Ultra-rapid glutathionylation of chymotrypsinogen in its molten globule-like conformation: A comparison to archaeal proteins.

Chymotrypsinogen, when reduced and taken to its molten globule-like conformation, displays a single cysteine with an unusual kinetic propensity toward oxidized glutathione (GSSG) and other organic thiol reagents. A single residue, identified by mass spectrometry like Cys1, reacts with GSSG about 1400 times faster than an unperturbed protein cysteine. A reversible protein-GSSG complex and a low pKa (8.1 ± 0.1) make possible such astonishing kinetic property which is absent toward other natural disulfides like cystine, homocystine and cystamine. An evident hyper-reactivity toward 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) and 1-chloro-2,4-dinitrobenzene (CDNB) was also found for this specific residue. The extraordinary reactivity toward GSSG is absent in two proteins of the thermophilic archaeon Sulfolobus solfataricus, an organism lacking glutathione: the Protein Disulphide Oxidoreductase (SsPDO) and the Bacterioferritin Comigratory Protein 1 (Bcp1) that displays Cys residues with an even lower pKa value (7.5 ± 0.1) compared to chymotrypsinogen. This study, which also uses single mutants in Cys residues for Bcp1, proposes that this hyper-reactivity of a single cysteine, similar to that found in serum albumin, lysozyme, ribonuclease, may have relevance to drive the "incipit" of the oxidative folding of proteins from organisms where the glutathione/oxidized glutathione (GSH/GSSG) system is present.

Isolation and analysis of a non-protein low molecular weight thiol-mercurial adduct from human prostate lymph node cells (LNCaP).

Thiol compounds present in human malignant prostate cells (LNCaP) were investigated after reaction with a mercurial blocking reagent. After extracting the cellular glutathione and some other low molecular weight (LMW) thiols using trichloroacetic acid the resulting the protein precipitate was extracted with buffered 8 M urea containing 2-chloromercuri-4-nitrophenol in an equimolar amount to that of the thiol present. After removing the insoluble chromatin fraction the urea soluble labeled adducts formed were chromatographed on G15 Sephadex. Three yellow coloured (A410 nm) fractions were obtained; first, the excluded protein fraction containing 16.0 ± 4.1% of the applied label followed by an intermediate fraction containing 5.9 ± 1.2%. Finally a LMW fraction emerged which contained 77.2 ± 3.7% of the total label applied and this was further analyzed by column chromatography, first on an anion exchange column and then on a PhenylSepharose 6 column to give what appeared to be a single component. LC-MS analysis of this component gave a pattern of mercuri-clusters, formed on MS ionization showing possible parent ions at 704 or 588 m/z, the former indicating that a thiol fragment of molecular weight approximately 467 could be present. No fragments with a single sulfur adduct (a 369 m/z fragment) were observed The adduct was analyzed for cysteine and other amino acids, nucleic acid bases, ribose and deoxyribose sugars, selenium and phosphorus; all were negative leading to the conclusion that a new class of unknown LMW thiol is present concealed in the protein matrices of these cells.

Design and Synthetic Strategies for Helical Peptides.

Abnormal protein-protein interactions (PPIs) are the basis of multiple diseases, and the large and shallow PPI interfaces make the target "undruggable" for traditional small molecules. Peptides, emerging as a new therapeutic modality, can efficiently mimic PPIs with their large scaffolds. Natural peptides are flexible and usually have poor serum stability and cell permeability, features that limit their further biological applications. To satisfy the clinical application of peptide inhibitors, many strategies have been developed to constrain peptides in their bioactive conformation. In this report, we describe several classic methods used to constrain peptides into a fixed secondary structure which could significantly improve their biophysical properties.

Synthesis and Bioconjugation of Thiol-Reactive Reagents for the Creation of Site-Selectively Modified Immunoconjugates.

