Thiol-reactive Probes Research Articles

Oxygen-Induced Conformational Changes in the PAS-Heme Domain of the Pseudomonas aeruginosa Aer2 Receptor.

The Aer2 receptor from Pseudomonas aeruginosa has an O2-binding PAS-heme domain that stabilizes O2 via a Trp residue in the distal heme pocket. Trp rotates ∼90° to bond with the ligand and initiate signaling. Although the isolated PAS domain is monomeric, both in solution and in a cyanide-bound crystal structure, an unliganded structure forms a dimer. An overlay of the two structures suggests possible signaling motions but also predicts implausible clashes at the dimer interface when the ligand is bound. Moreover, in a full-length Aer2 dimer, PAS is sandwiched between multiple N- and C-terminal HAMP domains, which would feasibly restrict PAS motions. To explore the PAS dimer interface and signal-induced motions in full-length Aer2, we introduced Cys substitutions and used thiol-reactive probes to examine in vivo accessibility and residue proximities under both aerobic and anaerobic conditions. In vivo, PAS dimers were retained in full-length Aer2 in the presence and absence of O2, and the dimer interface was consistent with the isolated PAS dimer structure. O2-mediated changes were also consistent with structural predictions in which the PAS N-terminal caps move apart and the C-terminal DxT region moves closer together. The DxT motif links PAS to the C-terminal HAMP domains and was critical for PAS-HAMP signaling. Removing the N-terminal HAMP domains altered the distal PAS dimer interface and prevented signaling, even after signal-on lesions were introduced into PAS. The N-terminal HAMP domains thus facilitate the O2-dependent shift of PAS to the signal-on conformation, clarifying their role upstream of the PAS-sensing domain.

A quantitative thiol reactivity profiling platform to analyze redox and electrophile reactive cysteine proteomes.

Cysteine is unique among all protein-coding amino acids, owing to its intrinsically high nucleophilicity. The cysteinyl thiol group can be covalently modified by a broad range of redox mechanisms or by various electrophiles derived from exogenous or endogenous sources. Measuring the response of protein cysteines to redox perturbation or electrophiles is critical for understanding the underlying mechanisms involved. Activity-based protein profiling based on thiol-reactive probes has been the method of choice for such analyses. We therefore adapted this approach and developed a new chemoproteomic platform, termed 'QTRP' (quantitative thiol reactivity profiling), that relies on the ability of a commercially available thiol-reactive probe IPM (2-iodo-N-(prop-2-yn-1-yl)acetamide) to covalently label, enrich and quantify the reactive cysteinome in cells and tissues. Here, we provide a detailed and updated workflow of QTRP that includes procedures for (i) labeling of the reactive cysteinome from cell or tissue samples (e.g., control versus treatment) with IPM, (ii) processing the protein samples into tryptic peptides and tagging the probe-modified peptides with isotopically labeled azido-biotin reagents containing a photo-cleavable linker via click chemistry reaction, (iii) capturing biotin-conjugated peptides with streptavidin beads, (iv) identifying and quantifying the photo-released peptides by mass spectrometry (MS)-based shotgun proteomics and (v) interpreting MS data by a streamlined informatic pipeline using a proteomics software, pFind 3, and an automatic post-processing algorithm. We also exemplified here how to use QTRP for mining H2O2-sensitive cysteines and for determining the intrinsic reactivity of cysteines in a complex proteome. We anticipate that this protocol should find broad applications in redox biology, chemical biology and the pharmaceutical industry. The protocol for sample preparation takes 3 d, whereas MS measurements and data analyses require 75 min and <30 min, respectively, per sample.

Probing the DNA-binding center of the MutL protein from the Escherichia coli mismatch repair system via crosslinking and Förster resonance energy transfer

As no crystal structure of full-size MutL bound to DNA has been obtained up to date, in the present work we used crosslinking and Förster resonance energy transfer (FRET) assays for probing the putative DNA-binding center of MutL from Escherichia coli. Several single-cysteine MutL variants (scMutL) were used for site-specific crosslinking or fluorophore modification. The crosslinking efficiency between scMutL proteins and mismatched DNA modified with thiol-reactive probes correlated with the distances from the Cys residues to the DNA calculated from a model of MutS–MutL–DNA complex. FRET-based investigation of DNA binding with different scMutL variants clearly showed that the highest signals were detected for the variants MutL(T218C) and MutL(A251C) indicating closeness of the positions 218 and 251 to DNA in the MutL–DNA complex. Indeed, the Cys218 and Cys251 of scMutL were crosslinked to the reactive DNA with the highest yield demonstrating their proximity to DNA in the MutL–DNA complex. The presence of MutS increased the yield of conjugate formation between the MutL variants and the modified DNA due to tighter MutL–DNA interactions caused by MutS binding to MutL.

