scholarly journals Reversibly Cross-Linking Polymer Brushes Using Interchain Disulfide Bonds

2020 ◽  
Vol 53 (2) ◽  
pp. 731-740 ◽  
Author(s):  
Piotr Mocny ◽  
Harm-Anton Klok
Keyword(s):  
Disulfide Bonds ◽  
Polymer Brushes ◽  
Cross Linking

scholarly journals Spontaneous Cross-link of Mutated α1 Subunits during GABAA Receptor Assembly

2006 ◽  
Vol 282 (7) ◽  
pp. 4354-4363 ◽  
Author(s):  
Isabella Sarto-Jackson ◽  
Roman Furtmueller ◽  
Margot Ernst ◽  
Sigismund Huck ◽  
Werner Sieghart
Keyword(s):  
Disulfide Bonds ◽  
Early Stage ◽  
Cross Linking ◽  
Cross Link ◽  
Chloride Currents ◽  
Assembly Mechanism ◽  
Acid Type ◽  
Starting Point ◽  
Separate Pathway ◽  
Receptor Assembly

γ-Aminobutyric acid, type A (GABAA) receptor α1 subunits containing a cysteine mutation at a position in the channel mouth (H109C) surprisingly formed a spontaneous cross-link with each other in receptors composed of α1H109C, β3, and γ2 subunits. Cross-linking of two α1H109C subunits did not significantly change the affinity of [3H]muscimol or [3H]Ro15-1788 binding in α1H109Cβ3γ2 receptors, but GABA displayed a reduced potency for activating chloride currents. On reduction of the disulfide bond, however, GABA activation as well as diazepam modulation was similar in mutated and wild-type receptors, suggesting that these receptors exhibited the same subunit stoichiometry and arrangement. Disulfide bonds could not be reoxidized by copper phenanthroline after having been reduced in completely assembled receptors, suggesting that cross-linking can only occur at an early stage of assembly. The cross-link of α1H109C subunits and the subsequent transport of the resulting homodimers to the cell surface caused a reduction of the intracellular pool of α1H109C subunits and a reduced formation of completely assembled receptors. The formation of α1H109C homodimers as well as of correctly assembled GABAA receptors containing cross-linked α1H109C subunits could indicate that homodimerization of α1 subunits via contacts located in the channel mouth might be one starting point of GABAA receptor assembly. Alternatively the assembly mechanism might have started with the formation of heterodimers followed by a cross-link of mutated α1 subunits at the heterotrimeric stage. The formation of cross-linked α1H109C homodimers would then have occurred independently in a separate pathway.

scholarly journals Crosslinked Recombinant-Ara h 1 Catalyzed by Microbial Transglutaminase: Preparation, Structural Characterization and Allergic Assessment

Foods ◽  
2020 ◽  
Vol 9 (10) ◽  
pp. 1508
Author(s):  
Yang Tian ◽  
Chenglong Liu ◽  
Wentong Xue ◽  
Zhongfu Wang
Keyword(s):  
Disulfide Bonds ◽  
Structural Changes ◽  
Structural Features ◽  
Microbial Transglutaminase ◽  
Enzyme Linked Immunosorbent Assay ◽  
Cross Linking ◽  
Intrinsic Structure ◽  
Ara H 1 ◽  
Molecular Forces ◽  
The One

As the one of the major allergens in peanut, the allergenicity of Ara h 1 is influenced by its intrinsic structure, which can be modified by different processing. However, molecular information in this modification has not been clarified to date. Here, we detected the influence of microbial transglutaminase (MTG) catalyzed cross-linking on the recombinant peanut protein Ara h 1 (rAra h 1). Electrophoresis and spectroscopic methods were used to analysis the structural changes. The immunoreactivity alterations were characterized by enzyme linked immunosorbent assay (ELISA), immunoblotting and degranulation test. Structural features of cross-linked rAra h 1 varied at different reaction stages. Hydrogen bonds and disulfide bonds were the main molecular forces in polymers induced by heating and reducing. In MTG-catalyzed cross-linking, ε-(γ-glutamyl) lysine isopeptide bonds were formed, thus inducing a relatively stable structure in polymers. MTG catalyzed cross-linking could modestly but significantly reduce the immunoreactivity of rAra h 1. Decreased content of conserved secondary structures led to a loss of protection of linear epitopes. Besides, the reduced surface hydrophobic index and increased steric hindrance of rAra h 1 made it more difficult to bind with antibodies, thus hindering the subsequent allergic reaction.

