Intramolecular Disulfide Formation Research Articles

Rapid Formation of Intramolecular Disulfide Bridges using Light: An Efficient Method to Control the Conformation and Function of Bioactive Peptides.

Disulfide bonds are widely found in natural peptides and play a pivotal role in stabilizing their secondary structures, which are highly associated with their biological functions. Herein, we introduce a light-mediated strategy to effectively control the formation of disulfides. Our strategy is based on 2-nitroveratryl (oNv), a widely used photolabile motif, which serves both as a photocaging group and an oxidant (after photolysis). We demonstrated that irradiation of oNv-caged thiols with UV light could release free thiols that are rapidly oxidized by locally released byproduct nitrosoarene, leading to a "break-to-bond" fashion. This strategy is highlighted by the in situ restoration of the antimicrobial peptide tachyplesin I (TPI) from its external disulfide-caged analogue TPI-1. TPI-1 exhibits a distorted structure and a diminished function. However, upon irradiation, the β-hairpin structure and membrane activity of TPI were largely restored via rapid intramolecular disulfide formation. Our study proposes a powerful method to regulate the conformation and function of peptides in a spatiotemporal manner, which has significant potential for the design of disulfide-centered light-responsive systems.

Folding-Assisted Peptide Disulfide Formation and Dimerization.

Disulfide bonds form covalent bonds between distal regions of peptides and proteins to dramatically impact their folding, stability, and oligomerization. Given the prevalence of disulfide bonds in many natural products, considerable effort has been invested in site-selective disulfide bond formation approaches to control the folding of chemically synthesized peptides and proteins. Here, we show that the careful choice of thiol oxidation conditions can lead to monomeric or dimeric species from fully deprotected linear bisthiol peptides. Starting from a p53-derived peptide, we found that oxidation under aqueous (nondenaturing) conditions produces antiparallel dimers with enhanced α-helical character, while oxidation under denaturing conditions promotes formation of a nonhelical intramolecular disulfide species. Examination across peptide variants suggests that intramolecular disulfide formation is robust across diverse peptide sequences, while dimerization is sensitive to both the α-helical folding of the linear peptide and aromatic residues at the dimerization interface. All disulfide species are more resistant to protease degradation than the linear peptide but are easily reduced to restore the initial bisthiol peptide. Both disulfide formation approaches are compatible with α-helix-stabilizing cross-linkers. These results provide an approach for using disulfide bonds to control peptide folding and oligomerization to better understand how folding influences interactions with diverse molecular targets.

Oligodeoxynucleotides Modified with 2'-O-(Cysteinylaminobutyl)carbamoylethylribothymidine Residues for Native Chemical Ligation with Peptide at Internal Positions.

We used native chemical ligation (NCL) to synthesize a 2'-O-{N-[N-(S-tert-butylthiocysteinyl)aminobutyl]carbamoylethyl} (CysBCE) ribothymidine-derived oligonucleotide to expand the variety of peptide conjugation sites, allowing the incorporation of peptides at the 2'-hydroxy group when the oligonucleotide forms a duplex with the complementary strand. The NCL reaction with a peptide thioester and the modified oligonucleotide proceeded smoothly even when the CysBCE modification was in the middle of the oligonucleotide sequence. In addition, we incorporated two CysBCEs into an oligonucleotide to conjugate two peptides to one oligonucleotide. The results indicated that the tandem NCL reactions proceeded efficiently when the oligonucleotide hybridized to the complementary strand to avoid intramolecular disulfide formation between the two CysBCE groups. This method could be useful for peptide conjugation on the 2'-position.

HaloTag Forms an Intramolecular Disulfide.

HaloTag is a modified haloalkane dehalogenase used for many applications in chemical biology including protein purification, cell-based imaging, and cytosolic penetration assays. While working with purified, recombinant HaloTag protein, we discovered that HaloTag forms an internal disulfide bond under oxidizing conditions. In this work, we describe this internal disulfide formation and the conditions under which it occurs, and we identify the relevant cysteine residues. Further, we develop a mutant version of HaloTag, HaloTag8, that maintains activity while avoiding internal disulfide formation altogether. While there is no evidence that HaloTag is prone to disulfide formation in intracellular environments, researchers using recombinant HaloTag, HaloTag expressed on the cell surface, or HaloTag in the extracellular space might consider using HaloTag8 to avoid intramolecular disulfide formation.