Chemical Proteomic Characterization of a Covalent KRASG12C Inhibitor.

The KRASG12C protein product is an attractive, yet challenging, target for small molecule inhibition. One option for therapeutic intervention is to design small molecule ligands capable of binding to and inactivating KRASG12C via formation of a covalent bond to the sulfhydryl group of cysteine 12. In order to better understand the cellular off-target interactions of Compound 1, a covalent KRASG12C inhibitor, we have completed a series of complementary chemical proteomics experiments in H358 cells. A new thiol reactive probe (TRP) was designed and used to construct a cellular target occupancy assay for KRASG12C. In addition, the thiol reactive probes allowed us to profile potential off-target interactions of Compound 1 with over 3200 cysteine residues. In order to complement the TRP data we designed Compound 2, an alkyne containing version of Compound 1, to serve as bait in competitive chemical proteomics experiments. Herein, we describe and compare data from both the TRP and the click chemistry probe pull down experiments.

Fluoreszenzsonden mit mehreren Bindungsstellen unterscheiden zwischen Cys, Hcy und GSH

AbstractThiole wie Cystein (Cys), Homocystein (Hcy) und Glutathion (GSH) spielen für das Redoxgleichgewicht in biologischen Systemen eine entscheidende Rolle. Dieser Kurzaufsatz fasst aktuelle Aspekte aus dem Bereich der reaktiven Thiolsonden für die biomedizinische Forschung und Diagnostik zusammen und betont die verbleibenden Herausforderungen für Chemiker im Bereich der selektiven Sonden und Sensoren. Der Fortschritt bei Sonden mit mehreren Bindungsstellen stellt eine kreative neue Richtung des Feldes dar, die gleichzeitige und akkurate ratiometrische Messungen ermöglichen kann. Neue Strategien zur Entwicklung von Sonden können helfen, aktuelle Aufgaben anzugehen. Dazu zählt das Monitoring von Krankheiten, die durch abweichende GSH‐, Cys‐ und Hcy‐Spiegel gekennzeichnet sind, wie etwa Autismus und chronische Erkrankungen, die auf oxidativem Stress beruhen.

Fluorescent Probes with Multiple Binding Sites for the Discrimination of Cys, Hcy, and GSH.

Biothiols such as cysteine (Cys), homocysteine (Hcy), and glutathione (GSH) play crucial roles in maintaining redox homeostasis in biological systems. This Minireview summarizes the most significant current challenges in the field of thiol-reactive probes for biomedical research and diagnostics, emphasizing the needs and opportunities that have been under-investigated by chemists in the selective probe and sensor field. Progress on multiple binding site probes to distinguish Cys, Hcy, and GSH is highlighted as a creative new direction in the field that can enable simultaneous, accurate ratiometric monitoring. New probe design strategies and researcher priorities can better help address current challenges, including the monitoring of disease states such as autism and chronic diseases involving oxidative stress that are characterized by divergent levels of GSH, Cys, and Hcy.

Spatial positioning of CFTR's pore-lining residues affirms an asymmetrical contribution of transmembrane segments to the anion permeation pathway.

