Bicycle synthesis through peptide macrocyclization using aziridine aldehydes followed by late stage disulfide bond installation

MedChemComm ◽  
2013 ◽  
Vol 4 (7) ◽  
pp. 1124-1128 ◽  
Author(s):  
Benjamin K. W. Chung ◽  
Jennifer L. Hickey ◽  
Conor C. G. Scully ◽  
Serge Zaretsky ◽  
Andrei K. Yudin
Keyword(s):  
Disulfide Bond ◽  
Late Stage ◽  
Disulfide Bonds ◽  
Aziridine Aldehydes

We present a method that can be applied to generate medium-sized peptidomimetic macrocycles equipped with disulfide bonds.

scholarly journals Characterization of Soluble, Disulfide Bond-Stabilized, Prokaryotically Expressed Human Thyrotropin Receptor Ectodomain*

Endocrinology ◽  
1997 ◽  
Vol 138 (2) ◽  
pp. 588-593 ◽  
Author(s):  
Y. Bobovnikova ◽  
P. N. Graves ◽  
H. Vlase ◽  
T. F. Davies
Keyword(s):  
Amino Acids ◽  
Disulfide Bond ◽  
Disulfide Bonds ◽  
Thioredoxin Reductase ◽  
Biologically Active ◽  
Receptor Protein ◽  
Enzyme Linked Immunosorbent Assay ◽  
Fusion Partner ◽  
E Coli ◽  
Recombinant Receptor

Abstract To study the interaction of TSH receptor (TSHR) autoantibodies with receptor protein, it is necessary first to express the receptor in the proper conformation including the formation of correct disulfide bridges. However, the reducing environment of the Escherichia coli (E. coli) cytoplasm prevents the generation of protein disulfide bonds and limits the solubility and immunoreactivity of recombinant human TSHR (hTSHR) products. To circumvent these limitations, hTSHR complementary DNA encoding the extracellular domain (hTSHR-ecd; amino acids 21–415) was inserted into the vector pGEX-2TK by directional cloning and used to transform the thioredoxin reductase mutant strain of E. coli (Ad494), which allowed formation of disulfide bonds in the cytoplasm. After induction, the expressed soluble hTSHR-ecd fusion protein was detected by Western blot analysis using a monoclonal antibody directed against hTSHR amino acids 21–35. This showed that over 50% of the expressed hTSHR-ecd was soluble in contrast to expression in a wild-type E. coli (strain αF′), where the majority of the recombinant receptor was insoluble. The soluble recombinant receptor was affinity purified and characterized. Under nonreducing SDS-PAGE conditions, the soluble hTSHR-ecd migrated as refolded, disulfide bond-stabilized, multimeric species, whose formation was independent of fusion partner protein. This product was found to be biologically active as evidenced by the inhibition of the binding of 125I-TSH to the full-length hTSHR expressed in transfected CHO cells and was used to develop a competitive capture enzyme-linked immunosorbent assay for mapping of hTSHR antibody epitopes. Hence, hTSHR-ecd produced in bacteria with a thioredoxin reductase mutation was found to be highly soluble and biologically relevant.

scholarly journals Optimised production of disulfide-bonded fungal effectors in E. coli using CyDisCo and FunCyDisCo co-expression approaches

2021 ◽  
Author(s):  
Daniel Yu ◽  
Megan A Outram ◽  
Emma Creen ◽  
Ashley Smith ◽  
Yi-Chang Sung ◽  
...  
Keyword(s):  
Disulfide Bond ◽  
Disulfide Bonds ◽  
Fungal Pathogens ◽  
Sequence Similarity ◽  
Expression System ◽  
Pathogenic Fungi ◽  
Efficient Production ◽  
E Coli ◽  
Functional Studies ◽  
Folded Structure