scholarly journals Site-Directed Disulfide Cross-Linking Shows that Cleft Flexibility in the Periplasmic Domain Is Needed for the Multidrug Efflux Pump AcrB of Escherichia coli

2007 ◽  
Vol 189 (23) ◽  
pp. 8677-8684 ◽  
Author(s):  
Yumiko Takatsuka ◽  
Hiroshi Nikaido
Keyword(s):  
Escherichia Coli ◽  
Conformational Changes ◽  
Disulfide Bonds ◽  
Drug Transport ◽  
Site Directed Mutagenesis ◽  
Cross Linking ◽  
Cysteine Residues ◽  
Multidrug Efflux ◽  
Specific Chemical ◽  
Periplasmic Domain

ABSTRACT Escherichia coli AcrB is a multidrug efflux transporter that recognizes multiple toxic chemicals having diverse structures. Recent crystallographic studies of the asymmetric trimer of AcrB suggest that each protomer in the trimeric assembly goes through a cycle of conformational changes during drug export. However, biochemical evidence for these conformational changes has not been provided previously. In this study, we took advantage of the observation that the external large cleft in the periplasmic domain of AcrB appears to become closed in the crystal structure of one of the three protomers, and we carried out in vivo cross-linking between cysteine residues introduced by site-directed mutagenesis on both sides of the cleft, as well as at the interface between the periplasmic domains of the AcrB trimer. Double-cysteine mutants with mutations in the cleft or the interface were inactive. The possibility that this was due to the formation of disulfide bonds was suggested by the restoration of transport activity of the cleft mutants in a dsbA strain, which had diminished activity to form disulfide bonds in the periplasm. Furthermore, rapidly reacting, sulfhydryl-specific chemical cross-linkers, methanethiosulfonates, inactivated the AcrB transporter with double-cysteine residues in the cleft expressed in dsbA cells, and this inactivation could be observed within a few seconds after the addition of a cross-linker in real time by increased ethidium influx into the cells. These observations indicate that conformational changes, including the closure of the external cleft in the periplasmic domain, are required for drug transport by AcrB.

scholarly journals Improved Gel Properties of Whey Protein-Stabilized Emulsions by Ultrasound and Enzymatic Cross-Linking

Gels ◽  
2021 ◽  
Vol 7 (3) ◽  
pp. 135
Author(s):  
Yanli Zhao ◽  
Shiqi Xue ◽  
Xinyue Zhang ◽  
Tiehua Zhang ◽  
Xue Shen
Keyword(s):  
Whey Protein ◽  
Disulfide Bonds ◽  
Noncovalent Interactions ◽  
Textural Properties ◽  
Industrial Applications ◽  
Cross Linking ◽  
Similar Frequency ◽  
Soluble Aggregate ◽  
Gelation Behavior ◽  
Cross Links

This study investigated the effects of high-intensity ultrasound (HUS) and transglutaminase pretreatment on the gelation behavior of whey protein soluble aggregate (WPISA) emulsions. HUS pretreatment and TGase-mediated cross-linking delayed the onset of gelation but significantly increased (p < 0.05) the gel firmness (G′) both after gel formation at 25 °C and during storage at 4 °C. The frequency sweep test indicated that all gels had a similar frequency dependence at 4 and 25 °C, and the elasticity and viscosity of the WPISA-stabilized emulsion gel were significantly enhanced by HUS pretreatment and TGase-mediated cross-linking (p < 0.05). HUS and TGase-mediated cross-linking greatly improved the textural properties of WPISA-stabilized emulsion gels, as revealed by their increases in gel hardness, cohesiveness, resilience, and chewiness. HUS pretreatment and TGase-mediated cross-linking significantly increased the water-holding capacity but decreased the swelling ratios of the gels (p < 0.05). Interactive force analysis confirmed that noncovalent interactions, disulfide bonds, and TGase-induced covalent cross-links were all involved in the formation of gel networks. In conclusion, the combination of HUS and TGase-mediated cross-linking were beneficial for improving the gelation properties of WPISA-stabilized emulsion as a controlled release vehicle for potential food industrial applications.

