Impact of an easily reducible disulfide bond on the oxidative folding rate of multi‐disulfide‐containing proteins

Abstract: The burial of native disulfide bonds, formed within stable structure in the regeneration of multi‐disulfide‐containing proteins from their fully reduced states, is a key step in the folding process, as the burial greatly accelerates the oxidative folding rate of the protein by sequestering the native disulfide bonds from thiol‐disulfide exchange reactions. Nevertheless, several proteins retain solvent‐exposed disulfide bonds in their native structures. Here, we have examined the impact of an easily reducible native disulfide bond on the oxidative folding rate of a protein. Our studies reveal that the susceptibility of the (40–95) disulfide bond of Y92G bovine pancreatic ribonuclease A (RNase A) to reduction results in a reduced rate of oxidative regeneration, compared with wild‐type RNase A. In the native state of RNase A, Tyr 92 lies atop its (40–95) disulfide bond, effectively shielding this bond from the reducing agent, thereby promoting protein oxidative regeneration. Our work sheds light on the unique contribution of a local structural element in promoting the oxidative folding of a multi‐disulfide‐containing protein.     

Thiol–Disulfide Exchange in Peptides Derived from Human Growth Hormone

Disulfide bonds stabilize proteins by cross‐linking distant regions into a compact three‐dimensional structure. They can also participate in hydrolytic and oxidative pathways to form nonnative disulfide bonds and other reactive species. Such covalent modifications can contribute to protein aggregation. Here, we present experimental data for the mechanism of thiol–disulfide exchange in tryptic peptides derived from human growth hormone in aqueous solution. Reaction kinetics was monitored to investigate the effect of pH (6.0–10.0), temperature (4–50°C), oxidation suppressants [ethylenediaminetetraacetic acid (EDTA) and N₂ sparging], and peptide secondary structure (amide cyclized vs. open form). The concentrations of free thiol containing peptides, scrambled disulfides, and native disulfide‐linked peptides generated via thiol–disulfide exchange and oxidation reactions were determined using reverse‐phase HPLC and liquid chromatography–mass spectrometry. Concentration versus time data were fitted to a mathematical model using nonlinear least squares regression analysis. At all pH values, the model was able to fit the data with R² ≥ 0.95. Excluding oxidation suppressants (EDTA and N₂ sparging) resulted in an increase in the formation of scrambled disulfides via oxidative pathways but did not influence the intrinsic rate of thiol–disulfide exchange. In addition, peptide secondary structure was found to influence the rate of thiol–disulfide exchange. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 103:1032–1042, 2014     

Statine and its derivatives. Conformational studies using nuclear magnetic resonance spectrometry and energy calculations

The conformations of derivatives of 3(S)-hydroxy-4(S)-amino-6-methylheptanoic acid (statine) and its analogs have been studied by n.m.r. in chloroform and in dimethyl sulfoxide, and by molecular mechanics calculations. The data obtained from these studies indicate that: 1) the coupling constant between NH and C₄H is large, suggesting that the dihedral angle (θ) is near 165° or 0°; 2) the coupling constant between C₄H-C₃H is small, indicating a vicinal bond angle of approximately 90°; 3) the hydrogen deuterium exchange rate of statine amide protons is slow; however, the rate is dependent upon the electron withdrawing substituents adjacent to the amide NH's; 4) intramolecular hydrogen bonds involving the NH of the statine amide group do not stabilize conformations of single amino acid derivatives. Based on the n.m.r. results, four possible conformations of Boc-statine-OMe in solution are possible. MM1 calculations indicate one conformation is especially likely.     

STUDIES ON TRYPSIN INHIBITORS Part IX. Synthesis and Trypsin Inhibitory Activity of the Duopentacontapeptide Corresponding to the Amino Acid Sequence of Porcine Pancreatic Secretory Trypsin Inhibitor II (Kazal)*

The synthesis of the protected duopentacontapeptide corresponding to the entire amino acid sequence 1–52 of porcine pancreatic secretory trypsin inhibitor II (Kazal type) is described. The benzyloxycarbonyltetradecapeptide tert-butyloxycarbonylhydrazide (sequence 1–14) was selectively deblocked with trifluoroacetic acid and used to acylate, by the azide procedure, the peptide free base corresponding to the sequence 15–52. The isolated material was purified by ion exchange chromatography and the protecting groups were removed by successive treatments with anhydrous hydrogen fluoride, 1M piperidine and mercuric acetate. Folding and formation of the disulfide bonds was accomplished by air oxidation in 0.02M phosphate buffer, pH8. Determination of the inhibitory capacity indicated that the synthetic material is about 50% effective, at 30:1 inhibitor:trypsin molar ratio, in inhibiting the tryptic hydrolysis of Nα-benzoyl-dl -arginine-4-nitroanilide. Full inhibition was achieved at a higher inhibitor:trypsin molar ratio. The stability constants and the standard free energy of binding of the complex between trypsin and the synthetic inhibitor have been determined.     

