Intact Disulfide Bonds Research Articles

The Marine Antimicrobial Peptide AOD with Intact Disulfide Bonds Has Remarkable Antibacterial and Anti-Biofilm Activity.

American Oyster Defensin (AOD) is a marine peptide that is derived from North American mussels. It has been demonstrated to exhibit potent antimicrobial activity and high safety in both in vitro and in vivo models. In this study, to facilitate synthesis, mutants of AOD with fewer disulfide bonds were designed and subjected to structural, antimicrobial, and anti-biofilm analysis. The antimicrobial activity of AOD-derived peptides decreased after reduction in the disulfide bond, and among its three derivatives, only AOD-1 inhibited very few bacteria with a MIC value of 64 μg/mL, whereas the others had no inhibitory effect on pathogenic bacteria. The findings demonstrated that full disulfide bonds are indispensable for bactericidal activity, with the α-helix playing a pivotal role in inhibiting bacterial membranes. Furthermore, the results of the ATP, ROS, membrane potential, and membrane fluidity assays demonstrated that intracellular ATP, reactive oxygen species, and membrane fluidity were all increased, while membrane potential was reduced. This indicated that AOD resulted in the impairment of membrane fluidity and induced metabolic disorders, ultimately leading to bacterial death. The inhibitory effect of AOD on the biofilm of S. epidermidis G-81 was determined through the crystal violet and confocal microscopy. The results demonstrated that AOD exhibited a notable inhibitory impact on the biofilm of S. epidermidis G-81. The minimum biofilm inhibitory concentration of AOD on S. epidermidis G-81 was 16 μg/mL, and the minimum biofilm scavenging concentration was 32 μg/mL, which exhibited superior efficacy compared to that of lincomycin. The inhibitory effect on the primary biofilm was 90.3%, and that on the mature biofilm was 82.85%, with a dose-dependent inhibition effect. Concurrently, AOD cleared intra-biofilm organisms and reduced the number of biofilm-holding bacteria by six orders of magnitude. These data indicate that disulfide bonds are essential to the structure and activity of AOD, and AOD may potentially become an effective dual-action antimicrobial and anti-biofilm agent.

B-247 A Missing Peptide Link: A Case Study Involving Detection of an Unusual IGF-1 Protein Form Using High-Resolution Mass Spectrometry

Abstract Background Previously, we described a method for monitoring insulin-like growth factor-1 (IGF-1) variants using liquid chromatography coupled with high-resolution, accurate-mass, mass spectrometry (LC-HRAM-MS). The method split s variants in 4 Variant Groups (VG) and characterizes them by the Isotopic Peak index (IPi) and relative retention time (rRT). Uncommon variants are analyzed by tandem mass-spectrometry (MS/MS) and specimens suspected to have a novel variant are deidentified and routed for DNA sequencing. A potentially novel species was detected that did not match characteristics of any previously identified IGF-1 variant. Methods LC-HRAM-MS was used for the detection of wild type (WT) IGF-1 protein and its variants in a serum specimen. Results The patient’s IGF-1 WT concentration was 210 ng/mL and z-score was -1.5, which were within normal reference ranges values for their sex and age. LC-HRAM-MS also revealed the isotopic distribution and charge for the variant, but with new characteristics (VG2:IP2, rRT= 0.01 min). The area under curve for the variant was 36% of that of the WT, which is a stark contrast to the usual 100% found in a typical heterozygous patient. MS/MS of the suspected variant showed 2 sets of b- and y-ions, which has not been seen in our laboratory. Sequencing of the fragments revealed that one set of C- and N-terminal amino acids were from the WT (C-terminus GPETL sequence and N-terminus PLKPAKSA sequence). The intact molecular weight (MW) of the species (monoisotopic m/z 1095.5250 at charge 7+) was 18 Da heavier than that of the WT (monoisotopic m/z 1092.9482 at charge 7+), so further investigation was warranted. The other set of b- and y-ions sequenced were manually identified as GL(or I)VD and PTGYG, respectively. BLAST search did not identify relevant proteins with these N- and C-termini. However, closer inspection revealed that identified sequences were found in the WT protein, albeit not at its termini. The rest of the amino acids were found that complete termini: C-terminal b-ions sequence RAPQTGIVD corresponded to positions 37-45 of the WT sequence, while N-terminal y-ions sequence KPTGYGSSSR corresponded to positions 27-36. All evidence indicates that the species is the WT protein, but with loss of a peptide bond between 2 arginines at positions 36-37 resulting in a gain of a water molecule to “cap” the newly formed termini, which is confirmed by its observed intact m/z. Conclusions We identified an extremely rare IGF-1 species formed through loss of a peptide bond between arginines at positions 36 and 37 resulting in a “missing link” in IGF-1 amino acid sequence. Despite the cleavage, the variant was a single protein molecule with intact disulfide bonds and 2 sets of C- and N-termini. The processes involved in generating this species (e.g., hydrolysis or enzymatic cleavage) and potential consequences for the patient are unknown. Because binding to receptors and binding proteins are potentially affected, and the total level of active WT IGF-1 is reduced, we are working with patient’s physician to establish whether this unusual form of IGF-1 protein may contribute to the etiology of the patient’s condition.

