Effect Of Disulfide Bridges Research Articles

Fabrication of GSH‐Responsive Poly(Tertiary Amine‐Oxide)‐Based Nanomedicines for Enhanced Anticancer Drug Release

AbstractPoly(tertiary amine‐oxide)‐based polymeric nanocarriers have showed more functionalities than stealthy PEG‐based analogs due to the structure of phospholipid affinitive N‐oxides groups, which has attracted considerable attention to the synthesis of various zwitterionic copolymers with tertiary amine‐oxide grafts for enhanced anticancer drug delivery. However, poly(tertiary amine‐oxide)‐based amphiphilic copolymers with tumor intracellular microenvironment sensitivities, to the knowledge, have been rarely reported likely due to the lack of a controlled synthetic strategy. Herein, a reducible zwitterionic copolymer, poly(ε‐caprolactone)25‐SS‐poly(2‐(N‐oxide‐N,N‐diethylamino)ethyl methacrylate)30 (PCL25‐SS‐OPDEA30) is designed and synthesized successfully via combination of controlled polymerization techniques and post polymerization modification. Specifically, postoxidation of poly(tertiary amine) is confirmed to be a better synthetic strategy toward a well‐defined polymer structure relative to direct polymerization of N,N‐diethylaminoethyl methacrylate (ODEA). The effect of disulfide bridges on the self‐assembly behaviors, in vitro drug loading and drug release properties is investigated in detail. Notablely, the resulting micelles self‐assembled from PCL25‐SS‐OPDEA30 show much greater colloidal stability, higher drug loading content (DLC), and better in vitro tumor cell inhibition than the reduction‐insensitive counterparts. Overall, this study reports a robust strategy toward zwitterionic nanomedicines for on‐demand anticancer drug delivery.

Effect of Disulfide Bridge on the Binding of SARS-CoV-2 Fusion Peptide to Cell Membrane: A Coarse-Grained Study.

In this paper, we present the parameterization of the CAVS coarse-grained (CG) force field for 20 amino acids, and our CG simulations show that the CAVS force field could accurately predict the amino acid tendency of the secondary structure. Then, we used the CAVS force field to investigate the binding of a severe acute respiratory syndrome-associated coronavirus fusion peptide (SARS-CoV-2 FP) to a phospholipid bilayer: a long FP (FP-L) containing 40 amino acids and a short FP (FP-S) containing 26 amino acids. Our CAVS CG simulations displayed that the binding affinity of the FP-L to the bilayer is higher than that of the FP-S. We found that the FP-L interacted more strongly with membrane cholesterol than the FP-S, which should be attributed to the stable helical structure of the FP-L at the C-terminus. In addition, we discovered that the FP-S had one major and two minor membrane-bound states, in agreement with previous all-atom molecular dynamics (MD) studies. However, we found that both the C-terminal and N-terminal amino acid residues of the FP-L can strongly interact with the bilayer membrane. Furthermore, we found that the disulfide bond formed between Cys840 and Cys851 stabilized the helices of the FP-L at the C-terminus, enhancing the interaction between the FP-L and the bilayer membrane. Our work indicates that the stable helical structure is crucial for binding the SARS-CoV-2 FP to cell membranes. In particular, the helical stability of FP should have a significant influence on the FP–membrane binding.

Effect of disulfide bridge on hydrolytic characteristics of xylanase from Penicillium janthinellum

Highly efficient and stable enzymes are required for application in biotechnology, to meet the technical, environmental, and economic industrial demands. Xylanases are hemicellulolytic enzymes that degrade the heteroxylan constituent of the lignocellulosic plant cell wall. In this study, an acidic xylanase designated Pjxyn (pH 4.0) from Penicillium janthinellum was engineered by the introduction of a disulfide bridge. This strategy exploited the influence of the bridge on hydrolysis characteristics and enhanced hydrolysis was achieved. Three mutants [PjxynS(27)S(39), PjxynS(27)S(186), and PjxynS(39)S(186)] produced more xylose and xylobiose as hydrolysis products compared with the wild-type Pjxyn, when commercial xylans and lab-prepared water-insoluble corncob-xylan were used as the substrates, especial for the PjxynS(27)S(39) mutant, the content of xylose and xylobiose was 87.62% (using beechwood xylan) and 69.91% (using oat-spelt xylan) higher than that in the hydrolysis products of Pjxyn. Moreover, each mutant combined with the xylanase mutant T-XynFM effectively decreased the production of xylose with an optimum xylobiose yield. The findings demonstrate the potential industrial value of engineering xylanase to improve its hydrolytic properties and thermostability.

