Conformations and vibrational properties of disulfide bridges: Potential energy distribution in the model system diethyl disulfide

Disulfide bridges are important structural elements in proteins. It is well-known that the position of the characteristic disulfide band at ca. 500 cm⁻¹ in the vibrational spectra varies with the conformation around the disulfide unit. In our computational study on the model system diethyl disulfide, both wavenumber and normal mode composition are analyzed simultaneously as a function of conformation. For the disulfide band, a negative correlation between the calculated vibrational wavenumber and the SS stretching contribution is detected. This trend in the normal mode composition provides an explanation for experimentally observed wavenumber shifts of the disulfide band.     

A Disulfide Switch Providing Absolute Handedness Control in Double Helices via Conversion from the Antiparallel to Parallel Helical Pattern

A strategy to reversibly switch the parallel/antiparallel helical conformation of aromatic double helices through the formation/breakage of a disulfide bond is presented. Single-crystal X-ray structures, NMR, and circular dichroism spectroscopy demonstrate that the double helices with terminal thiol groups favor an antiparallel helical arrangement both in the solid state and in solution, while the P/M bias of helicity induced by chiral segments from another extremity of the sequence is weak in this structural motif. The antiparallel helices can be rearranged to parallel helices through the disulfide connection of the sequences. This change enhances the bias of helical handedness and results in absolute chirality control of the double helices. The handedness-mediated process can be governed by the oxidation-reduction cycle, thereby switching the structural arrangement and the enhancement of chiral bias. In addition, we find that the sequences can dimerize into an intermolecular double helix with the disulfide connection. And the helical handedness is also fully controlled due to the head-to-head structural motif.     

HYDROPEROXYNAPHTHALIMIDE DERIVATIVE-MEDIATED OXIDATION OF LYSOZYME

Abstract— Lysozyme was photoirradiated in the presence of photo-Fenton reagents (hydroperoxynaph-thalimide derivatives) at 366 nm. Enzymatic activities of photoirradiated lysozymes were lower than that of native lysozyme. Taking account of the results of amino acid analysis and of fluorescence spectra, it was probably that Trp residues in the photoirradiated lysozyme were oxidized with hydroxyl radicals. The reagents formed complexes with lysozyme as proved by the inhibitory effects of the reagents on the enzymatic activities ( K ₁= 4.7 ± 1.2 × 10⁴ M for HPO II, a hydroperoxide derivative of naphthalimide), which suggested that these reagents were bound to the active site cleft of lysozyme, and the Trp residues located in or near the active site cleft were photooxidized. Fluorescence-difference spectra of photoirradiated lysozymes showed that Trp 62 was preferentially photooxidized. Furthermore, sodium dodecyl sulfatepolyacrylamide gel electrophoresis and circular dichroism spectra showed that the photooxidation examined here induced no significant change in the molecular size but a slight change in the conformation of lysozyme, which suggests the usefulness of the reagents in the site-selective oxidation of biopolymers.     

用蛋白电泳和高效凝胶排阻色谱法分析还原脲变性蛋白溶菌酶稀释复性过程的集聚体

本文利用蛋白电泳和高效凝胶排阻层析法分析了还原脲变性蛋白溶菌酶稀释复性过程中的集聚体。当用复性液稀释复性还原脲变性蛋白溶菌酶时,会迅速产生可观量的沉淀。沉淀和上清液的不连续十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和高效凝胶排阻层析分析结果表明,还原脲变性蛋白溶菌酶在稀释复性过程中除了能够复性成天然态蛋白溶菌酶分子外,还会形成可溶的蛋白溶菌酶分子二聚体和三聚体,二聚体和三聚体主要是靠分子间二硫键的错配连接而成的;可溶的蛋白溶菌酶分子二聚体之间通过非共价键相互作用而形成集聚体沉淀,而可溶的三聚体溶菌酶分子则仍处于复性液上清液中。     

Thermodynamic analysis of denatured lysozyme folded on moderately hydrophobic surface at 298 K

Both calorimetric determination of displacement adsorption enthalpies ΔH and measurement of adsorbed amounts of lysozyme (Lyz) denatured by 1.8 mol L⁻¹ guanidine hydrochloride (GuHCl) on a moderately hydrophobic packings at 298 K, pH 7.0 and various salt concentrations were carried out. Based on the thermodynamics of stoichiometric displacement theory (SDT) the fractions of thermodynamic functions, which related to four subprocesses of denatured protein refolding on the surface, were calculated and thermodynamic analysis that which one of the subprocesses plays major role for contribution to the thermodynamic fractions was made in detail. The moderately hydrophobic surface can provide denatured Lyz energy and make it gain more conformation with surface coverage or salt concentration increment. The displacement adsorptions of denatured Lyz onto PEG-600 surface are exothermic, more structure-ordered and enthalpy driven processes.     