Effects of variable domain orientation on anti-HER2 single-chain variable fragment antibody expressed in the Escherichia coli cytoplasm.

Single-chain variable fragment (scFv) antibodies have great potential for a range of applications including as diagnostic and therapeutic agents. However, production of scFvs is challenging because proper folding and activity depend on the formation of two intrachain disulfide bonds that do not readily form in the cytoplasm of living cells. Functional expression in bacteria therefore involves targeting to the more oxidizing periplasm, but yields in this compartment can be limiting due to secretion bottlenecks and the relatively small volume compared to the cytoplasm. In the present study, we evaluated an anti-HER2 scFv, which is specific for human epidermal growth receptor 2 (HER2) overexpressed in breast cancer, for functional expression in the cytoplasm of Escherichia coli strains BL21(DE3) and SHuffle T7 Express, the latter of which is genetically engineered for cytoplasmic disulfide bond formation. Specifically, we observed much greater solubility and binding activity with SHuffle T7 Express cells, which likely resulted from the more oxidative cytoplasm in this strain background. We also found that SHuffle T7 Express cells were capable of supporting high-level soluble production of anti-HER2 scFvs with intact disulfide bonds independent of variable domain orientation, providing further evidence that SHuffle T7 Express is a promising host for laboratory and preparative expression of functional scFv antibodies.

Top-Down Characterization of Proteins with Intact Disulfide Bonds Using Activated-Ion Electron Transfer Dissociation.

Here we report the fragmentation of disulfide linked intact proteins using activated-ion electron transfer dissociation (AI-ETD) for top-down protein characterization. This fragmentation method is then compared to the alternative methods of beam-type collisional activation (HCD), electron transfer dissociation (ETD), and electron transfer and higher-energy collision dissociation (EThcD). We analyzed multiple precursor charge states of the protein standards bovine insulin, α-lactalbumin, lysozyme, β-lactoglobulin, and trypsin inhibitor. In all cases, we found that AI-ETD provides a boost in protein sequence coverage information and the generation of fragment ions from within regions enclosed by disulfide bonds. AI-ETD shows the largest improvement over the other techniques when analyzing highly disulfide linked and low charge density precursor ions. This substantial improvement is attributed to the concurrent irradiation of the gas phase ions while the electron-transfer reaction is taking place, mitigating nondissociative electron transfer, helping unfold the gas phase protein during the electron transfer event, and preventing disulfide bond reformation. We also show that AI-ETD is able to yield comparable sequence coverage information when disulfide bonds are left intact relative to proteins that have been reduced and alkylated. This work demonstrates that AI-ETD is an effective fragmentation method for the analysis of proteins with intact disulfide bonds, dramatically enhancing sequence ion generation and total sequence coverage compared to HCD and ETD.

Influence of reaction conditions on the oxidation of thiol groups in model peptidomimetic oligoureas.