scholarly journals Developing a Slow-release Nitrogen Fertilizer from Organic Sources: II. Using Poultry Feathers

1996 ◽  
Vol 121 (4) ◽  
pp. 634-638 ◽  
Author(s):  
Jong-Myung Choi ◽  
Paul V. Nelson
Keyword(s):  
Enzymatic Hydrolysis ◽  
Disulfide Bonds ◽  
Slow Release ◽  
Cross Linking ◽  
The Third ◽  
Slow Release Fertilizer ◽  
Microbial Hydrolysis ◽  
N Release ◽  
Poultry Feathers ◽  
Modest Reduction

The structure of feather keratin protein was modified in attempts to develop a slow-release N fertilizer of 12 weeks duration or longer by steam hydrolysis to break disulfide bonds, enzymatic hydrolysis with Bacillus licheniformis (Weigmann) to break polypeptide bonds, and steam hydrolysis (autoclaving) to hasten mineralization followed by cross-linking of the protein by a formaldehyde reaction to control the increased rate of mineralization. Release of N in potting substrate within elution columns from ground, but otherwise untreated, raw feathers occurred mainly during the first 5 weeks with a much smaller release occurring from weeks 8 to 12. Steam hydrolysis resulted in an increase of N during the first 5 weeks and a decrease during weeks 8 to 11. Cumulative N release over 11 weeks increased from 12% in raw feathers to 52% for feathers steam hydrolyzed for 90 minutes. This favored an immediately available fertilizer but not a slow-release fertilizer. Microbial hydrolysis with B. licheniformis resulted in a modest reduction of N release during the first 5 weeks and a small increase during weeks 8 to 11. Both shifts, while not desirable for an immediately available fertilizer, enhanced the slow-release fertilizer potential of feathers but not sufficiently to result in a useful product. Steam hydrolyzed feathers cross-linked with quantities of formaldehyde equal to 5% and 10% of the feather weight released less N during the first 5 weeks, more during weeks 6 and 7, and less during weeks 9 to 12 compared to raw feathers. The first two shifts were favorable for a slow-release fertilizer while the third was not.

Cross-Linking Highly Lubricious Phosphocholinated Polymer Brushes: Effect on Surface Interactions and Frictional Behavior

2017 ◽  
Vol 50 (18) ◽  
pp. 7361-7371 ◽  
Author(s):  
Noa Iuster ◽  
Odeya Tairy ◽  
Michael J. Driver ◽  
Steven P. Armes ◽  
Jacob Klein
Keyword(s):  
Polymer Brushes ◽  
Surface Interactions ◽  
Cross Linking ◽  
Frictional Behavior

scholarly journals Hydrogen/Deuterium Exchange Mass Spectrometry and Site-Directed Disulfide Cross-Linking Suggest an Important Dynamic Interface between the Two Lysostaphin Domains

2013 ◽  
Vol 57 (4) ◽  
pp. 1872-1881 ◽  
Author(s):  
Hai-Rong Lu ◽  
Mei-Gang Gu ◽  
Qiang Huang ◽  
Jin-Jiang Huang ◽  
Wan-Ying Lu ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Disulfide Bonds ◽  
Deuterium Exchange ◽  
Full Length ◽  
Cross Linking ◽  
Hydrogen Deuterium Exchange ◽  
Content Type ◽  
Exchange Mass Spectrometry ◽  
Hydrogen Deuterium ◽  
Deuterium Exchange Mass Spectrometry