The structural composition of CFTR's anion permeation pathway has been proposed to consist of a short narrow region, flanked by two wide inner and outer vestibules, based on systematic cysteine scanning studies using thiol-reactive probes of various sizes. Although these studies identified several of the transmembrane segments (TMs) as pore lining, the exact spatial relationship between pore-lining elements remains under debate. Here, we introduce cysteine pairs in several key pore-lining positions in TM1, 6, and 12 and use Cd(2+) as a probe to gauge the spatial relationship of these residues within the pore. We find that inhibition of single cysteine CFTR mutants, such as 102C in TM1 or 341C in TM6, by intracellular Cd(2+) is readily reversible upon removal of the metal ion. However, the inhibitory effect of Cd(2+) on the double mutant 102C/341C requires the chelating agent dithiothreitol (DTT) for rapid reversal, indicating that 102C and 341C are close enough to the internal edge of the narrow region to coordinate one Cd(2+) ion between them. We observe similar effects of extracellular Cd(2+) on TM1/TM6 cysteine pairs 106C/337C, 107C/337C, and 107C/338C, corroborating the idea that these paired residues are physically close to each other at the external edge of the narrow region. Although these data paint a picture of relatively symmetrical contributions to CFTR's pore by TM1 and TM6, introducing cysteine pairs between TM6 and TM12 (348C/1141C, 348C/1144C, and 348C/1145C) or between TM1 and TM12 (95C/1141C) yields results that contest the long-held principle of twofold pseudo-symmetry in the assembly of ABC transporters' TMs. Collectively, these findings not only advance our current understanding of the architecture of CFTR's pore, but could serve as a guide for refining computational models of CFTR by imposing physical constraints among pore-lining residues.

Deficient expression of aldehyde dehydrogenase 1A1 is consistent with increased sensitivity of Gorlin syndrome patients to radiation carcinogenesis

Human phenotypes that are highly susceptible to radiation carcinogenesis have been identified. Sensitive phenotypes often display robust regulation of molecular features that modify biological response, which can facilitate identification of the pathways/networks that contribute to pathophysiological outcomes. Here we interrogate primary dermal fibroblasts isolated from Gorlin syndrome patients (GDFs), who display a pronounced inducible tumorigenic response to radiation, in comparison to normal human dermal fibroblasts (NHDFs). Our approach exploits newly developed thiol reactive probes to define changes in protein thiol profiles in live cell studies, which minimizes artifacts associated with cell lysis. Redox probes revealed deficient expression of an apparent 55 kDa protein thiol in GDFs from independent Gorlin syndrome patients, compared with NHDFs. Proteomics tentatively identified this protein as aldehyde dehydrogenase 1A1 (ALDH1A1), a key enzyme regulating retinoic acid synthesis, and ALDH1A1 protein deficiency in GDFs was confirmed by Western blot. A number of additional protein thiol differences in GDFs were identified, including radiation responsive annexin family members and lamin A/C. Collectively, candidates identified in our study have plausible implications for radiation health effects and cancer susceptibility.

Two Salt Bridges Differentially Contribute to the Maintenance of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Channel Function

Previous studies have identified two salt bridges in human CFTR chloride ion channels, Arg(352)-Asp(993) and Arg(347)-Asp(924), that are required for normal channel function. In the present study, we determined how the two salt bridges cooperate to maintain the open pore architecture of CFTR. Our data suggest that Arg(347) not only interacts with Asp(924) but also interacts with Asp(993). The tripartite interaction Arg(347)-Asp(924)-Asp(993) mainly contributes to maintaining a stable s2 open subconductance state. The Arg(352)-Asp(993) salt bridge, in contrast, is involved in stabilizing both the s2 and full (f) open conductance states, with the main contribution being to the f state. The s1 subconductance state does not require either salt bridge. In confirmation of the role of Arg(352) and Asp(993), channels bearing cysteines at these sites could be latched into a full open state using the bifunctional cross-linker 1,2-ethanediyl bismethanethiosulfonate, but only when applied in the open state. Channels remained latched open even after washout of ATP. The results suggest that these interacting residues contribute differently to stabilizing the open pore in different phases of the gating cycle.

Optically-Resolved Conformational Rearrangements of the Voltage-Sensing Domains of the Human CaV1.2 Channel