Working with thiols is associated with the risk of unwanted oxidation reactions, which includes the formation of disulfides. Here, the use of short peptidomimetic oligoureas as a model system to study the influence of reaction conditions on the thiol oxidation process is described. The formation of disulfide bond is usually performed in aqueous solutions; in this study, it was shown that the oxidation reaction occurs also in organic medium. It was discovered that the rate of the oxidation reaction strongly depends on the type of solvent, which is the consequence of the change of the oxidant (air or DMSO), the temperature, and the presence (or absence) of base. The details reported here may help those working with thiols to find suitable conditions to control the oxidation behaviour in organic solvents, ensuring that ─SH groups either remain intact or form disulfide bonds as desired.

Cu/Zn Superoxide Dismutase Forms Amyloid Fibrils under Near-Physiological Quiescent Conditions: The Roles of Disulfide Bonds and Effects of Denaturant.

Cu/Zn superoxide dismutase (SOD1) forms intracellular aggregates that are pathological indicators of amyotrophic lateral sclerosis. A large body of research indicates that the entry point to aggregate formation is a monomeric, metal-ion free (apo), and disulfide-reduced species. Fibril formation by SOD1 in vitro has typically been reported only for harsh solvent conditions or mechanical agitation. Here we show that monomeric apo-SOD1 in the disulfide-reduced state forms fibrillar aggregates under near-physiological quiescent conditions. Monomeric apo-SOD1 with an intact intramolecular disulfide bond is highly resistant to aggregation under the same conditions. A cysteine-free variant of SOD1 exhibits fibrillization behavior and fibril morphology identical to those of disulfide-reduced SOD1, firmly establishing that intermolecular disulfide bonds or intramolecular disulfide shuffling are not required for aggregation and fibril formation. The decreased lag time for fibril formation resulting from reduction of the intramolecular disulfide bond thus primarily reflects the decreased stability of the folded state relative to partially unfolded states, rather than an active role of free sulfhydryl groups in mediating aggregation. Addition of urea to increase the amount of fully unfolded SOD1 increases the lag time for fibril formation, indicating that the population of this species does not dominate over other factors in determining the onset of aggregation. Our results contrast with previous results obtained for agitated samples, in which case amyloid formation was accelerated by denaturant. We reconcile these observations by suggesting that denaturants destabilize monomeric and aggregated species to different extents and thus affect nucleation and growth.

Studies on the Detection, Expression, Glycosylation, Dimerization, and Ligand Binding Properties of Mouse Siglec-E

CD33-related Siglecs are a family of proteins widely expressed on innate immune cells. Binding of sialylated glycans or other ligands triggers signals that inhibit or activate inflammation. Immunomodulation by Siglecs has been extensively studied, but relationships between structure and functions are poorly explored. Here we present new data relating to the structure and function of Siglec-E, the major CD33-related Siglec expressed on mouse neutrophils, monocytes, macrophages, and dendritic cells. We generated nine new rat monoclonal antibodies specific to mouse Siglec-E, with no cross-reactivity to Siglec-F. Although all antibodies detected Siglec-E on transfected human HEK-293T cells, only two reacted with mouse bone marrow neutrophils by flow cytometry and on spleen sections by immunohistochemistry. Moreover, whereas all antibodies recognized Siglec-E-Fc on immunoblots, binding was dependent on intact disulfide bonds and N-glycans, and only two antibodies recognized native Siglec-E within spleen lysates. Thus, we further investigated the impact of Siglec-E homodimerization. Homology-based structural modeling predicted a cysteine residue (Cys-298) in position to form a disulfide bridge between two Siglec-E polypeptides. Mutagenesis of Cys-298 confirmed its role in dimerization. In keeping with the high level of 9-O-acetylation found in mice, sialoglycan array studies indicate that this modification has complex effects on recognition by Siglec-E, in relationship to the underlying structures. However, we found no differences in phosphorylation or SHP-1 recruitment between dimeric and monomeric Siglec-E expressed on HEK293A cells. Phylogenomic analyses predicted that only some human and mouse Siglecs form disulfide-linked dimers. Notably, Siglec-9, the functionally equivalent human paralog of Siglec-E, occurs as a monomer.

Human Islet Amyloid Polypeptide N-Terminus Fragment Self-Assembly: Effect of Conserved Disulfide Bond on Aggregation Propensity.