Discovery of a Macropinocytosis‐Inducing Peptide Potentiated by Medium‐Mediated Intramolecular Disulfide Formation

AbstractMacropinocytosis is a ubiquitous cellular uptake mechanism of peptide‐based intracellular delivery. This entry pathway shows promise as a route for the intracellular uptake of biomacromolecules and nanoparticles. In this work, we obtained the 8‐residue analogue P4A bearing higher macropinocytosis induction ability. P4A contains vital cysteine residues in its sequence, which immediately reacts with cystine in culture medium to convert into its oxidized forms, including the intramolecularly oxidized form (oxP4A) as the dominant and active species. The conjugate of oxP4A and the membrane lytic peptide LK15 delivered bioactive proteins into cells; notably, this peptide delivered functional proteins fused with a negatively charged protein tag at a significantly reduced amount (up to nanomolar range) without compromising the delivery efficiency and the cellular activities of delivered proteins.

Discovery of a Macropinocytosis-Inducing Peptide Potentiated by Medium-Mediated Intramolecular Disulfide Formation.

Macropinocytosis is a ubiquitous cellular uptake mechanism of peptide-based intracellular delivery. This entry pathway shows promise as a route for the intracellular uptake of biomacromolecules and nanoparticles. In this work, we obtained the 8-residue analogue P4A bearing higher macropinocytosis induction ability. P4A contains vital cysteine residues in its sequence, which immediately reacts with cystine in culture medium to convert into its oxidized forms, including the intramolecularly oxidized form (oxP4A) as the dominant and active species. The conjugate of oxP4A and the membrane lytic peptide LK15 delivered bioactive proteins into cells; notably, this peptide delivered functional proteins fused with a negatively charged protein tag at a significantly reduced amount (up to nanomolar range) without compromising the delivery efficiency and the cellular activities of delivered proteins.

Redox sensor properties of human cytoglobin allosterically regulate heme pocket reactivity

Cytoglobin is a conserved hemoprotein ubiquitously expressed in mammalian tissues, which conducts electron transfer reactions with proposed signaling functions in nitric oxide (NO) and lipid metabolism. Cytoglobin has an E7 distal histidine (His81), which unlike related globins such as myoglobin and hemoglobin, is in equilibrium between a bound, hexacoordinate state and an unbound, pentacoordinate state. The His81 binding equilibrium appears to be allosterically modulated by the presence of an intramolecular disulfide between two cysteines (Cys38 and Cys83). The formation of this disulfide bridge regulates nitrite reductase activity and lipid binding. Herein, we attempt to clarify the effects of defined thiol oxidation states on small molecule binding of cytoglobin heme, using cyanide binding to probe the ferric state. Cyanide binding kinetics to wild-type cytoglobin reveal at least two kinetically distinct subpopulations, depending on thiol oxidation states. Experiments with covalent thiol modification by NEM, glutathione, and amino acid substitutions (C38S, C83S and H81A), indicate that subpopulations ranging from fully reduced thiols, single thiol oxidation, and intramolecular disulfide formation determine heme binding properties by modulating the histidine-heme affinity and ligand binding. The redox modulation of ligand binding is sensitive to physiological levels of hydrogen peroxide, with a functional midpoint redox potential for the native cytoglobin intramolecular disulfide bond of −189 ± 4 mV, a value within the boundaries of intracellular redox potentials. These results support the hypothesis that Cys38 and Cys83 on cytoglobin serve as sensitive redox sensors that modulate the cytoglobin distal heme pocket reactivity and ligand binding.