Voltage-gated Ca2+ channels (CaV) consist of four tandem transmembrane repeats (I-IV), each including two pore-forming helices and four segments that make up a voltage-sensing domain (VSD). To understand voltage-dependent gating in human CaV1.2 channels, we have optically tracked the activation of the S4 helices of repeats I, III and IV by site-directed fluorescent labeling of introduced Cysteines with thiol-reactive probes. The channels were co-expressed with their modulatory subunits β3 and α2δ in Xenopus oocytes. Ionic current and fluorescence emission were simultaneously recorded using the cut-open oocyte voltage clamp fluorometry technique. Prior voltage-clamp, oocytes were injected with 100 nl 80 mM BAPTA.4K, to prevent the activation of endogenous Ca2+-gated Cl− channels. The extracellular solution contained 2 mM Ba2+ or 10 mM Ca2+ as charge carrier. Gating currents were recorded by replacing Ca2+ or Ba2+ with Co2+. 0.1 mM ouabain was added to abolish Na+/K+ ATPase non-linear charge movement. Voltage-dependent fluorescence changes (ΔF) were reported by fluorophores attached to substituted Cysteines at the extracelular end of the S4 segment in repeats I, III and IV, tracking local, voltage-dependent conformational rearrangements. The voltage dependence of observed fluorescence deflections preceded ionic activation, reporting repeat-specific VSD transitions taking place during shut states of the channel. Prolonged sojourns at depolarized potentials (+50 mV) shifted the voltage-dependence of reported ΔF to more hyperpolarized potentials by >50mV, recapitulating previously-characterized gating current properties. In summary, we report the first optical characterization of VSD conformational changes in a human voltage-gated Ca2+ channel, revealing repeat-specific voltage- and time-dependent properties.

Alternating Access to the Transmembrane Domain of the ATP-binding Cassette Protein Cystic Fibrosis Transmembrane Conductance Regulator (ABCC7)

The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is a member of the ATP-binding cassette (ABC) protein family, most members of which act as active transporters. Actively transporting ABC proteins are thought to alternate between "outwardly facing" and "inwardly facing" conformations of the transmembrane substrate pathway. In CFTR, it is assumed that the outwardly facing conformation corresponds to the channel open state, based on homology with other ABC proteins. We have used patch clamp recording to quantify the rate of access of cysteine-reactive probes to cysteines introduced into two different transmembrane regions of CFTR from both the intracellular and extracellular solutions. Two probes, the large [2-sulfonatoethyl]methanethiosulfonate (MTSES) molecule and permeant Au(CN)(2)(-) ions, were applied to either side of the membrane to modify cysteines substituted for Leu-102 (first transmembrane region) and Thr-338 (sixth transmembrane region). Channel opening and closing were altered by mutations in the nucleotide binding domains of the channel. We find that, for both MTSES and Au(CN)(2)(-), access to these two cysteines from the cytoplasmic side is faster in open channels, whereas access to these same sites from the extracellular side is faster in closed channels. These results are consistent with alternating access to the transmembrane regions, however with the open state facing inwardly and the closed state facing outwardly. Our findings therefore prompt revision of current CFTR structural and mechanistic models, as well as having broader implications for transport mechanisms in all ABC proteins. Our results also suggest possible locations of both functional and dysfunctional ("vestigial") gates within the CFTR permeation pathway.

Reversible Inter- and Intra-Microgel Cross-Linking Using Disulfides

Thermoresponsive hydrogel nanoparticles composed of poly(N-isopropylmethacrylamide) (pNIPMAm) and the disulfide-based cross-linker N,N'-bis(acryloyl)cystamine (BAC) have been prepared using a redox-initiated, aqueous precipitation polymerization approach, leading to improved stability of the disulfide bond compared to traditional thermally-initiated methods. The resultant particles demonstrate complete erosion in response to reducing conditions or thiol competition. This stands in contrast to the behavior of thermally-initiated particles, which retain a cross-linked network following disulfide cleavage due to uncontrolled chain-branching and self-cross-linking side reactions. The synthetic strategy has also been combined with the non-degradable cross-linker N,N-methylenebisacrylamide (BIS) to generate "co-cross-linked" pNIPMAm-BAC-BIS microgels. These particles are redox-responsive, swell upon BAC cross-link scission and present reactive thiols. This pendant thiol functionality was demonstrated to be useful for conjugation of thiol-reactive probes and in reversible network formation by assembling particles cross-linked by disulfide linkages.