Amyloid formation by human islet amyloid polypeptide (hIAPP) has long been implicated in the pathogeny of type 2 diabetes mellitus (T2DM) and failure of islet transplants, but the mechanism of IAPP self-assembly is still unclear. Numerous fragments of hIAPP are capable of self-association into oligomeric aggregates, both amyloid and non-amyloid in structure. The N-terminal region of IAPP contains a conserved disulfide bond between cysteines at position 2 and 7, which is important to hIAPP's in vivo function and may play a role in in vitro aggregation. The importance of the disulfide bond in this region was probed using a combination of ion mobility-based mass spectrometry experiments, molecular dynamics simulations, and high-resolution atomic force microscopy imaging on the wildtype 1-8 hIAPP fragment, a reduced fragment with no disulfide bond, and a fragment with both cysteines at positions 2 and 7 mutated to serine. The results indicate the wildtype fragment aggregates by a different pathway than either comparison peptide and that the intact disulfide bond may be protective against aggregation due to a reduction of inter-peptide hydrogen bonding. Graphical Abstract ᅟ.

Disulfide-bond scrambling promotes amorphous aggregates in lysozyme and bovine serum albumin.

Disulfide bonds are naturally formed in more than 50% of amyloidogenic proteins, but the exact role of disulfide bonds in protein aggregation is still not well-understood. The intracellular reducing agents and/or improper use of antioxidants in extracellular environment can break proteins disulfide bonds, making them unstable and prone to misfolding and aggregation. In this study, we report the effect of disulfide-reducing agent dithiothreitol (DTT) on hen egg white lysozyme (lysozyme) and bovine serum albumin (BSA) aggregation at pH 7.2 and 37 °C. BSA and lysozyme proteins treated with disulfide-reducing agents form very distinct amorphous aggregates as observed by scanning electron microscope. However, proteins with intact disulfide bonds were stable and did not aggregate over time. BSA and lysozyme aggregates show unique but measurable differences in 8-anilino-1-naphthalenesulfonic acid (ANS) and 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (bis-ANS) fluorescence, suggesting a loose and flexible aggregate structure for lysozyme but a more compact aggregate structure for BSA. Scrambled disulfide-bonded protein aggregates were observed by nonreducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) for both proteins. Similar amorphous aggregates were also generated using a nonthiol-based reducing agent, tris(2-carboxyethyl)phosphine (TCEP), at pH 7.2 and 37 °C. In summary, formation of distinct amorphous aggregates by disulfide-reduced BSA and lysozyme suggests an alternate pathway for protein aggregation that may be relevant to several proteins.

A trypsin inhibitor from rambutan seeds with antitumor, anti-HIV-1 reverse transcriptase, and nitric oxide-inducing properties.

Nephelium lappaceum L., commonly known as "rambutan," is a typical tropical tree and is well known for its juicy and sweet fruit which has an exotic flavor. Chemical studies on rambutan have led to the identification of various components such as monoterpene lactones and volatile compounds. Here, a 22.5-kDa trypsin inhibitor (N . lappaceum trypsin inhibitor (NLTI)) was isolated from fresh rambutan seeds using liquid chromatographical techniques. NLTI reduced the proteolytic activities of both trypsin and α-chymotrypsin. Dithiothreitol reduced the trypsin inhibitory activity of NLTI at a concentration of 1 mM, indicating that an intact disulfide bond is essential to the activity. NLTI inhibited HIV-1 reverse transcriptase with an IC50 of 0.73 μM. In addition, NLTI manifested a time- and dose-dependent inhibitory effect on growth in many tumor cells. NLTI is one of the few trypsin inhibitors with nitric oxide-inducing activity and may find application in tumor therapy.