Receptor-independent modulation of cAMP-dependent protein kinase and protein phosphatase signaling in cardiac myocytes by oxidizing agents

The contraction and relaxation of the heart is controlled by stimulation of the β1-adrenoreceptor (AR) signaling cascade, which leads to activation of cAMP-dependent protein kinase (PKA) and subsequent cardiac protein phosphorylation. Phosphorylation is counteracted by the main cardiac protein phosphatases, PP2A and PP1. Both kinase and phosphatases are sensitive to intramolecular disulfide formation in their catalytic subunits that inhibits their activity. Additionally, intermolecular disulfide formation between PKA type I regulatory subunits (PKA-RI) has been described to enhance PKA's affinity for protein kinase A anchoring proteins, which alters its subcellular distribution. Nitroxyl donors have been shown to affect contractility and relaxation, but the mechanistic basis for this effect is unclear. The present study investigates the impact of several nitroxyl donors and the thiol-oxidizing agent diamide on cardiac myocyte protein phosphorylation and oxidation. Although all tested compounds equally induced intermolecular disulfide formation in PKA-RI, only 1-nitrosocyclohexalycetate (NCA) and diamide induced reproducible protein phosphorylation. Phosphorylation occurred independently of β1-AR activation, but was abolished after pharmacological PKA inhibition and thus potentially attributable to increased PKA activity. NCA treatment of cardiac myocytes induced translocation of PKA and phosphatases to the myofilament compartment as shown by fractionation, immunofluorescence, and proximity ligation assays. Assessment of kinase and phosphatase activity within the myofilament fraction of cardiac myocytes after exposure to NCA revealed activation of PKA and inhibition of phosphatase activity thus explaining the increase in phosphorylation. The data suggest that the NCA-mediated effect on cardiac myocyte protein phosphorylation orchestrates alterations in the kinase/phosphatase balance.

A suitable and effective stepwise oxidative refolding procedure for highly‐cationic tetrameric avidin in nucleic acid free conditions

Optimized conditions are needed to refold recombinant proteins from bacterial inclusion bodies into their biologically active conformations. In this study, we found two crucial requirements for efficient refolding of cationic tetrameric chicken avidin. The first step is to eliminate nucleic acid contaminants from the bacterial inclusion body. The electrostatic interactions between the remaining nucleic acids and proteins strongly enhanced protein aggregation during the refolding process. The cysteine specific reversible S-cationization procedure was successfully employed for large-scale preparation of nucleic acid free denatured protein without purification tag system. The second step is the intramolecular disulfide formation prior to refolding in dialysis removing denaturant. Disulfide intact monomeric avidin showed efficient formation of biologically active tetrameric conformation during the refolding process. Using this optimized refolding procedure, highly cationic avidin derivative designed as an intracellular delivery carrier of biotinylated protein was successfully prepared.

Oxidative misfolding of Cu/Zn-superoxide dismutase triggered by non-canonical intramolecular disulfide formation

Misfolded Cu/Zn-superoxide dismutase (SOD1) is a pathological species in a subset of amyotrophic lateral sclerosis (ALS). Oxidative stress is known to increase in affected spinal cords of ALS and is thus considered to cause damages on SOD1 leading to the misfolding and aggregation. Despite this, it still remains elusive what triggers misfolding of SOD1 under oxidizing environment. Here, we show that a thiol group of Cys111 in SOD1 is oxidized to a sulfenic acid with hydrogen peroxide and reveal that further dissociation of the bound metal ions from the oxidized SOD1 allows another free Cys residue (Cys6) to nucleophilically attack the sulfenylated Cys111. As a result, an intra-molecular disulfide bond forms between Cys6 and Cys111. Such an abnormal SOD1 with the non-canonical disulfide bond was conformationally extended with significant cytotoxicity as well as high propensity to aggregate. Taken together, we propose a new model of SOD1 misfolding under oxidizing environment, in which formation of the non-canonical intramolecular disulfide bond plays a pivotal role.