Adsorption Reaction Dynamics of Systems Lysozyme and Nanodiamond/Nanosilica at pH=7-13

Adsorption reactions between surfaces of nanodiamond and nanosilica with diameter of 100 nm prepared as suspension solutions of 0.25 μg/μL and lysozyme molecule with different concentrations of 7 mmol/L PPBS at pH=7, 9, 11, and 13 have been investigated by fluores-cence spectroscopy. Adsorption reaction constants and coverages of lysozyme with different concentrations of 0-1000 nmol/L under the influences of different pH values have been ob-tained. Helicities and conformations of the adsorbed lysozyme molecules, free spaces of every adsorbed lysozyme molecule on the surfaces of nanoparticles at different concentrations and pH values have been deduced and discussed. The highest adsorption capabilities for both sys-tems and conformational efficiency of the adsorbed lysozyme molecule at pH=13 have been obtained. Lysozyme molecules can be prepared, adsorbed and carried with optimal activity and helicity, with 2 and 10 mg/m2 on unit nanosurface, 130 and 150 mg/g with respect to the weight of nanoparticle, within the linear regions of the coverages at around 150-250 nmol/L and four pH values for nanodiamond and nanosilica, respectively. They can be prepared inthe tightest packed form, with 20 and 55 mg/m2, 810-1680 and 580-1100 mg/g at threshold concentrations and four pH values for nanodiamond and nanosilica, respectively.     

Effect of Native Disulfide Bonds on the Denaturation of Proteins in 6 mol/L Guanidinium Chloride——A Fourier Transformed Infrared Study

The unfolding of bovine serum albumin, lysozyme and ribonuclease A denatured in 6 mol/L GuHCl with their disulfide bridges intact and reduced have been compared by FTIR studies. The peak positions and heights in the deconvolved spectra of amide I bands of the above denatured proteins with native disulfide bonds show marked differences whereas those for the denatured proteins without disulfide linkages are closely similar. The above and other evidence suggest that denatured proteins with intact disulfides still have considerable ordered conformation even in 6 mol/L GuHCl.     

A Thermodynamic Study on the Binding of PEG-Stearic Acid Copolymer with Lysozyme

The thermodynamics of the interaction between a copolymer of polyethyleneglycol400-stearic acid, S400, and lysozyme was investigated at pH=7.0 and 27 °C in phosphate buffer by isothermal titration calorimetry, ITC. The extended solvation model was used to reproduce the enthalpies of the S400 + lysozyme interactions. The solvation parameters recovered from the extended solvation model are attributed to the structural change of lysozyme and its biological activity. The binding parameters found for the interaction of S400 with lysozyme indicate that at low concentrations of S400, the lysozyme structure was destabilized but at higher concentrations of S400 lysozyme it was stabilized by S400. It is suggested that S400 interacts with a set of three identical binding sites on lysozyme.     

Hydrogen-Bonded Double Strands: Crystal Structure and Spectroscopic Propertiesof a 2,2′-Dipyrryl Ketone

Summary.  The synthesis, crystal structure determination, conformational analysis, and spectroscopic properties of 3,3′-diethyl-4,4′-dimethyl-2,2′-dipyrryl ketone (1) are reported. The dipyrryl ketone is a model for the dipyrrole core of 10-oxobilirubin, a presumed metabolite in alternate pathways of excretion of the yellow pigment of jaundice, bilirubin. In the crystal, 1 adopts a helical conformation, with a molecule of one helicity being hydrogen-bonded to two molecules of the opposite helicity. Thus, 1 self-assembles via hydrogen bonding into supramolecular double-stranded arrays, where molecules of the same helicity comprise one strand and are paired through hydrogen bonding to molecules of opposite helicity in the second strand. In the observed molecular conformation each pyrrole ring and adjacent carbonyl group are rotated into an sc conformation (torsion angle ∼29 °), with each N-H pointing in the same direction as the C*O. Molecular mechanics/dynamics calculations predict the sc,sc conformation, absent hydrogen bonding, to be the most stable, but only by a few tenths of a kj/mol. In CHCl₃, 1 is monomeric according to vapor pressure osmometry studies (). ¹H NMR NH chemical shifts in CDCl₃ suggest a predominantly anti orientation of the C=O and pyrrole NHs, which is opposite to the orientation observed in the crystal. Received February 4, 2000. Accepted February 14, 2000     