Maleimide-bearing bifunctional probes have been employed for decades for the site-selective modification of thiols in biomolecules, especially antibodies. Yet maleimide-based conjugates display limited stability in vivo because the succinimidyl thioether linkage can undergo a retro-Michael reaction. This, of course, can lead to the release of the radioactive payload or its exchange with thiol-bearing biomolecules in circulation. Both of these processes can produce elevated activity concentrations in healthy organs as well as decreased activity concentrations in target tissues, resulting in reduced imaging contrast and lower therapeutic ratios. In 2018, we reported the creation of a modular, stable, and easily accessible phenyloxadiazolyl methyl sulfone reagent - dubbed 'PODS' - as a platform for thiol-based bioconjugations. We have clearly demonstrated that PODS-based site-selective bioconjugations reproducibly and robustly create homogenous, well-defined, highly immunoreactive, and highly stable radioimmunoconjugates. Furthermore, preclinical experiments in murine models of colorectal cancer have shown that these site-selectively labeled radioimmunoconjugates exhibit far superior in vivo performance compared to radiolabeled antibodies synthesized via maleimide-based conjugations. In this protocol, we will describe the four-step synthesis of PODS, the creation of a bifunctional PODS-bearing variant of the ubiquitous chelator DOTA (PODS-DOTA), and the conjugation of PODS-DOTA to the HER2-targeting antibody trastuzumab.

Simple and Novel Assay of the Host-Guest Complexation of Homocysteine with Cucurbit[7]uril.

This paper introduces three ways to determine host-guest complexation of cucurbit[7]uril (CB[7]) with homocysteine (Hcy). After preincubating Hcy and cysteine (Cys) with CB[7], Ellman's reagent (DTNB) was used to detect Hcy and Cys. Only Cys reacted with DTNB and Hcy gave a retarded color change. This suggests that the -SH group of Hcy is buried inside CB[7]. Human cystathionine γ-lyase (hCGL) decreased the level of Hcy degradation after preincubating Hcy and CB[7]. These results suggest that the amount of free Hcy available was decreased by the formation of a Hcy-CB[7] complex. The immunological signal of anti-Hcy monoclonal antibody was decreased significantly by preincubating CB[7] with Hcy. The ELISA results also show that ethanethiol group (-CH2CH2SH) of Hcy, which is an epitope of anti-Hcy monoclonal antibody, was blocked by the cavity in CB[7]. Overall, CB[7] can act as a host by binding selectively with Hcy, but not Cys. The calculated half-complexation formation concentration of CB[7] was 58.2 nmol using Ellman's protocol, 97.9 nmol using hCGL assay and 87.7 nmol using monoclonal antibody. The differing binding abilities of Hcy and Cys towards the CB[7] host may offer a simple and useful method for determining the Hcy concentration in plasma or serum.

A Cysteine-Reactive Small Photo-Crosslinker Possessing Caged-Fluorescence Properties: Binding-Site Determination of a Combinatorially-Selected Peptide by Fluorescence Imaging/Tandem Mass Spectrometry.

To determine the binding-site of a combinatorially-selected peptide possessing a fluoroprobe, a novel cysteine reactive small photo-crosslinker that can be excited by a conventional long-wavelength ultraviolet handlamp (365 nm) was synthesized via Suzuki coupling with three steps. The crosslinker is rationally designed, not only as a bioisostere of the fluoroprobe, but as a caged-fluorophore, and the photo-crosslinked target protein became fluorescent with a large Stokes-shift. By introducing the crosslinker to a designated sulfhydryl (SH) group of a combinatorially-selected peptide, the protein-binding site of the targeted peptide was deduced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)/fluorescence imaging followed by matrix-assisted laser desorption ionization-time of flight tandem mass spectrometry (MALDI-TOF-MS/MS) analysis.

Automated radiosynthesis of [18F]FBEM, a sulfhydryl site specific labeling agent for peptides and proteins.