ABSTRACTLysostaphin is a peptidoglycan hydrolase secreted byStaphylococcus simulans. It can specifically lyseStaphylococcus aureusand is being tested as a novel antibacterial agent. The protein contains an N-terminal catalytic domain and a C-terminal cell wall targeting domain. Although the two domains from homologous enzymes were structurally determined, the structural organization of lysostaphin domains remains unknown. We used hydrogen/deuterium exchange mass spectrometry (H/DX-MS) and site-directed disulfide cross-linking to probe the interface between the lysostaphin catalytic and targeting domains. H/DX-MS-mediated comparison of peptides from full-length lysostaphin and the separated domains identified four peptides of lower solvent accessibility in the full-length protein. Cross-linking analysis using cysteine pair substitutions within those peptides showed that two pairs of cysteines can form disulfide bonds, supporting the domain association role of the targeted peptides. The cross-linked mutant exhibited a binding capacity toS. aureusthat was similar to that of the wild-type protein but reduced bacteriolytic activity probably because of restraint in conformation. The diminished activity was further reduced with increasing NaCl concentrations that can cause contractions of bacterial peptidoglycan. The lytic activity, however, could be fully recovered by reducing the disulfide bonds. These results suggest that lysostaphin may require dynamic association of the two domains for coordinating substrate binding and target cleavage on the elastic peptidoglycan. Our study will help develop site-specific PEGylated lysostaphin to treat systemicS. aureusinfections.

scholarly journals Enhancing protein stability with extended disulfide bonds

2016 ◽  
Vol 113 (21) ◽  
pp. 5910-5915 ◽  
Author(s):  
Tao Liu ◽  
Yan Wang ◽  
Xiaozhou Luo ◽  
Jack Li ◽  
Sean A. Reed ◽  
...  
Keyword(s):  
Amino Acids ◽  
Disulfide Bond ◽  
Disulfide Bonds ◽  
Building Blocks ◽  
Side Chain ◽  
Cross Linking ◽  
Noncanonical Amino Acids ◽  
Genetic Encoding ◽  
Selection Experiments ◽  
Significant Distance

Disulfide bonds play an important role in protein folding and stability. However, the cross-linking of sites within proteins by cysteine disulfides has significant distance and dihedral angle constraints. Here we report the genetic encoding of noncanonical amino acids containing long side-chain thiols that are readily incorporated into both bacterial and mammalian proteins in good yields and with excellent fidelity. These amino acids can pair with cysteines to afford extended disulfide bonds and allow cross-linking of more distant sites and distinct domains of proteins. To demonstrate this notion, we preformed growth-based selection experiments at nonpermissive temperatures using a library of random β-lactamase mutants containing these noncanonical amino acids. A mutant enzyme that is cross-linked by one such extended disulfide bond and is stabilized by ∼9 °C was identified. This result indicates that an expanded set of building blocks beyond the canonical 20 amino acids can lead to proteins with improved properties by unique mechanisms, distinct from those possible through conventional mutagenesis schemes.

scholarly journals Mechanism of Laccase-assisted Tyrosine Grafting on Keratin using BSA as a Model Protein

2021 ◽  
Author(s):  
Yu Li ◽  
Jing Su ◽  
Tarsila G. Castro ◽  
Artur Cavaco-Paulo
Keyword(s):  
Disulfide Bonds ◽  
Solvent Accessibility ◽  
Strength Loss ◽  
Cross Linking ◽  
Operational Conditions ◽  
Model Protein ◽  
Pre Treatment ◽  
Dynamics Simulations ◽  
User Friendly ◽  
Protein Molecular Dynamics

Commercial hair perming uses strong reducing agents and is harmful to hair fiber’s quality even human health. In this study, tyrosine is adopted as a cross-linking agent between thiols as the shape-changing of hair involves breakage of disulfide bonds and the rearrangement of new bonds between keratin molecules. To investigate the mechanism of the cross-linking, bovine serum albumin (BSA) is used as a model protein. Molecular dynamics simulations give an insight on Cys solvent accessibility and protein stability for the wild type BSA and a designed BSA presenting the three broken disulfide bonds. A new cross-linked peptide, NECFLSHK-Tyrosine-Tyrosine-GACLLPK, inter- or intra- BSA monomers is formed, whose reactive cysteine residues are Cys-101 and Cys-176. Moreover, curling of Asian hair is conducted using tyrosine as a perming agent by laccase-assisted reaction. The optimized operational conditions are hair with cysteine pre-treatment (50.0 mM) followed by grafting with 3.0 mM tyrosine. The reshaped hair performed a better perming performance than commercial perming product before washing, although a lower perming efficiency after washing, however without strength loss and could be easily accomplished with a blow-drier. Hence, this new methodology may lead to the development of a gentle and user-friendly approach in the hair care industry.

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