Functional Siglec lectin domains from soluble expression in the cytoplasm of Escherichia coli

Siglecs (sialic acid-binding immunoglobulin-like lectins) are a family of mammalian cell-surface receptors that are involved in cell–cell interactions and signaling functions, primarily expressed on cells of the immune system. Key to their function is their specific binding of distinct sialylated glycan ligands mediated via an N-terminal carbohydrate recognition (lectin) domain. Studies concerning the molecular basis of their individual carbohydrate specificities are rare due to the absence of suitable recombinant expression methods for producing these disulfide-containing proteins in sufficient quantities required for their in-depth in vitro characterization. We established an efficient E. coli-based expression and purification method for Siglec lectin domains, utilizing the trxB gor suppressor strain Rosetta-gami B (DE3) in which proper folding with intact disulfide bonds was achieved in the cytoplasm. The approach is demonstrated for human Siglec-7, -8 and -9 lectin domains and works equally well for expression in nutrient-rich (LB) or minimal growth medium, allowing stable-isotope labeling for NMR studies. The recombinant proteins were properly folded as proven by 2D 1H–15N HSQC NMR spectroscopy and by thermal unfolding followed by CD spectroscopy, and functionally active as confirmed by monitoring ligand binding using NMR titration experiments. Our method enables efficient production of homogeneous and active protein samples in milligram quantities. Its implementation will significantly enhance future structure–function studies of this important class of immune-modulating receptors and will support a variety of applications including screening for natural and synthetic ligands or the development of fluorescently-labeled molecular tools for glycan ligand detection or flow-cytometric cell sorting.

Conformation and Dynamics of Interchain Cysteine-Linked Antibody-Drug Conjugates as Revealed by Hydrogen/Deuterium Exchange Mass Spectrometry

Antibody-drug conjugates (ADCs) are protein therapeutics in which a target specific monoclonal antibody (mAb) is conjugated with drug molecules. The manufacturing of ADCs involves additional conjugation steps, which are carried out on the parent mAbs, and it is important to evaluate how the drug conjugation process impacts the conformation and dynamics of the mAb. Here, we present a comparative study of interchain cysteine linked IgG1 ADCs and the corresponding mAb by hydrogen/deuterium exchange mass spectrometry (HDX-MS). We found that ∼90% of the primary sequence of the ADC conjugated with either monomethyl auristatin E or F (vcMMAE/mcMMAF) displayed the same HDX kinetics as the mAb, indicating the ADCs and mAbs share very similar conformation and dynamics in solution. Minor increases in HDX kinetic rates were observed in two Fc regions in the ADCs relative to the mAb which indicated that both regions become more structurally dynamic and/or more solvent-accessible in the ADCs. The findings led to a subsequent inquiry into whether the local conformational changes were due to the presence of drugs on the interchain cysteine residues or the absence of intact interchain disulfides or both. To address this question, a side-by-side HDX comparison of ADCs, mAbs, reduced mAbs (containing 8 reduced interchain cysteine thiols), and partially reduced mAbs (conjugation process intermediate) was performed. Our results indicated that the slight increase in conformational dynamics detected at the two regions in the ADCs was due to the absence of intact interchain disulfide bonds and not the presence of vcMMAE or mcMMAF on the alkylated interchain cysteine residues. These results highlight the utility of HDX-MS for interrogating the higher-order structure of ADCs and other protein therapeutics.

The Effect of Magnesium on the Thermodynamics of Nucleic Acid Tertiary Contact Formation

Functional RNAs fold into compact, well-defined tertiary structures despite strong electrostatic repulsion both within and between helices. To achieve these compact structures, many RNAs employ structural divalent cations, typically Mg2+. The simplest tertiary contact in nucleic acids is two helices, joined by some non-helical contact. To explore the fundamental characteristics of tertiary contact formation in nucleic acids we studied a system of two DNA helices tethered via a short PEG linker by both computational and experimental methods. Computationally, we predict the electrostatic repulsion between these helices as a function of Mg2+ and Na+ concentration. Experimentally, we linked the distal termini of these helices via a disulfide linker. Using small-angle x-ray scattering, we measured the fraction of intact disulfide bonds as a function of reducing strength of the buffer at a range of Mg2+ and Na+ concentration. Using these data we can extrapolate the magnitude of the strain on the disulfide bond, and thus the repulsion between the helices. Previous results show that the conformational ensemble is narrowly distributed around an extended, co-linear conformation at low salt and becomes more relaxed at high salt, but is unable to isolate a conformation in which the helices are stacked. Furthermore, previous results also suggest that specific interactions between Mg2+ and the phosphate backbone strongly are more important than simple ionic strength in determining the magnitude of the repulsive potential between the helices. In this project, we hope to experimentally demonstrate the energy of helix stacking and the different role of ionic strength and ionic specific interactions.