Effectors are a key part of the arsenal of plant pathogenic fungi and promote pathogen virulence and disease. Effectors typically lack sequence similarity to proteins with known functional domains and motifs, limiting our ability to predict their functions and understand how they are recognised by plant hosts. As a result, cross-disciplinary approaches involving structural biology and protein biochemistry are often required to decipher and better characterise effector function. These approaches are reliant on high yields of relatively pure protein, which often requires protein production using a heterologous expression system. For some effectors, establishing an efficient production system can be difficult, particularly those that require multiple disulfide bonds to achieve their naturally folded structure. Here, we describe the use of a co-expression system within the heterologous host E. coli termed CyDisCo (cytoplasmic disulfide bond formation in E. coli) to produce disulfide bonded fungal effectors. We demonstrate that CyDisCo and a naturalised co-expression approach termed FunCyDisCo (Fungi-CyDisCo) can significantly improve the production yields of numerous disulfide bonded effectors from diverse fungal pathogens. The ability to produce large quantities of functional recombinant protein has facilitated functional studies and crystallisation of several of these reported fungal effectors. We suggest this approach could be broadly useful in the investigation of the function and recognition of a broad range of disulfide-bond containing effectors.

scholarly journals Disulfide Bond Formation at the C Termini of Vaccinia Virus A26 and A27 Proteins Does Not Require Viral Redox Enzymes and Suppresses Glycosaminoglycan-Mediated Cell Fusion

2009 ◽  
Vol 83 (13) ◽  
pp. 6464-6476 ◽  
Author(s):  
Yao-Cheng Ching ◽  
Che-Sheng Chung ◽  
Cheng-Yen Huang ◽  
Yu Hsia ◽  
Yin-Liang Tang ◽  
...  
Keyword(s):  
Vaccinia Virus ◽  
Cell Fusion ◽  
Disulfide Bond ◽  
Protein Complex ◽  
Disulfide Bonds ◽  
Bond Formation ◽  
In Vitro Mutagenesis ◽  
Disulfide Bond Formation ◽  
Infected Cells

ABSTRACT Vaccinia virus A26 protein is an envelope protein of the intracellular mature virus (IMV) of vaccinia virus. A mutant A26 protein with a truncation of the 74 C-terminal amino acids was expressed in infected cells but failed to be incorporated into IMV (W. L. Chiu, C. L. Lin, M. H. Yang, D. L. Tzou, and W. Chang, J. Virol 81:2149-2157, 2007). Here, we demonstrate that A27 protein formed a protein complex with the full-length form but not with the truncated form of A26 protein in infected cells as well as in IMV. The formation of the A26-A27 protein complex occurred prior to virion assembly and did not require another A27-binding protein, A17 protein, in the infected cells. A26 protein contains six cysteine residues, and in vitro mutagenesis showed that Cys441 and Cys442 mediated intermolecular disulfide bonds with Cys71 and Cys72 of viral A27 protein, whereas Cys43 and Cys342 mediated intramolecular disulfide bonds. A26 and A27 proteins formed disulfide-linked complexes in transfected 293T cells, showing that the intermolecular disulfide bond formation did not depend on viral redox pathways. Finally, using cell fusion from within and fusion from without, we demonstrate that cell surface glycosaminoglycan is important for virus-cell fusion and that A26 protein, by forming complexes with A27 protein, partially suppresses fusion.

Galectin-13/placental protein 13: redox-active disulfides as switches for regulating structure, function and cellular distribution

Glycobiology ◽  
2019 ◽  
Vol 30 (2) ◽  
pp. 120-129 ◽  
Author(s):  
Tong Yang ◽  
Yuan Yao ◽  
Xing Wang ◽  
Yuying Li ◽  
Yunlong Si ◽  
...  
Keyword(s):  
Disulfide Bond ◽  
Disulfide Bonds ◽  
Bond Formation ◽  
Gel Filtration ◽  
Wild Type ◽  
High Concentration ◽  
Redox Active ◽  
Placental Protein ◽  
The Relationship ◽  
Low Expression