An Amphiphilic Selenide Catalyst Behaves Like a Hybrid Mimic of Protein Disulfide Isomerase and Glutathione Peroxidase 7

Protein disulfide isomerase (PDI) and glutathione peroxidase 7 (GPx7) cooperatively promote the oxidative folding of disulfide (SS)‐containing proteins in endoplasmic reticulum by recognizing the nascent proteins to convert them into the native folds by means of SS formation and SS isomerization and by catalyzing reoxidation of reduced PDI with H₂O₂, respectively. In this study, new amphiphilic selenides with a long‐chain alkyl group were designed as hybrid mimics of PDI and GPx7 and were applied to the refolding of reduced hen egg‐white lysozyme (HEL‐R). Competitive SS formation at pH 4 using HEL‐R and glutathione (GSH) in the presence of the selenide catalyst and H₂O₂ showed that the amphiphilic selenides can preferentially catalyze SS formation of HEL‐R, probably on account of hydrophobic interactions between the protein and the catalyst. In contrast, simple water‐soluble selenides did not exhibit such behavior. In addition, when the pH of the solution was adjusted to 8.5 after the SS formation, surviving GSH promoted the SS isomerization of misfolded HEL to recover the native SS linkages. Thus, the amphiphilic selenides designed here could mimic the function of the PDI‐GPx7 system. The combination of a water‐soluble selenide and a long‐chain alkyl group would be a useful motif in designing medicines for both protein misfolding diseases and antioxidant therapy.     

Effects of temperature and pH on the helicity of a peptide adsorbed to colloidal silica

The conformation of a cationic -helical peptide (DDDDAAAARRRRR) adsorbed to anionic colloidal silica has been investigated by circular dichroism (CD) spectroscopy as a function of temperature and pH in order to examine how the structure of an adsorbed molecule responds to two simultaneous perturbations. Increased temperature destabilizes the helicity of the peptide in solution, while pH changes alter the substrate surface charge and the corresponding strength of the interaction with the peptide. Near neutral pH, the helicity of the adsorbed peptide, which is determined from the intensity of the CD signal at 222 nm, decreases with increasing temperature, similarly to the temperature-dependent behavior observed for the peptide in aqueous solution. By contrast, at basic pH and a strongly negative surface charge, the helicity of the adsorbed peptide increases with temperature. In order to elucidate the origin of the reversal of the temperature dependence of helicity, a statistical model for the conformation of the adsorbed peptide has been formulated based on the Lifson–Roig model for the helix–coil transition of the peptide in solution. The model provides insight into the trends in fractional helicity and reveals that the temperature dependence of the helicity of the adsorbed peptide results from a competition between the intramolecular interactions that promote helicity and the intermolecular interactions with the surface. The statistical model also enables estimation of the free energy contributions from specific aspects of the adsorption process. Through identification of a connection between the conformation of adsorbed peptide and the interactions of the peptide with the surface, this work suggests a route for the control of adsorbate conformation through peptide and surface engineering.     

Release of lysozyme from the branched polyelectrolyte-lysozyme complexation

On the basis of the discretely charged sphere model of lysozyme, the release behavior of lysozyme from the branched polyelectrolyte-lysozyme complexation is investigated by adding salt and changing the pH values of the solution. It is found that, with the increase of the salt ionic strength of the solution, the lysozymes are gradually released from the oppositely charged polyelectrolyte as a result of the screening of electrostatic attraction between the two ionic species by adding the salt. Interestingly, there exists a critical salt ionic strength at which all proteins are released from the branched polyelectrolyte, and the polyelectrolyte-protein complexation is broken completely. Beyond the critical value, the increase of the salt ionic strength causes self-association of the proteins released from the branched polyelectrolyte-protein complexation. The self-association of the protein is detrimental in biological systems. By calculating the second virial coefficient, we found that the optimal salt content for the dispersion of proteins coincides with the critical ionic strength, because the second virial coefficient reaches its maximum at the critical ionic strength. Similarly, increasing the pH value of the solution can also release the lysozymes from the polyelectrolyte, because the increase of pH value of the solution changes the charge distribution and net charge of the lysozyme, weakens the attraction between lysozymes mediated by polyelectrolyte, and finally leads to the dissolution of the complexation of branched polyelectrolyte with lysozymes in strong alkaline solution. In addition, by exploring the effect of architecture of the polyelectrolyte on the release behavior of proteins, we found that it is more difficult to release proteins from the branched polyelectrolyte than from the linear polyelectrolyte.     