In the process of developing [18F]FBEM coupled target peptide, we have instituted a robust automated synthesis of [18F]FBEM, a sulfhydryl (-SH) site specific agent for radiolabeling of peptides and proteins. The radiosynthesis generated 1.67-3.89 GBq (45.1-105.1 mCi, 7.5-18.8% non-decay corrected yield) of [18F]FBEM from 22.2 GBq (600 mCi) of starting [18F]fluoride with molar activity of 31.8 ±â€¯5.3 GBq/µmol (0.86 ±â€¯0.14 mCi/nmol) (n = 3) at the end of synthesis. Radiochemical purity was greater than 98%, and total synthesis time was ~90 min.

Three unrelated and unexpected amino acids determine the susceptibility of the interface cysteine to a sulfhydryl reagent in the triosephosphate isomerases of two trypanosomes.

Proteins with great sequence similarity usually have similar structure, function and other physicochemical properties. But in many cases, one or more of the physicochemical or functional characteristics differ, sometimes very considerably, among these homologous proteins. To better understand how critical amino acids determine quantitative properties of function in proteins, the responsible residues must be located and identified. This can be difficult to achieve, particularly in cases where multiple amino acids are involved. In this work, two triosephosphate isomerases with very high similarity from two related human parasites were used to address one such problem. We demonstrate that a seventy-fold difference in the reactivity of an interface cysteine to the sulfhydryl reagent methylmethane sulfonate in these two enzymes depends on three amino acids located far away from this critical residue and which could not have been predicted using other current methods. Starting from previous observations with chimeric proteins involving these two triosephosphate isomerases, we developed a strategy involving additive mutant enzymes and selected site directed mutants to locate and identify the three amino acids. These three residues seem to induce changes in the interface cysteine in reactivity by increasing (or decreasing) its apparent pKa. Some enzymes with four to seven mutations also exhibited altered reactivity. This study completes a strategy for identifying key residues in the sequences of proteins that can have applications in future protein structure-function studies.

Analysis of endogenous H2S and H2Sn in mouse brain by high-performance liquid chromatography with fluorescence and tandem mass spectrometric detection.

Previous studies indicated that bound sulfur species (BSS), including hydrogen polysulfide (H2Sn), have various physiological functions in mammalian cells. Although H2Sn molecules have been considered as secondary metabolites derived from hydrogen sulfide (H2S) based on in vitro studies or predetermined reaction formula, the physiological form of BSS and their endogenous concentration remain unclear. In the present study, we aimed to improve the usual method using monobromobimane (mBB) followed by high performance liquid chromatographic (HPLC) analysis for HS－ for simultaneous determination of H2S, H2S2, H2S3 and cysteine persulfide in biological samples. We demonstrated that mBB derivatization of H2S and H2Sn standards under alkaline conditions (pH 9.5) induced significant decreases in H2S2 and H2S3 levels and a significant increase in the H2S level in an incubation time-dependent manner. Conversely, the derivatization of mBB adducts of H2S2 and H2S3 were stable under neutral conditions (pH 7.0), which is physiologically relevant. Therefore, we re-examined the method using mBB and applied an improved method for the evaluation of H2S, H2S2, and H2S3 in mouse brain under physiological pH conditions. The concentrations of H2S and H2S2 were 0.030 ± 0.004µmol/g protein and 0.026 ± 0.002µmol/g protein, respectively. Although the level of H2S3 was below the quantification limit of this method, H2S3 was detected in mouse brain. Using the method established here, we reveal for the first time the existence of endogenous H2S2 and H2S3 in mammalian brain tissues. H2S2 and H2S3 exert anti-oxidant activity and anti-carbonyl stress effects through the regulation of redox balance in neuronal cells. Thus, our observations provide novel insights into the physiological functions of BSS in the brain and into neuronal diseases involved in redox imbalance.

Discovery of Heteroaromatic Sulfones As a New Class of Biologically Compatible Thiol-Selective Reagents.