Abstract Galectin-13 (Gal-13) plays numerous roles in regulating the relationship between maternal and fetal tissues. Low expression levels or mutations of the lectin can result in pre-eclampsia. The previous crystal structure and gel filtration data show that Gal-13 dimerizes via formation of two disulfide bonds formed by Cys136 and Cys138. In the present study, we mutated them to serine (C136S, C138S and C136S/C138S), crystalized the variants and solved their crystal structures. All variants crystallized as monomers. In the C136S structure, Cys138 formed a disulfide bond with Cys19, indicating that Cys19 is important for regulation of reversible disulfide bond formation in this lectin. Hemagglutination assays demonstrated that all variants are inactive at inducing erythrocyte agglutination, even though gel filtration profiles indicate that C136S and C138S could still form dimers, suggesting that these dimers do not exhibit the same activity as wild-type (WT) Gal-13. In HeLa cells, the three variants were found to be distributed the same as with WT Gal-13. However, a Gal-13 variant (delT221) truncated at T221 could not be transported into the nucleus, possibly explaining why women having this variant get pre-eclampsia. Considering the normally high concentration of glutathione in cells, WT Gal-13 should exist mostly as a monomer in cytoplasm, consistent with the monomeric variant C136S/C138S, which has a similar ability to interact with HOXA1 as WT Gal-13.

Preparation of Water-Soluble Soy Protein-Based Surfactants by Oxidizing Disulfide Bonds

2011 ◽  
Vol 236-238 ◽  
pp. 63-66
Author(s):  
Jun Sheng Li ◽  
Wei Jing ◽  
Liu Juan Yan ◽  
Li Na Li
Keyword(s):  
Molecular Structure ◽  
Surface Activity ◽  
Disulfide Bond ◽  
Soy Protein ◽  
Disulfide Bonds ◽  
Water Soluble ◽  
Performic Acid ◽  
Protein Surface ◽  
Emulsifying Property ◽  
Oxidation Damage

In this paper, the correlations between the cleavage degree of disulfide bonds and soy protein surface activity had been studied in order to show the surface activity of soy protein. The disulfide bonds of soy protein were oxidized to sulfonic groups by performic acid. The distribution of polar and nonpolar groups, and the molecular structure of soy protein were changed because of the oxidation damage of disulfide bonds, and these changes led to changes in surface activity of soy protein. The results showed that the emulsifying property of soy protein was improved by oxidizing the disulfide bonds of protein compared with that of natural soy protein. The change of soy protein emulsifying property is closely related with the degree of the disulfide bond oxidation damage,and that was also an effective way to prepare the protein-based surfactant.

scholarly journals Glycoprotein Ibα forms disulfide bonds with 2 glycoprotein Ibβ subunits in the resting platelet

Blood ◽  
2006 ◽  
Vol 109 (2) ◽  
pp. 603-609 ◽  
Author(s):  
Shi-Zhong Luo ◽  
Xi Mo ◽  
Vahid Afshar-Kharghan ◽  
Sankaranarayanan Srinivasan ◽  
José A. López ◽  
...  
Keyword(s):  
Disulfide Bond ◽  
Cho Cells ◽  
Disulfide Bonds ◽  
Transmembrane Domain ◽  
Chinese Hamster ◽  
Human Platelets ◽  
Sequence Polymorphism ◽  
Size Difference ◽  
Receptor Complex ◽  
Subunit Stoichiometry

Abstract It is widely accepted that glycoprotein (GP) Ib contains one Ibα and one Ibβ subunit that are connected by a disulfide bond. It is unclear which Cys residue in Ibα, C484 or C485, forms the disulfide bond with Ibβ. Using mutagenesis studies in transfected Chinese hamster ovary (CHO) cells, we found that both C484 and C485 formed a disulfide bond with C122 in Ibβ. In the context of isolated peptides containing the Ibα or Ibβ transmembrane domain and nearby Cys residue, C484 and C485 in the Ibα peptide were both capable of forming a disulfide bond with the Ibβ peptide. Furthermore, coimmunoprecipitation of epitope-tagged subunits showed that at least 2 Ibβ subunits but only 1 Ibα and 1 IX subunit were present in the GP Ib-IX complex. Finally, the size difference between GP Ib from transfected CHO cells and human platelets was attributed to a combination of sequence polymorphism and glycosylation difference in Ibα, not the number of Ibβ subunits therein. Overall, these results demonstrate that Ibα is covalently connected to 2 Ibβ subunits in the resting platelet, necessitating revision of the subunit stoichiometry of the GP Ib-IX-V complex. The αβ2 composition in GP Ib may provide the basis for possible disulfide rearrangement in the receptor complex.