Isolation and characterization of monodeamidated derivatives of bovine pancreatic Ribonuclease A

The isolation and characterization of the initial intermediates formed during the irreversible acid denaturation of enzyme Ribonuclease A are described. The products obtained when RNase A is maintained in 0.5 M HCl at 30° for periods up to 20 h have been analyzed by ion-exchange chromatography on Amberlite XE-64. Four distinct components were found to elute earlier to RNase A; these have been designated RNase Aa₂, Aa_1c, Aa_1b, and Aa_1a in order of their elution. With the exception of RNase Aa₂, the other components are nearly as active as RNase A. Polyacrylamide gel electrophoresis at near-neutral pH indicated that RNase Aa_1a, Aa_1b, and Aa_1c are monodeamidated derivatives of RNase A; RNase Aa₂ contains, in addition, a small amount of a dideamidated component. RNase Aa₂, which has 75% enzymic activity as compared to RNase A, consists of dideamidated and higher deamidated derivatives of RNase A. Except for differences in the proteolytic susceptibilities at an elevated temperature or acidic pH, the monodeamidated derivatives were found to have very nearly the same enzymic activity and the compact folded structure as the native enzyme. Fingerprint analyses of the tryptic peptides of monodeamidated derivatives have shown that the deamidations are restricted to an amide cluster in the region 67–74 of the polypeptide chain. The initial acid-catalyzed deamidation occurs in and around the 65–72 disulfide loop giving rise to at least three distinct monodeamidated derivatives of RNase A without an appreciable change in the catalytic activity and conformation of the ribonuclease molecule. Significance of this specific deamidation occurring in highly acidic conditions, and the biological implications of the physiological deamidation reactions of proteins are discussed.     

Studies of repeating synthetic peptides designed to adopt a cross-β conformation

The conformational preferences of peptides forming parts of the sequence Val-Val-Asn-Pro-Gly-Gln-Val-Val have been studied by circular dichroism (CD) and nuclear magnetic resonance (n.m.r.) spectroscopy. The sequence is intended as a model for cross-β structures and keratins. CD spectra characteristic of type II β-bends are produced by Pro-Gly sequences: these have a maximum at 196–203 nm and a single minimum at 222–225 nm. Peptides with the sequence Asn-Pro-Gly have a CD spectrum attributable to a type I(III) β-bend with two minima at 200–205 nm and 220 nm respectively. N.m.r. data support the suggestion that the type I(III) bend is stabilised by H-bonding between the oxygen of the side-chain amide of Asn and the hydrogen of the main chain NH of the Gly (or other third) residue. Thus, sequences where such H-bonding is possible show shielding of the Asn amide protons, non-equivalence of C^βH₂ protons and low temperature dependencies of NH protons of the third residue for each peptide examined. Calculations of rotamer populations for the Asn amide side chain support these conclusions.     

Roles of disulfide bridges in scorpion toxin BmK M1 analyzed by mutagenesis

Abstract: The unique fold of scorpion toxins is a natural scaffold for protein engineering, in which multiple disulfide bonds are crucial structural elements. To understand the respective roles of these disulfide bridges, a mutagenesis analysis for the four disulfide bonds, 12–63, 16–36, 22–46 and 26–48, of a representative toxin BmK M1 from the scorpion Buthus martensii Karsch was carried out. All cysteines were replaced by serine with double mutations. The recombinant mutants were expressed in the Saccharomyces cerevisiae S‐78 system. Toxic activities of the expressed mutants were tested on ICR mice in vivo and on neuronal Na⁺ channels (rNa_v1.2) by electrophysiological analysis. Recombinant variants M1 (C22S,C46S) and M1 (C26S,C48S) were not expressed at all; M1 (C16S,C36S) could be expressed at trace levels but was extremely unstable. Variant M1 (C12S,C63S) could be expressed in an amount comparable with that of unmodified rBmK M1, but had no detectable bioactivities. The results indicated that among the four disulfide bonds for long‐chain scorpion toxins, loss of either bridge C22?C46 or C26?C48 is fatal for the general folding of the molecule. Bridge C16?C36 mainly contributes to the global stability of the folded scaffold, and bridge C12?C63 plays an essential role in the functional performance of scorpion toxins.     