Recovery and detection of botulinum toxin type A from drinking water

s Toxins 2011 / Toxicon 68 (2013) 60–123 94 Double anchorage to the membrane and intact interchain disulfide bond are required for the lowpH-induced entry of tetanus and botulinum neurotoxins into neurons M. Pirazzini , O. Rossetto , P. Bolognese , C.C. Shone , C. Montecucco a Dipartimento di Scienze Biomediche and Istituto CNR di Neuroscienze, Universita di Padova, Padova, Italy Health Protection Agency, Porton Down, Salisbury, UK E-mail address: marcopiraz@yahoo.it (M. Pirazzini). Purpose of study: To study themembrane translocation of the clostridial neurotoxins in neurons. Methods used: We have developed a protocol based on an experiment first performed with diphtheria toxin. Conditions were found that by-pass the SV endocytosis step and induce the entry of the L chain directly from the plasma membrane. The neurotoxin, bound to the plasma membrane in the cold, was exposed to a warm, low pH extracellular medium, and the entry of the L chain was monitored by measuring its specific metalloprotease activity with a ratiometric method. Summary of results: We found that the neurotoxin has tobebound to themembraneviaat least twoanchorage sites in order for a productive low-pH-induced structural change to take place. In addition, this process can only occur if the single inter-chain disulfide bond is intact. The pH dependence of the conformational change of tetanus neurotoxin and botulinum neurotoxins B, C and D is similar and take places in the same slightly acidic range, which comprises that present inside synaptic vesicles. Furthermore, using PROPKA3.0 software, we found that tetanus and botulinum neurotoxins share a pool of conserved acid residues that are predicted to be protonated in the pH range 4.5-6. Conclusions: Tetanus and botulinum B, C and D neurotoxins undergo to a conformational change in a very similar pH range (4.5-6), which leads to light chain internalization. These neurotoxins share a pool of acidic residues which could be involved in the initial steps of the pHdependent conformational change. http://dx.doi.org/10.1016/j.toxicon.2012.07.099 Recovery and detection of botulinum toxin type A from drinking water B.H. Raphael , M. Lautenschlager , A. Kahler , S. Pai , B.A. Parks , S. Kalb , S. Maslanka , S. Shah , M. Magnuson , V. Hill a Division of Foodborne, Waterborne, and Environmental Diseases, National Center of Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, GA, USA Division of Laboratory Science, National Center of Environmental Health, Centers for Disease Control and Prevention, Atlanta, GA, USA National Homeland Security Research Center, U.S. Environmental Protection Agency, Cincinnati, OH, USA E-mail address: braphael@cdc.gov (B.H. Raphael). Purpose of study: Botulinum neurotoxin (BoNT) represents a possible biothreat agent for drinking water systems. However, a standard method for monitoring drinking water potentially contaminated with BoNT is not currently available to response laboratories. Methods used: Both the botulinum toxin ELISA and Endopep-MS (a mass spectrometric-based endopeptidase method) were adapted to quantify complex BoNT/A in deionized water and in chlorine-demand-free (CDF) water. Summary of results: The method detection limit (MDL) of the ELISA and theMSmethods in CDF water was 500 and 50 pg/ml of BoNT/A complex toxin, respectively. While adapting these methods to municipal tap water samples, residual chlorine in such samples was neutralized with 50 mg/L sodium thiosulfate prior to toxin spiking because free chlorine rapidly (<5 minutes) inactivates BoNT. Prior to testing by either ELISA or MS, municipal tap water samples from 5 U.S. states having distinct water quality properties were processed using ultrafiltration (UF) to concentrate the toxin by up to 200-fold. In order to evaluate the efficiency of toxin concentration using UF, 100 L tap water samples were spiked with 5 ug BoNT/A, which represents a final concentration equivalent to the MDL of the Endopep-MS method (i.e., 50 pg/ml). Recovery efficiencies of the UF method for BoNT/A complex, quantified by ELISA, ranged from 11%-36%, while efficiencies quantified by MS ranged from 26%-55%. Conclusions: Both the botulinum toxin ELISA and the Endopep-MS methods were effective for the quantification of BoNT in municipal tap water samples of varying properties. Additionally, UF was capable of significantly increasing the BoNT/A concentration in tap water samples for analysis by either ELISA or EndopepMS. http://dx.doi.org/10.1016/j.toxicon.2012.07.100 Simultaneous detection and differentiation of botulinum toxin serotypes A and B by internallyquenched fluorogenic peptides