The kinetics of in vitro reoxidation and reduction of the inter heavy–light chain disulfide bond in an unusual murine immunoglobulin G myeloma protein lacking inter-heavy chain disulfide bonds

1979 ◽  
Vol 57 (3) ◽  
pp. 279-285 ◽  
Author(s):  
Maire E. Percy ◽  
Lebe Chang ◽  
Catherine Demoliou ◽  
Reuben Baumal
Keyword(s):  
Disulfide Bond ◽  
Disulfide Bonds ◽  
Peptide Mapping ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sedimentation Coefficient ◽  
Disulfide Bridge ◽  
Myeloma Protein ◽  
Kinetics Of

After 5 years of subcutaneous transfer in Balb/C mice, our MOPC 173 myeloma tumour line (originally an IgG2a,κ H2L2-producer) exclusively synthesized an unusual IgG2b,κ protein lacking inter-heavy (H) chain disulfide bonds. This protein was designated MOPC 173B. On sodium dodecyl sulfate – polyacrylamide gel electrophoresis, it migrated with an apparent molecular weight of 77 000; following complete reduction and alkylation, the mobilities of its constituent H and light (L) chains were found to differ slightly from those of MOPC 173 H2L2. MOPC 173B was serologically identical to another typical IgG2b,κ myeloma protein, MOPC 195, and peptide mapping studies showed that it possessed only the inter H–L disulfide bond characteristic of typical IgG2b,κ proteins. In a nondissociating solvent, the sedimentation coefficient of the protein was 6.3S even at concentrations as low as 0.2 mg/ml, indicating that noncovalent interactions existed between two half-molecule subunits. Since this unusual IgG myeloma protein contained only a single category of interchain disulfide bridge, the inter H–L bond, it was an ideal model system for characterization of the kinetics of formation and reduction of interchain disulfide bonds. The kinetics of the glutathione-catalyzed reoxidation of the inter H–L disulfide bridge in MOPC 173B followed an apparent second-order rate equation. In contrast, reduction of its inter H–L bridge under anaerobic conditions with dithioerythritol in excess, was strictly a first-order process and not a simple reversal of the reoxidation. These studies provide the basis for the more complex mathematical models that describe the reoxidation and reduction of typical immunoglobulin molecules.

scholarly journals Effects of the addition of gluten with different disulfide bonds and sulfhydryl concentrations on Chinese white noodle quality

2018 ◽  
Vol 36 (No. 3) ◽  
pp. 246-254 ◽  
Author(s):  
Li Cuicui ◽  
Lu Qiyu ◽  
Liu Zipeng ◽  
Yan Huili
Keyword(s):  
Cross Section ◽  
Disulfide Bond ◽  
Disulfide Bonds ◽  
Sodium Sulfite ◽  
Structural Characteristics ◽  
Cooking Time ◽  
Disulphide Bonds ◽  
Chinese White ◽  
Noodle Quality

The disulfide bonds and sulfhydryl in gluten were evaluated to investigate the effects of structural characteristics on the quality of white noodles. The free sulfhydryl concentration increased significantly, but disulphide bonds decreased initially, and then became stable with increasing sodium sulfite concentration. With a decrease in disulfide bond concentration from 56.78 μmol/g to 20.01 μmol/g in gluten added to noodles, microstructural graphs verified that the cross-section and surface of noodles became rougher and looser with more holes generated; optimal cooking time decreased from 4.4 min to 3.3 min for fresh noodles and from 10.11 min to 9.19 min for dried samples; water absorption and cooking loss increased from 159% to 203% and from 5.26% to 9.06%, respectively. A decreasing trend and marked differences were observed for hardness, springiness, chewiness as well as resilience of fresh and cooked noodles, but the cohesiveness of fresh noodles exhibited no significant changes (P < 0.05).