Replacement of the disulfide bond in oxytocin by an amide group. Synthesis and some biological properties of (cyclo-(1-L-aspartic acid,6-L-alpha,beta-diaminopropionic acid))oxytocin

As part of a continuing investigation of the steric and electronic functions of the disulfide group in neurohypophyseal hormones on their biological activity, the synthesis of "oxytocin lactam", [cyclo-(1-aspartic acid,6-alpha,beta-diaminopropionic acid)]oxytocin, has been undertaken. The protected nonapeptide was prepared in a stepwise manner by solution techniques; after removal of side-chain protecting groups, formation of the briding amide bonds was accomplished by oxidation-reduction condensation. The analogue possesses rat uterotonic, avian vasodepressor, and rat antidiuretic potencies of 16 +/- 2, 6.6 +/- 0.6, and 5.6 +/- 3.8 units/mg, respectively.     

Novel Pentapeptide GLP‐1 (32–36) Amide Inhibits β‐Cell Apoptosis In Vitro and Improves Glucose Disposal in Streptozotocin‐Induced Diabetic Mice

We proposed that a pentapeptide, LVKGR amide, GLP‐1 (32–36) amide, derived from the gluco‐incretin hormone, glucagon‐like peptide‐1 (GLP‐1), might possess favorable actions against diabetes. Therefore, GLP‐1 (32–36) amide was synthesized and the effects of it were examined in INS‐1 cell and streptozotocin‐induced diabetic mice model. To determine the protective effects of GLP‐1 (32–36) amide on INS‐1 cell viability and apoptosis, cells were exposed to 1 μm streptozotocin (STZ) and GLP‐1 (32–36) amide for 24 h. Results showed that GLP‐1 (32–36) amide treatment decreased apoptosis rate and significantly retained cell viability compared with saline‐treated controls. Then, GLP‐1 (32–36) amide was administered intraperitoneally to streptozotocin‐induced diabetic mice with normal mice used as control. Body weight, energy intake, plasma glucose, and histopathology of the pancreas were assessed. Results showed that GLP‐1 (32–36) amide protected β‐cell viability and apoptosis against STZ‐induced toxicity, inhibited weight gain, and relieved symptoms of polydipsia. Moreover, GLP‐1 pentapeptide‐treated mice showed a slight trend toward reduced glucose excursions in intraperitoneal glucose tolerance test at the end of the experiment. GLP‐1 (32–36) amide exerted favorable protective actions in streptozotocin‐induced diabetic mice. The peptide curtailed weight gain and alleviates symptoms of polydipsia. These findings suggested the probable utility of GLP‐1 (32–36) amide, a peptide mimetic derived there from GLP‐1, for adjuvant treatment of diabetes.     

Disulfide linkage to polyacrylic resin for automated Fmoc peptide synthesis. Immunochemical applications of peptide resins and mercaptoamide peptides

The use of disulfide bonds for peptide–resin linkage in solid-phase peptide synthesis was investigated using polyacrylic polymers (Expansin^TM) and automated Fmoc methodology. The disulfide moiety was bound to the support either by coupling a protected bifunctional handle or by an original stepwise procedure. Among the three different disulfide handles that were investigated, only the aminoethyldithio-2-isobutyric acid (AEDI) handle was stable enough to achieve peptide synthesis. A series of peptides of up to 10–20 amino acids were prepared in this manner, in good yield and purity. Rapid and quantitative peptide release was obtained by reduction with equimolecular amounts of dithiothreitol at pH 9 or tris(2-carboxymethyl) phosphine at pH 4.5. This allowed direct and rapid coupling of the released cysteamide peptides to an activated protein carrier and the use of free or resin-bound forms of the antigen in immunoassays.     

SMS 201–995, a very potent analogue of somatostatin. Assignment of the 1H 500 MHz n.m.r. spectra and conformational analysis in aqueous solution

The conformations of a cyclic analogue of somatostatin, SMS 201–995, have been studied by n.m.r. spectroscopy at 500 MHz in aqueous solution. Assignments were made by use of 2D-correlated methods, especially by detecting long-range connectivities in order to identify the aromic amino-acid and long-range couplings between α protons of consecutive residues. Measurements of temperature coefficients of amide protons and of NH-C_αH coupling constants enabled us to conclude that in water the molecule is rather flexible, with no evidence for a β turn structure involving Thr⁶. An equilibrium involving two γ turn conformations stabilized respectively by Cys²-d -Trp⁴and Phe³-Lys⁵hydrogen bonds, is responsible for the large upfield shift observed for the Lys⁵γ protons and is compatible with the measured J_NH-Cα_H coupling constants.     