Double anchorage to the membrane and intact interchain disulfide bond are required for the low-pH-induced entry of tetanus and botulinum neurotoxins into neurons

Double anchorage to the membrane and intact interchain disulfide bond are required for the low-pH-induced entry of tetanus and botulinum neurotoxins into neurons

The Native Structure and Composition of the Cruciferin Complex in Brassica napus

Globulins are an important group of seed storage proteins in dicotyledonous plants. They are synthesized during seed development, assembled into very compact protein complexes, and finally stored in protein storage vacuoles (PSVs). Here, we report a proteomic investigation on the native composition and structure of cruciferin, the 12 S globulin of Brassica napus. PSVs were directly purified from mature seeds by differential centrifugations. Upon analyses by blue native (BN) PAGE, two major types of cruciferin complexes of ∼ 300-390 kDa and of ∼470 kDa are resolved. Analyses by two-dimensional BN/SDS-PAGE revealed that both types of complexes are composed of several copies of the cruciferin α and β polypeptide chains, which are present in various isoforms. Protein analyses by two-dimensional isoelectric focusing (IEF)/SDS-PAGE not only revealed different α and β isoforms but also several further versions of the two polypeptide chains that most likely differ with respect to posttranslational modifications. Overall, more than 30 distinct forms of cruciferin were identified by mass spectrometry. To obtain insights into the structure of the cruciferin holocomplex, a native PSV fraction was analyzed by single particle electron microscopy. More than 20,000 images were collected, classified, and used for the calculation of detailed projection maps of the complex. In contrast to previous reports on globulin structure in other plant species, the cruciferin complex of Brassica napus has an octameric barrel-like structure, which represents a very compact building block optimized for maximal storage of amino acids within minimal space.

Identification of Disulfide Bonds in Wheat Gluten Proteins by Means of Mass Spectrometry/Electron Transfer Dissociation

Disulfide bonds within gluten proteins play a key role in the breadmaking performance of wheat flour. In the present study, disulfide bonds of wheat gluten proteins were identified by using a new liquid chromatography-mass spectrometry (LC-MS) technique with alternating electron transfer dissociation (ETD)/collision-induced dissociation (CID). Wheat flour was partially hydrolyzed with thermolysin (pH 6.5, 37 °C, 16 h), and the digest was subjected to LC-MS with alternating ETD/CID fragmentation. Whereas CID provided peptide fragments with intact disulfide bonds, cleavage of disulfide bonds was preferred over peptide backbone fragmentations in ETD. The simultaneous observation of disulfide-linked and disulfide-cleaved peptide ions in the mass spectra not only provided distinct interpretation with high confidence but also simplified the conventional approach for determination of disulfide bonds, which often requires two separate experiments with and without chemical reduction. By application of the new method 14 cystine peptides were identified. Eight peptides confirmed previously established disulfide bonds within gluten proteins, and the other six cystine peptides were identified for the first time. One of the newly identified cystine peptides represented a "head-to-tail" cross-link between high molecular weight glutenin subunits. This type of cross-link, which has been postulated as an integral part of glutenin models published previously, has now been proven experimentally for the first time. From the six remaining cystine peptides interchain disulfide bonds between α-gliadins, γ-gliadins, and low molecular weight glutenin subunits were established.