Construction of disulfide bonds in peptides via immobilized platinum(IV) complex oxidation

A novel TentaGel resin supported platinum(IV) complex named TentaGel-Pt(IV) was synthesized and characterized by SEM, XPS, ICP-MS and UV–vis spectrophotometry. The formation of intramolecular disulfides in eleven dithiol-containing peptides of variable lengths using TentaGel-Pt(IV) was investigated at room temperature. The disulfide-containing peptides were formed in excellent oxidation yields and were easily separated by filtration upon completion of the reaction. Excellent reusability of TentaGel-Pt(IV) was also achieved. The mechanism for construction of the disulfide bond in peptides via TentaGel-Pt(IV) oxidation was proposed.

Dehydroascorbic acid S-Thiolation of peptides and proteins: Role of homocysteine and glutathione

Ascorbic acid (vitamin C) plays a significant role in the prevention of oxidative stress. In this process, ascorbate is oxidized to dehydroascorbate (DHA). We have investigated the impact of DHA on peptide/protein intramolecular disulfide formation as well as S-glutathionylation and S-homocysteinylation. S-glutathionylation of peptides/proteins is a reversible, potential regulatory mechanism in oxidative stress. Although the exact role of protein S-homocysteinylation is unknown, it has been proposed to be of importance in pathobiological processes such as onset of cardiovascular disease. Using an in vitro model system, we demonstrate that DHA causes disulfide bond formation within the active site of recombinant human glutaredoxin (Grx-1). DHA also facilities the formation of S-glutathionylation and S-homocysteinylation of a model peptide (AcFHACAAK) as well as Grx-1. We discuss the possible mechanisms of peptide/protein S-thiolation, which can occur either via thiol exchange or a thiohemiketal intermediate. A thiohemiketal DHA-peptide adduct was detected by mass spectrometry and its location on the peptide/protein cysteinyl thiol group was unambiguously confirmed by tandem mass spectrometry. This demonstrates that peptide/protein S-thiolation mediated by DHA is not limited to thiol exchange reactions but also takes place directly via the formation of a thiohemiketal peptide intermediate. Finally, we investigated a potential reducing role of glutathione (GSH) in the presence of S-homocysteinylated peptide/protein adducts. S-homocysteinylated AcFHACAAK, human hemoglobin α-chain and Grx-1 were incubated with GSH. Both peptide and proteins were reduced, and homocysteine replaced with GS-adducts by thiol exchange, as a function of time.

Upstream signaling events leading to elevated production of pro-survival nitric oxide in photodynamically-challenged glioblastoma cells

Nitric oxide (NO) generated endogenously by inducible nitric oxide synthase (iNOS) promotes growth and migration/invasion of glioblastoma cells and also fosters resistance to chemotherapy and ionizing radiotherapy. Our recent studies revealed that glioblastoma cell iNOS/NO also opposes the cytotoxic effects of non-ionizing photodynamic therapy (PDT), and moreover stimulates growth/migration aggressiveness of surviving cells. These negative responses, which depended on PI3K/Akt/NF-κB activation, were strongly suppressed by blocking iNOS transcription with JQ1, a BET bromodomain inhibitor. In the present study, we sought to identify additional molecular events that precede iNOS transcriptional upregulation. Akt activation, iNOS induction, and viability loss in PDT-challenged glioblastoma U87 cells were all strongly inhibited by added l-histidine, consistent with primary involvement of photogenerated singlet oxygen (1O2). Transacetylase p300 not only underwent greater Akt-dependent activation after PDT, but greater interaction with NF-κB subunit p65, which in turn exhibited greater K310 acetylation. In addition, PDT promoted intramolecular disulfide formation and inactivation of tumor suppressor PTEN, thereby favoring Akt and p300 activation leading to iNOS upregulation. Importantly, deacetylase Sirt1 was down-regulated by PDT stress, consistent with the observed increase in p65-acK310 level, which fostered iNOS transcription. This study provides new mechanistic insights into how glioblastoma tumors can exploit iNOS/NO to not only resist PDT, but to attain a more aggressive survival phenotype.