The selective reaction of chemical reagents with reduced protein thiols is critical to biological research. This reaction is utilized to prevent cross-linking of cysteine-containing peptides in common proteomics workflows and is applied widely in discovery and targeted redox investigations of the mechanisms underlying physiological and pathological processes. However, known and commonly used thiol blocking reagents like iodoacetamide, N-ethylmaleimide, and others were found to cross-react with oxidized protein sulfenic acids (-SOH) introducing significant errors in studies employing these reagents. We have investigated and are reporting here a new heteroaromatic alkylsulfone, 4-(5-methanesulfonyl-[1,2,3,4]tetrazol-1-yl)-phenol (MSTP), as a selective and highly reactive -SH blocking reagent compatible with biological applications.

Universal and Quantitative Method To Evaluate Inhibitor Potency for Cysteinome Proteins Using a Nonspecific Activity-Based Protein Profiling Probe.

Recently, there have been a limited number of new, validated targets for small-molecule drug discovery in the pharmaceutical industry. Although there are approximately 30 000 genes in the human genome, only 2% are targeted by currently approved small-molecule drugs. One reason that many targets remain neglected by drug discovery programs is the absence of biochemical assays enabling evaluation of the potency of inhibitors in a quantitative and high-throughput manner. To overcome this issue, we developed a biochemical assay to evaluate the potency of both reversible and irreversible inhibitors using a nonspecific thiol-labeling fluorescent probe. The assay can be applied to any targets with a cysteine residue in a pocket that can accommodate small-molecule ligands. By constructing a mathematical model, we showed that the potency of compounds can be quantitatively evaluated by performing an activity-based protein profiling assay. In addition, the validity of the theory was confirmed experimentally using epidermal growth factor receptor kinase as a model target. This approach provides an assay system for targets for which biochemical assays cannot be developed. Our approach can potentially not only expand the number of exploitable targets but also accelerate the lead optimization process by providing quantitative structure-activity relationship information.

Redox status of serum apolipoprotein E and its impact on HDL cholesterol levels.

BACKGROUND: Apolipoprotein E (apoE) is closely involved in the pathogenesis of apoE-related diseases, such as Alzheimer's disease and cardiovascular disease. The redox modulation of cysteine-thiols in a protein is involved in various pathophysiological regulations; however, that of apoE has not been studied in detail. Herein, we devised an analytical method to determine the redox status of serum apoE and assessed its relation to serum cholesterol levels and apoE phenotype. METHODS: The present method was based on a band shift assay, using a photocleavable maleimide-conjugated polyethylene glycol. RESULTS: The basic characteristics of the present method were found to be satisfactory to determine the redox status of serum apoE quantitatively. Serum apoE was separated into its reduced-form (r-), non-reduced-form (nr-), apoE-AII complex, and homodimer using this method. R-apoE could be detected as a 40-kDa band, whereas nr-apoE remained as monomeric apoE. R-apoE displayed a preference for VLDL; however, the levels showed the correlation with HDL-cholesterol levels (p<0.005). Redox status of serum apoE was significantly different among apoE phenotypes. The quantitative ratios of nr-apoE to total apoE in serum from subjects with apoE4/E3 were higher than in serum from subjects with apoE3/E3 (p<0.0001) and apoE3/E2 (p<0.001). CONCLUSION: The redox status of serum apoE might be related to the synthesis of HDL. The information concerning the redox status of serum apoE provided by the present method may be a potent indicator to evaluate various apoE-related diseases.

The extreme hyper-reactivity of selected cysteines drives hierarchical disulfide bond formation in serum albumin.