NH resonances of Ribonuclease S-peptide in aqueous solution. Low temperature n.m.r. study

A detailed study of the NH resonances of Ribonuclease-S-peptide (1–19 N-terminal fragment of Ribonuclease A) has been carried out in H₂O, pH 3.0, in the temperature range 1–31, and ionic strength 0–1 M. Individual assignments of all NH amide signals have been achieved by means of extensive double resonance experiments. The folding of S-peptide at low temperature has been monitored by examination of the several NH resonance parameters: first, the non-linearity of chemical shift vs. temperature plots; second, the selective broadening observed for signals assigned to residues 3–13; and third, the decrease of ³J_HNCH coupling constants belonging to this region of the polypeptide chain. All these results are in agreement with the formation of a folded structure at low temperature, which is similar to the one found for the S-peptide in the RNase S crystal.     

Cystine peptides. Spectroscopic studies on the conformations of a cyclic pentapeptide disulfide

The model cyclic pentapeptide disulfide Boc-Cys-Ala-Aib-Gly-Cys-NHMe 1, has been synthesized. ¹H n.m.r. studies in (CD₃)₂SO and CDCl₃-(CD₃)₂SO mixtures establish the solvent exposed nature of the Cys(l) and Aib NH groups, while a moderate degree of shielding is observed for the other four NH groups. Nuclear Overhauser effects between Cα₁H and N_i+1H protons provide evidence for extended or semi-extended conformations (ø ± 130° ± 30°) at the Cys(l), Ala(2), Gly(4) and Cys(5) residues. The n.m.r. results are supportive of an intramolecular antiparallel β-sheet conformation at these residues, nucleated by a γ-turn centered at Aib(3). This conformation is not stabilized by strong transannular hydrogen bonds. CD studies establish solvent dependent structural changes of the disulfide linkage in methanol-dioxane mixtures. An unusual CD pattern is observed for the peptide chromophore.     

Efficient solution phase synthesis of [l-(β-mercapto-β, β-cyclopentamethylenepropionic acid)- 2-(O-ethyl-d-tyrosine)-4-valine-9-desglycine]arginine vasopressin

A convergent synthesis of the peptide [1-(β-mercapto-β,β-cyclopentamethylenepropionic acid)-2-(O-ethyl-d -tyrosine)-4-valine-9-desglycine]arginine vasopressin (1), based on the classical solution phase method, was developed. The molecule is assembled by a 3 + 4 coupling via the amide method; then the disulfide bridge is installed by iodine treatment of the bis-acetamidomethyl protected thiols, and the terminal arginine amide added by a 7 + 1 coupling. The method (see Scheme 1) has been used to prepare gram quantities of 1 in more than 98% purity and in 13% yield (based on tetrapeptide intermediate 13) after a single stage purification. The method appears to be particularly suitable for the large scale preparation of 1 and other vasopressin congeners. A novel, albeit low level, transfer of acetamidomethyl group from the sulfur of cysteine to the asparagine amide side-chain was detected following hydrogen chloride treatment of Boc-containing intermediates.     

Mapping the receptor binding regions of human chorionic gonadotropin (hCG) using disulfide peptides of its β‐subunit: possible involvement of the disulfide bonds Cys9−Cys57 and Cys23−Cys72 in receptor binding of the hormone

Abstract: Human chorionic gonadotropin (hCG) is a heterodimeric glycoprotein hormone essential for the establishment and maintenance of pregnancy. The α‐ and β‐subunits of hCG are highly cross‐linked internally by disulfide bonds which seem to stabilize the tertiary structures required for the noncovalent association of the subunits to generate hormonal activity. The purpose of this study was to delineate the role of the disulfide bonds of hCGβ in receptor binding of the hormone. Six disulfide peptides incorporating each of the six disulfide bonds of hCGβ were synthesized and screened, along with their linear counterparts, for their ability to competitively inhibit the binding of [¹²⁵I] hCG to sheep ovarian corpora luteal LH/CG receptor. Disulfide peptide Cys (9?57) was found to be ≈ 4‐fold more potent than the most active of its linear counterparts in inhibiting radiolabeled hCG from binding to its receptor. Similarly, disulfide peptide Cys (23?72) exhibited receptor binding inhibition activity, whereas the constituent linear peptides were found to be inactive. The results suggest the involvement of the disulfide bonds Cys⁹?Cys⁵⁷ and Cys²³?Cys⁷² of the β‐subunit of hCG in receptor binding of the hormone. This study is the first of its kind to use disulfide peptides rather than linear peptides to map the receptor binding regions of hCG.     