Uncovering the Contributions of Charge Regulation to the Stability of Single Alpha Helices**

The single alpha helix (SAH) is a recurring motif in biology. The consensus sequence has a di-block architecture that includes repeats of four consecutive glutamate residues followed by four consecutive lysine residues. Measurements show that the overall helicity of sequences with consensus E₄K₄ repeats is insensitive to a wide range of pH values. Here, we use the recently introduced q-canonical ensemble, which allows us to decouple measurements of charge state and conformation, to explain the observed insensitivity of SAH helicity to pH. We couple the outputs from separate measurements of charge and conformation with atomistic simulations to derive residue-specific quantifications of preferences for being in an alpha helix and for the ionizable residues to be charged vs. uncharged. We find a clear preference for accommodating uncharged Glu residues within internal positions of SAH-forming sequences. The stabilities of alpha helical conformations increase with the number of E₄K₄ repeats and so do the numbers of accessible charge states that are compatible with forming conformations of high helical content. There is conformational buffering whereby charge state heterogeneity buffers against large-scale conformational changes thus making the overall helicity insensitive to large changes in pH. Further, the results clearly argue against a single, rod-like alpha helical conformation being the only or even dominant conformation in the ensembles of so-called SAH sequences.     

Protein Engineering III: Computer Aided Design of Disulfide-Bond Free Cobra Venom Cardiotoxin with a Novel Conformation and Biological Activity from Synthetic Peptides

Using the crystal structure of cobra venom cardiotoxin as a templet, a computer designed peptide with a novel conformation and biological activity has been synthesized chemically. The designed peptide utilized two calcium coordination sites instead of disulfide bridges to hold the conformation. The coordination sites were introduced at the cleft of three β-sheet strands by replacing the residues of Leu-1, Leu-26, Ser-28, Leu-48, and Ser-55 with Glu and using their γ-carboxyl groups as legends. The residues of Cys at positions 3, 14, 21, 38, 42, 53, 54, and 59 of the four disulfide bridges were changed with Gly to remove all the disulfide bonds. Circular dichroism spectra showed that the synthesized peptide has a conformation similar to that of the native cardiotoxin of a defined structure only in aqueous solutions with the presence of calcium ions. Immunoprecipitation assay, using the anti-cardiotoxin V, showed that in the presence of calcium ion the peptide had same cross reaction as that of native cardiotoxin. Hemolysis assay in the presence of calcium ion (150–250 mmol) and phospholipase A₂ showed that the peptide had 65–70% as much cytolytic activity as the native toxin.     

Conformational study of a new SGTx1 neurotoxin from the spider Scodra griseipes using mass spectrometry

SGTx1 is a new neurotoxin from the venom of Scodra griseipes. Because of the small quantity of this natural peptide available, mass spectrometry was used to obtain information on its higher-order structure. The kinetics of reduction by 1,4-dithiothreitol (DTT) was monitored by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS), and showed that one of the three disulfide bridges was appreciably more accessible to the DTT. Studies based on the charge state distribution (CSD) and H/D exchange of the non-reduced peptide, under neutral and acidic conditions, were performed using electrospray mass spectrometry (ES-MS). In neutral solution, SGTx1 showed a maximum charge state of four compared with seven potentially protonated basic residues, and all labile hydrogens were exchanged. However, under acidic conditions, a maximum charge state of only five was observed, and four of the labile hydrogens could not be deuterated. These observations are interpreted in terms of a rigid structure maintained by the disulfide bridges, which can be temporarily relaxed by disulfide bridge scrambling only at higher pH values.     

A Cyclic Voltammetric Study of Some Porphyrazines

Summary.  Some porphyrazines with dimethylamino- or trimethylammoniumethylsulfanyl substituents were studied in dichloromethane or dimethylsulfoxide solution by cyclic voltammetric methods. The cyclic voltammogram of metal free octakis-(dimethyl-aminoethylsulfanyl)-porphyrazine is characterized by three one-electron reduction waves which show quasi-reversible behaviour at all sweep rates observed; the same holds for its cobalt(II) derivative. The quaternized octacationic derivative, however, exhibits four one-electron reduction waves which are reversible at all sweep rates. The reaction mechanisms and diffusion coefficients were investigated. Received October 17, 2000. Accepted (revised) December 11, 2000     

Qualitative and quantitative characterization of the arsenic‐binding behaviour of sulfur‐containing peptides and proteins by the coupling of reversed phase liquid chromatography to electrospray ionization mass spectrometry