Structural snapshots of OxyR reveal the peroxidatic mechanism of H2O2 sensing

Hydrogen peroxide (H2O2) is a strong oxidant capable of oxidizing cysteinyl thiolates, yet only a few cysteine-containing proteins have exceptional reactivity toward H2O2 One such example is the prokaryotic transcription factor OxyR, which controls the antioxidant response in bacteria, and which specifically and rapidly reduces H2O2 In this study, we present crystallographic evidence for the H2O2-sensing mechanism and H2O2-dependent structural transition of Corynebacterium glutamicum OxyR by capturing the reduced and H2O2-bound structures of a serine mutant of the peroxidatic cysteine, and the full-length crystal structure of disulfide-bonded oxidized OxyR. In the H2O2-bound structure, we pinpoint the key residues for the peroxidatic reduction of H2O2, and relate this to mutational assays showing that the conserved active-site residues T107 and R278 are critical for effective H2O2 reduction. Furthermore, we propose an allosteric mode of structural change, whereby a localized conformational change arising from H2O2-induced intramolecular disulfide formation drives a structural shift at the dimerization interface of OxyR, leading to overall changes in quaternary structure and an altered DNA-binding topology and affinity at the catalase promoter region. This study provides molecular insights into the overall OxyR transcription mechanism regulated by H2O2.

Direct Peptide Cyclization and One-Pot Modification Using the MeDbz Linker.

The one-pot synthesis and modification of cyclic peptides through a self-cleaving on-resin protocol is described. We apply Dawson's MeDbz linker to achieve direct intramolecular peptide cyclization by thioesterification followed by S → N acyl shift. This native chemical ligation approach requires no activating additive and allows direct modification of the crude cyclic peptides in one-pot. The strategy was applied to synthesize 5 cyclic peptide natural products of varying ring size. Finally, one-pot modifications include desulfurization, fluorophore conjugation, and intramolecular disulfide formation.

Synthesis and Characterization of the R27S Genetic Variant of Insulin-like Peptide 5.

We report the synthesis and in vitro bioactivity assessment for an insulin-like peptide 5 (INSL5) analogue that was recently discovered as a genetic mutation in an Amish population. The mutation was associated with improved metabolic status, and receptor-based antagonism was proposed as a potential mechanism for the altered phenotype. We determined the specific peptide analogue to be fully potent and of maximal efficacy at the human relaxin family peptide receptor 4 (RXFP4), suggesting an alternative basis for the observed effect. In preparation of this synthetically challenging hormone, we have introduced several improvements such as implementation of isoacyl chemistry for high-efficiency preparation of INSL5 B-chain and selective intramolecular A6-11 disulfide formation as a first step in sequential disulfide assembly.

A scaffold protein that chaperones a cysteine-sulfenic acid in H2O2 signaling.

In Saccharomyces cerevisiae, Yap1 regulates an H2O2-inducible transcriptional response that controls cellular H2O2 homeostasis. H2O2 activates Yap1 by oxidation through the intermediary of the thiol peroxidase Orp1. Upon reacting with H2O2, Orp1 catalytic cysteine oxidizes to a sulfenic acid, which then engages into either an intermolecular disulfide with Yap1, leading to Yap1 activation, or an intramolecular disulfide that commits the enzyme into its peroxidatic cycle. How the first of these two competing reactions, which is kinetically unfavorable, occurs was previously unknown. We show that the Yap1-binding protein Ybp1 brings together Orp1 and Yap1 into a ternary complex that selectively activates condensation of the Orp1 sulfenylated cysteine with one of the six Yap1 cysteines while inhibiting Orp1 intramolecular disulfide formation. We propose that Ybp1 operates as a scaffold protein and as a sulfenic acid chaperone to provide specificity in the transfer of oxidizing equivalents by a reactive sulfenic acid species.

Synthesis of Reusable Silica Nanosphere-Supported Pt(IV) Complex for Formation of Disulfide Bonds in Peptides.