Insights into the structure and assembly of Escherichia coli outer membrane protein A

Outer membrane protein A (OmpA) of Escherichia coli is a paradigm for the biogenesis of outer membrane proteins; however, the structure and assembly of OmpA have remained controversial. A review of studies to date supports the hypothesis that native OmpA is a single-domain large pore, while a two-domain narrow-pore structure is a folding intermediate or minor conformer. The in vitro refolding of OmpA to the large-pore conformation requires isolation of the protein from outer membranes with retention of an intact disulfide bond followed by sufficient incubation in lipids at temperatures of ≥ 26 °C to overcome the high energy of activation for refolding. The in vivo maturation of the protein involves covalent modification of serines in the eighth β-barrel of the N-terminal domain by oligo-(R)-3-hydroxybutyrates as the protein is escorted across the cytoplasm by SecB for post-translational secretion across the secretory translocase in the inner membrane. After cleavage of the signal sequence, protein chaperones, such as Skp, DegP and SurA, guide OmpA across the periplasm to the β-barrel assembly machinery (BAM) complex in the outer membrane. During this passage, a disulfide bond is formed between C290 and C302 by DsbA, and the hydrophobicity of segments of the C-terminal domain, which are destined for incorporation as β-barrels in the outer membrane bilayer, is increased by covalent attachment of oligo-(R)-3-hydroxybutyrates. With the aid of the BAM complex, OmpA is then assembled into the outer membrane as a single-domain large pore.

Identification of in Vivo Disulfide Conformation of TRPA1 Ion Channel

TRPA1 (transient receptor potential ankyrin 1) is an ion channel expressed in the termini of sensory neurons and is activated in response to a broad array of noxious exogenous and endogenous thiol-reactive compounds, making it a crucial player in chemical nociception. A number of conserved cysteine residues on the N-terminal domain of the channel have been identified as critical for sensing these electrophilic pungent chemicals, and our recent EM structure with modeled domains predicts that these cysteines form a ligand-binding pocket, allowing for the possibility of disulfide bonding between the cysteine residues. Here, we present a comprehensive mass spectrometry investigation of the in vivo disulfide bonding conformation and in vitro reactivity of 30 of the 31 cysteine residues in the TRPA1 ion channel. Four disulfide bonds were detected in the in vivo TRPA1 structure: Cys-666-Cys-622, Cys-666-Cys-463, Cys-622-Cys-609, and Cys-666-Cys-193. All of the cysteines detected were reactive to N-methylmaleimide (NMM) in vitro, with varying degrees of labeling efficiency. Comparison of the ratio of the labeling efficiency at 300 μM versus 2 mM NMM identified a number of cysteine residues that were outliers from the mean labeling ratio, suggesting that protein conformation changes rendered these cysteines either more or less protected from labeling at the higher NMM concentrations. These results indicate that the activation mechanism of TRPA1 may involve N-terminal conformation changes and disulfide bonding between critical cysteine residues.

Structure and Assembly of Escherichia Coli Outer Membrane Protein A

Outer membrane protein A (OmpA) of Escherichia coli is a paradigm for the biogenesis of outer membrane proteins; however, the structure and assembly of OmpA remain controversial. Studies to date support the hypothesis that native OmpA is a single-domain large pore, and a two-domain narrow pore conformer is a folding intermediate. The in vitro refolding of OmpA to the large pore conformation requires that the protein be isolated from outer membranes with an intact disulfide bond and then adequately incubated in lipids at temperatures ≥ 26 °C to overcome the energy of activation for refolding. The in vivo maturation of the protein involves covalent modification of serines of the N-terminal domain by oligo-(R)-3-hydroxybutyrates as the protein is escorted across the cytoplasm by SecB for post-translational secretion across the SEC translocase in the inner membrane. After cleavage of the signal sequence, protein chaperones, such a Skp, DegP and SurA, guide OmpA across the periplasm to the BAM complex in the outer membrane. During this passage, a disulfide bond is formed between C290 and C302 by DsbA, and the hydrophobicity of segments of the C-terminal domain which are destined for incorporation as β-barrels in the outer membrane bilayer is increased by covalent attachment of oligo-(R)-3-hydroxybutyrates. With the aid of the BAM complex, OmpA is then assembled into the outer membrane as a single-domain large pore.