cytochrome c, as shown by UV-vis spectrophotometry, cyclic voltammetry, differential-pulse voltammetry, EPR spectroscopy, and /sup 1/H NMR spectroscopy. The selectivity of Pt(trpy)Cl/sup +/ is entirely opposite from that of PtCl/sub 4//sup 2 -/ although both of them are platinum(II)-chloro complexes. Owing to an interplay between the steric and electronic effects of the terpyridyl ligand, the new reagent is unreactive toward methionine (a thio ether) and cystine (a disulfide), which are otherwise highly nucleophilic ligands, but very reactive toward imidazole, which is otherwise a relativelymore » weak ligand. Unusual and useful selectivity of preformed transition-metal complexes toward proteins evidently can be achieved by a judicious choice of ancillary ligands.« less

Efficient and highly selective covalent labeling of the estrogen receptor with [3H]tamoxifen aziridine.

Efficient and highly selective covalent labeling of the estrogen receptor with [3H]tamoxifen aziridine.

Essential sulfhydryl for reduced nicotinamide adenine dinucleotide binding in D-beta-hydroxybutyrate dehydrogenase.

Chemical derivatization studies have been directed at the sulfhydryl group of D-beta-hydroxybutyrate dehydrogenase, a lipid-requiring enzyme. Reaction with N-ethylmaleimide leads to progressive and parallel loss of both enzymic activity and coenzyme binding. Both functions are lost when 1 equiv of sulfhydryl is derivatized per mol of enzyme. Inactivation of the enzyme with methylmercury or with air oxidation also leads to loss of coenzyme binding. We conclude that a single "essential" sulfhydryl is required for coenzyme binding and consequently for enzymic activity. Only two "accessible" cysteine residues can be derivatized even at high levels of N-ethylmaleimide, whereas derivatization of the remaining three "inacessible" cysteines requires denaturation of the enzyme. The enzyme can apparently be labeled in the accessible, but nonessential, sulfhydryl in the presence of coenzyme which protects against inactivation by N-ethylmaleimide. Such selective covalent labeling of the nonessential sulfhydryl makes possible future biophysical studies of enzyme-phospholipid interaction of a functional enzyme using extrinsic probes.