After mild reduction of serum albumin, seven among the 34 cysteines forming the disulfide network displayed a surprising hyper-reactivity. Compared to the thiol group of glutathione, the average reactivity of these cysteines towards disulfides and thiol reagents was more than 100 times higher. Using mass spectrometry and kinetic data, we identified all these unusual residues, with Cys75, Cys123 and Cys264 showing the highest reactivity. This effect was mainly due to a low pKa of the sulfhydryl groups and may explain the very fast formation of early disulfides in the nascent protein suggesting the existence of a hierarchical propensity to form such covalent links in selected regions during oxidative folding. An identical pattern of hyper-reactive cysteines was found in albumins from six different mammals. This hyper-reactivity is much higher than the one found in other proteins containing multiple cysteines only devoted to structural disulfide bonds. It is possible that such hyper-reactive cysteines could also be present in other proteins, although their existence has been completely ignored so far.

Characterization of Reactions between Water-Soluble Trialkylphosphines and Thiol Alkylating Reagents: Implications for Protein-Conjugation Reactions.

Water-soluble trialkylphosphines such as tris(carboxyethyl)phosphine (TCEP) and trishydroxypropyl phosphine (THPP) are effective agents for reducing disulfide bonds in proteins and are increasingly becoming the reagents of choice for bioconjugation strategies that modify cysteine (thiol containing) amino acids. These reducing agents are often considered as being chemically compatible with Michael acceptors such as maleimides and, as such, are often not removed prior to performing protein conjugation reactions. Here, we demonstrate the rapid and irreversible reaction of both TCEP and THPP with derivatives of the commonly employed thiol alkylating groups, maleimide and vinyl sulfone. Mechanistic investigations revealed distinct differences between the reactions of TCEP and THPP with maleimide, leading to the production of either nonproductive ylenes or succidimidyl derivatives, respectively. Importantly, we also demonstrate the incorporation of nonproductive ylenes formed between maleimide and TCEP into the Pneumococcal capsular polysaccharide Pn6b following strategies employed toward the production of conjugate vaccines.

Novel technological strategies to enhance tropical thiol precursors in winemaking by-products.

Grape pomace is a winemaking by-product that can be used to extract oenological tannins. Recently, some grape skin tannins were shown to contain very high amounts of two polyfunctional thiol precursors (3-S-glutathionylhexan-1-ol, 3-S-cysteinylhexan-1-ol) whose free forms are responsible for appreciated tropical-like flavours. This study shows that an oxidative treatment (no SO2) of white grape pomace and the presence of grape leaves and stems can increase the content of the above mentioned precursors. Moreover, it shows significant differences between Sauvignon Blanc, Gewuerztraminer and Mueller-Thurgau grape pomace for the 3-mercaptohexan-1-ol precursors and 4-S-cysteinyl-4-methylpentan-2-one. The grape cultivar is crucial, but the technological ability of enhancing the level of the volatile thiol precursors simply by treating the grape marc in different ways is a promising and powerful tool for the production of potentially flavouring tannins intended for food and beverage industry.

Synthesis of a Novel Cysteine-Incorporated Anthraquinone Derivative and Its Structural Properties.

A novel cysteine-incorporated anthraquinone derivative was synthesized, and its molecular structure was determined by X-ray crystal analysis. Each mercapto group was located separately and did not form a disulfide bond, and hydrogen bondings and π-π interaction were observed from the packing structure.

The Fifth Transmembrane Segment of Cystic Fibrosis Transmembrane Conductance Regulator Contributes to Its Anion Permeation Pathway.