Synthetic amatoxin analogues

Amatoxin analogues with d and l -Ala substitutions in position 5 have been studied by means of 1- and 2-dimensional n.m.r. spectroscopy at 500 MHz. The assignment of all resonances for both analogues has been carried out mostly with the use of COSY and NOESY type experiments. Temperature coefficients for the amide NH protons have been measured and the data compared to known amatoxin structures. The results obtained demonstrate that the rigidity of the bicyclic amatoxin framework is preserved in the d and l -Ala⁵ analogues, although the temperature coefficients point to intramolecular hydrogen bonds stronger in the case of the l -Ala analogue. The 10-fold decrease of biological activity is discussed in terms of structural features involving also the Trp⁴ indole accessibility.     

The nature of the bond between peptide and carrier molecule determines the immunogenicity of the construct

Abstract: The influence of the nature of the bond between a peptide and a (lipidic) carrier molecule on the immunogenicity of that construct was investigated. As types of bonds a thioester‐, a disulfide‐, an amide‐ and a thioether bond were investigated. As carrier molecules a peptide, an N‐palmitoylated peptide or a C₁₆‐hydrocarbon chain were used. The biostability of the bond between peptide and carrier molecule is thioether > amide > disulfide ? thioester. However, the immunogenic potency of the constructs used was found to be thioester > disulfide > amide > thioether. In conclusion, a construct with a bond between peptide and (lipidic) carrier molecule that is more susceptible to biological degradation is more immunogenic when used in a peptide‐based vaccine than a bond that is less susceptible to biological degradation.     

SMS 201–995, an octapeptide somatostatin analogue.

The possible conformations of SMS 201–995, an active analogue of somastostatin, have been studied in dimethylsulfoxide solution by 500 MHz proton n.m.r. spectroscopy. The assignments have been made by use of 2D-correlated methods to detect long-range coupling connectivities in aromatic residues and between the α protons of consecutive residues. NOESY experiments enabled us to correlate amide and α protons of neighbouring amino acid residues, which indicate a less flexible situation than in water. Measurements of temperature coefficients of the amide protons, of NH-C_αH coupling constants and NOE effects are in favour of one predominant conformation with a β turn, of type II', involving amino acids Phe³to Thr⁶.     

Three-dimensional structure of fibrolase, the fibrinolytic enzyme from southern copperhead venom, modeled from the x-ray structure of adamalysin II and atrolysin C

The fibrinolytic enzyme from southern copperhead snake venom, fibrolase, contains 1 mole of zine per mole of protein, belongs to the major family of metalloproteinases known as the metzincins, and has been shown to degrade fibrin clots in vitro and in vivo. The purpose of this study was to develop a 3-dimensional model of fibrolase to investigate the geometry of conserved and variable sequences between members of the snake venom metalloproteinases. When compared to atrolysin C (form D) or adamalysin II (metzincins with completely different substrate specificity), fibrolase has approximately 60% overall sequence identity and nearly 100% sequence similarity in the active site. We used the crystal structure of adamalysin II to build a 3-dimensional homology model of fibrolase. Three disulfide bonds were constructed (the highly conserved disulfide bond [118–198] was maintained from the adamalysin II structure and 2 new disulfide bonds were introduced between residues 158–182 and 160–165). We used Sculpt 2.5 and HyperChem 5.0 to “dock” a substrate fragment octapeptide (HTEKLVTS), and a water molecule into the active site cleft. We calculated the differential average homology profile for fibrolase compared to 8 hemorrhagic and 5 nonhemorrhagic metzincins. We then determined the sequence regions that might be responsible for their substrate specificity. Our 3-dimensional homology model shows that the variable sequences lie on the periphery of the identified active site region containing the His triangle; this indicates that substrate specificity may depend on surface residues that are not directly associated with the active site.     

Nitroxyl-induced T1 relaxation rates as a probe of conformation in peptide hormones

Proton spin-lattice relaxation rates in the N-H region of the n.m.r. spectra of aqueous LHRH and angiotensin II were measured in the presence of varying concentrations of 2,2,6,6-tetramethylpiperidinoxyl. At peptide concentrations of 3–7 mM and nitroxyl concentrations up to three times the peptide concentration, the relaxation rate is linearly dependent on nitroxyl concentration. Second order rate constants for nitroxyl induced relaxation in water are in the 200–1000s^-1 M^-1 range, and are dependent more on conformational factors than on side chain bulk. Exclusion of the radical from the hydration sphere of imidazolium ion appears to occur. The measurements for LHRH agree with earlier observations of the effect of the nitroxyl on linewidths. A two- to threefold decrease in sensitivity of the amide protons of at least three of the residues in angiotensin II when the His⁶ imidazole is protonated indicates a conformational transition related to this ionization.