Labeling Protein with Thiol-reactive Probes

Labeling Protein with Thiol-reactive Probes

Nε‐(thiaprolyl)‐lysine as a handle for site‐specific protein conjugation

In this article, we introduce the use of a thiaproline-modified lysine side-chain [Lys(Thz)], as an unlockable handle that enables late-stage, site-selective modification of chemically synthesized proteins. The Lys(Thz) residue was incorporated into the murine chemokine RANTES to demonstrate its compatibility with Boc/Bzl solid phase peptide synthesis, native chemical ligation, and disulfide bond formation. After oxidative folding of the protein, the thiol was liberated under mild reaction conditions [0.2 M hydroxylamine (NH2OH) or O-methylhydroxylamine (MeONH2), pH 4] and was subsequently reacted with thiol-selective tags. This side chain protection strategy enables the use of readily available thiol-reactive probes for the modification of internally disulfide bonded proteins.

Cystic Fibrosis Transmembrane Conductance Regulator: Using Differential Reactivity toward Channel-Permeant and Channel-Impermeant Thiol-Reactive Probes To Test a Molecular Model for the Pore

The sixth transmembrane segment (TM6) of the CFTR chloride channel has been intensively investigated. The effects of amino acid substitutions and chemical modification of engineered cysteines (cysteine scanning) on channel properties strongly suggest that TM6 is a key component of the anion-conducting pore, but previous cysteine-scanning studies of TM6 have produced conflicting results. Our aim was to resolve these conflicts by combining a screening strategy based on multiple, thiol-directed probes with molecular modeling of the pore. CFTR constructs were screened for reactivity toward both channel-permeant and channel-impermeant thiol-directed reagents, and patterns of reactivity in TM6 were mapped onto two new, molecular models of the CFTR pore: one based on homology modeling using Sav1866 as the template and a second derived from the first by molecular dynamics simulation. Comparison of the pattern of cysteine reactivity with model predictions suggests that nonreactive sites are those where the TM6 side chains are occluded by other TMs. Reactive sites, in contrast, are generally situated such that the respective amino acid side chains either project into the predicted pore or lie within a predicted extracellular loop. Sites where engineered cysteines react with both channel-permeant and channel-impermeant probes occupy the outermost extent of TM6 or the predicted TM5−6 loop. Sites where cysteine reactivity is limited to channel-permeant probes occupy more cytoplasmic locations. The results provide an initial validation of two, new molecular models for CFTR and suggest that molecular dynamics simulation will be a useful tool for unraveling the structural basis of anion conduction by CFTR.

Methods for the determination and quantification of the reactive thiol proteome

Protein thiol modifications occur under both physiological and pathological conditions and have been shown to contribute to changes in protein structure, function, and redox signaling. The majority of protein thiol modifications occur on cysteine residues that have a low p K a; these nucleophilic proteins comprise the “reactive thiol proteome.” The most reactive members of this proteome are typically low-abundance proteins. Therefore, sensitive and quantitative methods are needed to detect and measure thiol modifications in biological samples. To accomplish this, we have standardized the usage of biotinylated and fluorophore-labeled alkylating agents, such as biotinylated iodoacetamide (IAM) and N-ethylmaleimide (NEM) and BODIPY-labeled IAM and NEM, for use in one- and two-dimensional proteomic strategies. Purified fractions of cytochrome c and glyceraldehyde-3-phosphate dehydrogenase were conjugated to a known amount of biotin or BODIPY fluorophore to create an external standard that can be run on standard SDS–PAGE gels, which allows for the quantification of protein thiols from biological samples by Western blotting or fluorescence imaging. A detailed protocol is provided for using thiol-reactive probes and making external standards for visualizing and measuring protein thiol modifications in biological samples.

Ricin A Chain Insertion into Endoplasmic Reticulum Membranes Is Triggered by a Temperature Increase to 37 °C

After endocytic uptake by mammalian cells, the heterodimeric plant toxin ricin is transported to the endoplasmic reticulum (ER), where the ricin A chain (RTA) must cross the ER membrane to reach its ribosomal substrates. Here, using gel filtration chromatography, sedimentation, fluorescence, fluorescence resonance energy transfer, and circular dichroism, we show that both fluorescently labeled and unlabeled RTA bind both to ER microsomal membranes and to negatively charged liposomes. The binding of RTA to the membrane at 0-30 °C exposes certain RTA residues to the nonpolar lipid core of the bilayer with little change in the secondary structure of the protein. However, major structural rearrangements in RTA occur when the temperature is increased. At 37 °C, membrane-bound toxin loses some of its helical content, and its C terminus moves closer to the membrane surface where it inserts into the bilayer. RTA is then stably bound to the membrane because it is nonextractable with carbonate. The sharp temperature dependence of the structural changes does not coincide with a lipid phase change because little change in fluorescence-detected membrane mobility occurred between 30 and 37 °C. Instead, the structural rearrangements may precede or initiate toxin retrotranslocation through the ER membrane to the cytosol. The sharp temperature dependence of these changes in RTA further suggests that they occur optimally in mammalian targets of the plant toxin.