Previous studies have identified several transmembrane segments (TMs), including TM1, TM3, TM6, TM9, TM11, and TM12, as pore-lining segments in cystic fibrosis transmembrane conductance regulator (CFTR), but the role of TM5 in pore construction remains controversial. In this study, we employed substituted cysteine accessibility methodology (SCAM) to screen the entire TM5 defined by the original topology model and its cytoplasmic extension in a Cysless background. We found six positions (A299, R303, N306, S307, F310, and F311) where engineered cysteines react to intracellular 2-sulfonatoethyl methanethiosulfonate (MTSES⁻). Quantification of the modification rate of engineered cysteines in the presence or absence of ATP suggests that these six residues are accessible in both the open and closed states. Whole-cell experiments with external MTSES⁻ identified only two positive positions (L323 and A326), resulting in a segment containing 11 consecutive amino acids, where substituted cysteines respond to neither internal nor external MTSES⁻, a unique feature not seen previously in CFTR's pore-lining segments. The observation that these positions are inaccessible to channel-permeant thiol-specific reagent [Au(CN)2]⁻ suggests that this segment of TM5 between F311 and L323 is concealed from the pore by other TMs and/or lipid bilayers. In addition, our data support the idea that the positively charged arginine at position 303 poses a pure electrostatic action in determining the single-channel current amplitude of CFTR and the effect of an open-channel blocker glibencalmide. Collectively, we conclude that the cytoplasmic portion of CFTR's TM5 lines the pore. Our functional data are remarkably consistent with predicted structural arrangements of TM5 in some homology models of CFTR.

Controlled heterogeneous stem cell differentiation on a shape memory hydrogel surface.

The success of stem cell therapies is highly dependent on the ability to control their programmed differentiation. So far, it is commonly believed that the differentiation behavior of stem cells is supposed to be identical when they are cultured on the same homogeneous platform. However, in this report, we show that this is not always true. By utilizing a double-ion-triggered shape memory effect, the pre-seeded hMSCs were controllably located in different growth positions. Here, we demonstrate for the first time that the differentiation behavior of hMSCs is highly sensitive to their growth position on a hydrogel scaffold. This work will not only enrich the mechanisms for controlling the differentiation of stem cells, but also offer a one-of-a-kind platform to achieve a heterogeneously differentiated stem cell-seeded hydrogel scaffold for complex biological applications.

Reconstitution of respiratory complex I on a biomimetic membrane supported on gold electrodes.

For the first time, respiratory complex I has been reconstituted on an electrode preserving its structure and activity. Respiratory complex I is a membrane-bound enzyme that has an essential function in cellular energy production. It couples NADH:quinone oxidoreduction to translocation of ions across the cellular (in prokaryotes) or mitochondrial membranes. Therefore, complex I contributes to the establishment and maintenance of the transmembrane difference of electrochemical potential required for adenosine triphosphate synthesis, transport, and motility. Our new strategy has been applied for reconstituting the bacterial complex I from Rhodothermus marinus onto a biomimetic membrane supported on gold electrodes modified with a thiol self-assembled monolayer (SAM). Atomic force microscopy and faradaic impedance measurements give evidence of the biomimetic construction, whereas electrochemical measurements show its functionality. Both electron transfer and proton translocation by respiratory complex I were monitored, simulating in vivo conditions.

Techniques for the analysis of cysteine sulfhydryls and oxidative protein folding.

SIGNIFICANCE: Modification of cysteine thiols dramatically affects protein function and stability. Hence, the abilities to quantify specific protein sulfhydryl groups within complex biological samples and map disulfide bond structures are crucial to gaining greater insights into how proteins operate in human health and disease. RECENT ADVANCES: Many different molecular probes are now commercially available to label and track cysteine residues at great sensitivity. Coupled with mass spectrometry, stable isotope-labeled sulfhydryl-specific reagents can provide previously unprecedented molecular insights into the dynamics of cysteine modification. Likewise, the combined application of modern mass spectrometers with improved sample preparation techniques and novel data mining algorithms is beginning to routinize the analysis of complex protein disulfide structures. CRITICAL ISSUES: Proper application of these modern tools and techniques, however, still requires fundamental understanding of sulfhydryl chemistry as well as the assumptions that accompany sample preparation and underlie effective data interpretation. FUTURE DIRECTIONS: The continued development of tools, technical approaches, and corresponding data processing algorithms will, undoubtedly, facilitate site-specific protein sulfhydryl quantification and disulfide structure analysis from within complex biological mixtures with ever-improving accuracy and sensitivity. Fully routinizing disulfide structure analysis will require an equal but balanced focus on sample preparation and corresponding mass spectral dataset reproducibility.