The Single Disulfide-Directed β-Hairpin Fold: Role of Disulfide Bond in Folding and Effect of an Additional Disulfide Bond on Stability

2020 ◽  
Vol 73 (4) ◽  
pp. 312
Author(s):  
Balasubramanyam Chittoor ◽  
Bankala Krishnarjuna ◽  
Rodrigo A. V. Morales ◽  
Raymond S. Norton
Keyword(s):  
Disulfide Bond ◽  
Disulfide Bonds ◽  
Solution Structure ◽  
Conformational Stability ◽  
Disulfide Bridge ◽  
Solution Nmr ◽  
Peptide Epitope ◽  
The Core ◽  
Native Fold

Disulfide bonds play a key role in the oxidative folding, conformational stability, and functional activity of many peptides. A few disulfide-rich peptides with privileged architecture such as the inhibitor cystine knot motif have garnered attention as templates in drug design. The single disulfide-directed β-hairpin (SDH), a novel fold identified more recently in contryphan-Vc1, has been shown to possess remarkable thermal, conformational, and chemical stability and can accept a short bioactive epitope without compromising the core structure of the peptide. In this study, we demonstrated that the single disulfide bond is critical in maintaining the native fold by replacing both cysteine residues with serine. We also designed an analogue with an additional, non-native disulfide bridge by replacing Gln1 and Tyr9 with Cys. Contryphan-Vc11–22[Q1C, Y9C] was synthesised utilising orthogonal cysteine protection and its solution structure determined using solution NMR spectroscopy. This analogue maintained the overall fold of native contryphan-Vc1. Previous studies had shown that the β-hairpin core of contryphan-Vc1 was resistant to proteolysis by trypsin and α-chymotrypsin but susceptible to cleavage by pepsin. Contryphan-Vc11–22[Q1C, Y9C] proved to be completely resistant to pepsin, thus confirming our design strategy. These results highlight the role of the disulfide bond in maintaining the SDH fold and provide a basis for the design of more stable analogues for peptide epitope grafting.

scholarly journals Identification and functional analysis of inter-subunit disulfide bonds of the F protein of Helicoverpa armigera nucleopolyhedrovirus

2014 ◽  
Vol 95 (12) ◽  
pp. 2820-2830 ◽  
Author(s):  
Feifei Yin ◽  
Manli Wang ◽  
Ying Tan ◽  
Fei Deng ◽  
Just M. Vlak ◽  
...  
Keyword(s):  
Disulfide Bond ◽  
Viral Entry ◽  
Helicoverpa Armigera ◽  
Disulfide Bonds ◽  
Site Directed Mutagenesis ◽  
Disulfonic Acid ◽  
Viral Infectivity ◽  
F Protein ◽  
Free Thiol ◽  
Helicoverpa Armígera

The major envelope fusion protein F of the budded virus of baculoviruses consists of two disulfide-linked subunits: an N-terminal F2 subunit and a C-terminal, membrane-anchored F1 subunit. There is one cysteine in F2 and there are 15 cysteines in F1, but their role in disulfide linking is largely unknown. In this study, the inter- and intra-subunit disulfide bonds of the Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus (HearNPV) F protein were analysed by site-directed mutagenesis. Results indicated that in a functional F protein, an inter-subunit disulfide bond exists between amino acids C108 (F2) and C241 (F1). When C241 was mutated, an alternative disulfide bond was formed between C108 and C232, rendering F non-functional. No inter-subunit bridge was observed in a double C232/C241 mutant of F1. C403 was not involved in the formation of inter-subunit disulfide bonding, but mutation of this amino acid decreased viral infectivity significantly, suggesting that it might be involved in intra-subunit disulfide bonds. The influence of reductant [tris(2-carboxyethyl) phosphine (TCEP)] and free-thiol inhibitors [4-acetamido-4′-maleimidylstilbene 2,2′-disulfonic acid (AMS) and 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB)] on the infectivity of HearNPV was tested. The results indicated that TCEP greatly decreased the infection of HzAm1 cells by HearNPV. In contrast, AMS and DTNB had no inhibitory effect on viral infectivity. The data suggested that free thiol/disulfide isomerization was not likely to play a role in viral entry and infectivity.

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