Functionality of Single-Cysteine Mutants in Subunit III of Rhodobacter sphaeroides Cytochrome c Oxidase

Cytochrome c oxidase (COX) catalyzes the reduction of oxygen to water using ferrocytochrome c and conserves the energy of this reaction by translocating protons across the bacterial or inner-mitochrondrial membrane. COX from Rhodobacter sphaeroides is a four subunit transmembrane protein that serves as a model for the mitochondrial enzyme. Subunit I and II contain the redox centers and proton pathways necessary for redox chemistry and proton translocation. The indispensable role of subunit III is an area still being investigated. This work examines the functionality of three mutant forms of COX - one in which all cysteines have been removed from the enzyme (CA1CS3), and two in which single cysteines are reintroduced into CA1CS3 at specific locals in subunit III (A4C, S187C). The single cysteine mutants provide a means to specifically target thiol-reactive probes to areas of interest in COX subunit III. The A4C mutant allows for a probe to be placed at the mouth of the D-channel - an important proton-conducting pathway necessary for the pumping and redox activities of COX. Bioconjugation of S187C would place a probe on an exterior loop which is thought to undergo redox-linked transient conformational changes. All three mutants were expressed and purified, and their absorbance spectra are identical to wildtype, indicating that the heme active centers are unperturbed. SDS-PAGE gels show that all three mutants retain wildtype subunit composition. The oxygen reduction activity of the mutants are also comparable to wildtype, with values between 1200-1600 e−/s∗mol at pH 7.4. In conclusion, these results indicate that the cysteine-free mutant and two mutants in which single cysteines are reintroduced at non-conserved locations retain wildtype functionality, indicating that cytochrome c oxidase subunit III is a candidate for cysteine scanning-mutagenesis studies utilizing thiol-reactive probes.

Fluorometric Assay for the Determination of Glutathione Reductase Activity

The determination of free sulfhydryl groups is important in many aspects of biotechnology, such as measuring the coupling efficiency of thiol-reactive probes, assaying cysteine-containing haptens, and assaying reductase/thiol transferase activity, as well as in various aspects of the food industry. This is generally achieved colorimetrically using Ellman's reagent, although the assay is relatively insensitive and Ellman's reagent is unstable. In this paper, we describe a highly sensitive fluorometric assay for free sulfhydryl groups based on FRET, which we have used to develop a sensitive assay for glutathione reductase activity. The assay exploits the specific increase in fluorescence intensity that occurs at 520 nm when a probe containing two molecules of fluorescein linked via a disulfide group is cleaved by glutathione. The assay is 2 orders of magnitude more sensitive than the commonly used colorimetric glutathione reductase assay and has a greater dynamic range.

Nonionic surfactant-capped gold nanoparticles as postcolumn reagents for high-performance liquid chromatography assay of low-molecular-mass biothiols

Gold nanoparticles (GNPs) have been stabilized with nonionic surfactant ligands, i.e., Brij @ 35, and their aggregation could be induced rapidly and selectively by biologically active low-molecular-mass thiols including sulphydryl-containing amino acids (cysteine and homocysteine) and small peptides (glutathione, cysteinylglycine, and glutamylcysteine). A new postcolumn detection method has been developed for high-performance liquid chromatography (HPLC) assay of these small biothiols based on the analyte-induced aggregation of the GNPs. Compared with conventional thiol-reactive probes, the GNP colloids are easier to prepare, much more stable in aqueous solution over a wide pH range and at ambient temperature, and exhibit relatively high selectivity toward small biothiols. The analysis of human urine samples demonstrated that the proposed method is promising in HPLC assay of the small thiol molecules in biological fluids.