Phenylarsenic‐substituted cysteine‐containing peptides and proteins were completely differentiated from their unbound original forms by the coupling of reversed phase liquid chromatography with electrospray ionization mass spectrometry. The analysis of biomolecules possessing structure‐stabilizing disulfide bridges after reduction provides new insights into requirements concerning the accessibility of cysteine residues for reducing agents as well as for arsenic compounds in a spatial protein structure. Complementary binding studies performed using direct ESI‐MS without chromatographic coupling in different solvent systems demonstrated that more than one binding site were activated for aprotinin and lysozyme in denaturing solvents because of a stronger defolding. From the intensities of the different charge states occurring in the mass spectra as well as from the LC elution behaviour, it can be deduced that the folding state of the arsenic‐bound protein species resembles the native, oxidized conformation. In contrast, although the milk protein α‐lactalbumin has several disulfide bridges, only one phenylarsenic moiety was bound under strongly denaturing conditions. Because of the charge state distribution in the ESI mass spectra, a conformational change to a molten globule structure is assumed. For the second considered milk protein ß‐lactoglobulin, a noncovalent interaction with phenylarsine oxide was detected. In general, smaller apparent binding constants for the condensation reactions of the biomolecules with phenylarsine oxide leading to covalent arsenic–sulfur bindings were determined from direct injection ESI‐MS measurements than from LC‐ESI‐MS coupling. The following order of binding affinities for one phenylarsenic group can be assumed from both ESI‐MS and LC‐ESI‐MS: nonapeptide vasopressin > nonapeptide vasotocin > lysozyme > aprotinin > α‐lactalbumin > thioredoxin. Kinetic investigations by LC‐ESI‐MS yielded a partial reaction order of 2 for vasopressin, Lys and α‐lactalbumin and corresponding half‐lives of 0.93, 2.56 and 123.5 min, respectively. Copyright © 2012 John Wiley & Sons, Ltd.     

On-Line Electrochemical Reduction of Disulfide Bonds: Improved FTICR-CID and -ETD Coverage of Oxytocin and Hepcidin

Particularly in the field of middle- and top-down peptide and protein analysis, disulfide bridges can severely hinder fragmentation and thus impede sequence analysis (coverage). Here we present an on-line/electrochemistry/ESI-FTICR-MS approach, which was applied to the analysis of the primary structure of oxytocin, containing one disulfide bridge, and of hepcidin, containing four disulfide bridges. The presented workflow provided up to 80 % (on-line) conversion of disulfide bonds in both peptides. With minimal sample preparation, such reduction resulted in a higher number of peptide backbone cleavages upon CID or ETD fragmentation, and thus yielded improved sequence coverage. The cycle times, including electrode recovery, were rapid and, therefore, might very well be coupled with liquid chromatography for protein or peptide separation, which has great potential for high-throughput analysis.      

Negative thermal expansibility change for dissociation of lysozyme variant amyloid protofibril

A disulfide‐deficient variant of hen lysozyme, 0SS, is known to form an amyloid protofibril spontaneously, and to dissociate into monomers at high hydrostatic pressure. We carried out native PAGE at various temperatures (20–35°C) and pressures (0.1–200 MPa), to characterize the dissociation equilibrium of disulfide‐deficient variant of hen lysozyme amyloid protofibril. Based on the density profiles, the partial molar volume and thermal expansibility changes for dissociation, Δv_D and Δe_D, were obtained to be ?74 cm³/mol at 25°C and ?2.3 cm³ mol^?1 K^?1, respectively. The dissociation of amyloid fibril destroys the cross β‐structure, and such conformational destruction in native protein fold rarely accompanies negative thermal expansibility change. We discussed the negative thermal expansibility change in terms of hydration and structural packing of the amyloid protofibril.     

Rapid and quantitative disulfide bond formation for a polypeptide chain using a cyclic selenoxide reagent in an aqueous medium

To elucidate the reaction mechanism of the disulfide (SS) bond formation reaction of a polypeptide molecule with a water‐soluble selenoxide reagent, trans‐3,4‐dihydroxyselenolane oxide (DHS^ox), short‐term oxidation experiments were carried out for the reduced state (R) of a recombinant hirudin CX‐397 variant at pH 7.0 and 25 °C. In the reaction, R was oxidized sequentially to one‐SS, two‐SS, and three‐SS intermediate ensembles within 1 min. The kinetic analysis revealed that the three second‐order rate constants for the SS formation are proportional to the number of thiol groups existing in the reactant SS intermediates, indicating the stochastic nature of the SS formation. Ab initio calculation at the HF/6‐31++G(d,p) level in water by using the polarizable continuum model suggested that the SS formation reaction is highly exothermic and proceeds via a reactive thioselenurane intermediate with a distorted linear O‐Se‐S linkage. The results clearly demonstrated that the rate‐determining step of the SS formation reaction is the first bimolecular process between a thiol substrate and DHS^ox rather than the subsequent process to release a SS product.