Some peptide-based drugs, including oxytocin, vasopressin, ziconotide, pramlintide, nesiritide, and octreotide, contain one intramolecular disulfide bond. A novel and reusable monodispersed silica nanosphere-supported Pt(IV) complex (SiO2@TPEA@Pt(IV)); TPEA: N-[3-(trimethoxysilyl)propyl]ethylenediamine) was synthesized via a four-step procedure and was used for the formation of intramolecular disulfide bonds in peptides. Transmission electron microscopy (TEM) and chemical mapping results for the Pt(II) intermediates and for SiO2@TPEA@Pt(IV) show that the silica nanospheres possess a monodisperse spherical structure and contain uniformly-distributed Si, O, C, N, Cl, and Pt. The valence state of Pt on the silica nanospheres was characterized by X-ray photoelectron spectroscopy (XPS). The Pt(IV) loaded on SiO2@TPEA@Pt(IV) was 0.15 mmol/g, as determined by UV-VIS spectrometry. The formation of intramolecular disulfides in six dithiol-containing peptides of variable lengths by the use of SiO2@TPEA@Pt(IV) was investigated, and the relative oxidation yields were determined by high-performance liquid chromatography (HPLC). In addition, peptide 1 (Ac-CPFC-NH2) was utilized to study the reusability of SiO2@TPEA@Pt(IV). No significant decrease in the relative oxidation yield was observed after ten reaction cycles. Moreover, the structure of SiO2@TPEA@Pt(IV) after being used for ten cycles was determined to be similar to its initial one, demonstrating the cycling stability of the complex.

Impact of posttranslational modifications of engineered cysteines on the substituted cysteine accessibility method: evidence for glutathionylation.

The substituted cysteine accessibility method (SCAM) is widely used to study the structure and function of channels, receptors and transporters. In its usual application, a cysteine residue is introduced into a protein which lacks native cysteines following which the accessibility of the residue to the aqueous compartment is assessed. Implicit, and generally assumed, is that if the cysteine-substituted residue is not available to react with sulfhydryl reagents it is not exposed to the extracellular compartment or within the aqueous translocation pathway. We demonstrate here, in a Hela-derived cell line, that some cysteine-substituted residues of the proton-coupled folate transporter (PCFT, SLC46A1) that are inaccessible to 2-((biotinoyl)amino)ethyl methanethiosulfonate are glutathionylated by biotinylated glutathione ethyl ester in the absence of an oxidizing agent. Intramolecular disulfide formation involving cysteine-substituted residues was also identified in some instances. These posttranslational modifications limit the accessibility of the cysteine residues to sulfhydryl-reactive reagents and can have a profound impact on the interpretation of SCAM but may not alter function. When a posttranslationally modified residue is used as a reference extracellular control, the high level of exposure required for detection on Western blot results in erroneous detection of otherwise inaccessible intracellular cysteine-substituted residues. The data indicate that in the application of SCAM, when a cysteine-substituted residue does not appear to be accessible to sulfhydryl-reactive reagents, the possibility of a posttranslational modification should be excluded. The data explain the discrepancies in the assessment, and confirm the localization, of the first intracellular loop of PCFT.

Sensing and signaling of oxidative stress in chloroplasts by inactivation of the SAL1 phosphoadenosine phosphatase

Intracellular signaling during oxidative stress is complex, with organelle-to-nucleus retrograde communication pathways ill-defined or incomplete. Here we identify the 3'-phosphoadenosine 5'-phosphate (PAP) phosphatase SAL1 as a previously unidentified and conserved oxidative stress sensor in plant chloroplasts. Arabidopsis thaliana SAL1 (AtSAL1) senses changes in photosynthetic redox poise, hydrogen peroxide, and superoxide concentrations in chloroplasts via redox regulatory mechanisms. AtSAL1 phosphatase activity is suppressed by dimerization, intramolecular disulfide formation, and glutathionylation, allowing accumulation of its substrate, PAP, a chloroplast stress retrograde signal that regulates expression of plastid redox associated nuclear genes (PRANGs). This redox regulation of SAL1 for activation of chloroplast signaling is conserved in the plant kingdom, and the plant protein has evolved enhanced redox sensitivity compared with its yeast ortholog. Our results indicate that in addition to sulfur metabolism, SAL1 orthologs have evolved secondary functions in oxidative stress sensing in the plant kingdom.