Effects of disulfide bridges and backbone connectivity on water sorption by protein matrices

Understanding the water sorption behavior of protein powders is important in applications such as the preservation of protein-based pharmaceuticals. Most globular proteins exhibit a characteristic sigmoidal water adsorption isotherm at ambient conditions. However, it is not well understood how water sorption behavior is influenced by intrinsic factors that are related to structural properties of proteins. We investigate computationally how structural constraints on proteins influence the water sorption isotherms of amorphous protein powders. Specifically, we study the effects of non-local disulfide linkages and backbone connectivity using pheromone ER-23 and lysozyme as model proteins. We find that non-local disulfide linkages can significantly restrict structural changes during hydration and dehydration, and this in turn greatly reduces the extent of hysteresis between the adsorption and desorption branches. Upon removing the backbone connectivity by breaking all peptide bonds in lysozyme, we find that the hysteresis shifts towards the lower humidity regime, and the water uptake capacity is significantly enhanced. We attribute these changes to the higher aggregation propensity of the constraint-free amino acids in dehydrated condition, and the formation of a spanning water network at high hydration levels.

Introduction of additional thiol groups into glucoamylase in Aspergillus awamori and their effect on the thermal stability and catalytic activity of the enzyme

Five mutant forms of glucoamylase (GA) from the filamentous fungus Aspergillus awamori with artificial disulfide bonds (4D-G137A\A14C, 6D-A14C\Y419C\G137A, 10D-V13C\G396C, 11D-V13C\G396C\A14C\Y419C\G137A, and 20D-G137A\A246C\A14C) were constructed using computer simulation and experimentally tested for thermostability. The introduction of two additional disulfide bonds between its first and thirteenth alpha-helices and that of the loop located close to a catalytic residue--E400--made it possible to assess the effects of disulfide bridges on protein thermostability. The mutant proteins with combined amino acid substitutions G137A\A14C, V13C\G396C\A14C\Y419C\G137A, and G137A\A246C\A14C showed higher thermal stability as compared to the wild-type protein. At the same time, new disulfide bridges in the mutant A14C\Y419C\G137A and V13C\G396C proteins led to the destabilization of their structure and the loss of thermal stability.

Crystal structure of c5321: a protective antigen present in uropathogenic Escherichia coli strains displaying an SLR fold

BackgroundIncreasing rates of antimicrobial resistance among uropathogens led, among other efforts, to the application of subtractive reverse vaccinology for the identification of antigens present in extraintestinal pathogenic E. coli (ExPEC) strains but absent or variable in non-pathogenic strains, in a quest for a broadly protective Escherichia coli vaccine. The protein coded by locus c5321 from CFT073 E. coli was identified as one of nine potential vaccine candidates against ExPEC and was able to confer protection with an efficacy of 33% in a mouse model of sepsis. c5321 (known also as EsiB) lacks functional annotation and structurally belongs to the Sel1-like repeat (SLR) family. Herein, as part of the general characterization of this potential antigen, we have focused on its structural properties.ResultsWe report the 1.74 Å-resolution crystal structure of c5321 from CFT073 E. coli determined by Se-Met SAD phasing. The structure is composed of 11 SLR units in a topological organisation that highly resembles that found in HcpC from Helicobacter pylori, with the main difference residing in how the super-helical fold is stabilised. The stabilising effect of disulfide bridges in HcpC is replaced in c5321 by a strengthening of the inter-repeat hydrophobic core. A metal-ion binding site, uncharacteristic of SLR proteins, is detected between SLR units 3 and 4 in the region of the inter-repeat hydrophobic core. Crystal contacts are observed between the C-terminal tail of one molecule and the C-terminal amphipathic groove of a neighbouring one, resembling interactions between ligand and proteins containing tetratricopeptide-like repeats.ConclusionsThe structure of antigen c5321 presents a mode of stabilization of the SLR fold different from that observed in close homologs of known structure. The location of the metal-ion binding site and the observed crystal contacts suggest a potential role in regulation of conformational flexibility and interaction with yet unidentified target proteins, respectively. These findings open new perspectives in both antigen design and for the identification of a functional role for this protective antigen.

Solution structure of the antimicrobial peptide gaegurin 4 by H and 15N nuclear magnetic resonance spectroscopy.

Gaegurin 4 (GGN4) is a 37-residue antimicrobial peptide isolated from the skin of a Korean frog, Rana rugosa. This peptide shows a broad range of activity against prokaryotic cells but shows very little hemolytic activity against human red blood cells. The solution structure of GGN4 was studied by using circular dichroism (CD) and NMR spectroscopy. CD investigations revealed that GGN4 adopts mainly an alpha-helical conformation in trifluoroethanol/water solution, in dodecylphosphocholine and in SDS micelles, but adopts random structure in aqueous solution. By using both homonuclear and heteronuclear NMR experiments, complete 1H and 15N resonance assignments were obtained for GGN4 in 50% trifluoroethanol/water solution. The calculated structures of GGN4 consist of two amphipathic alpha-helices extending from residues 2-10 and from residues 16-32. These two helices are connected by a flexible loop spanning between the residues 11 and 15. By using enzyme digestion and matrix-assisted laser desorption/ionization mass spectroscopy, we confirmed that GGN4 contains a disulfide bridge formed between the residues Cys31 and Cys37 in its C-terminus. The effect of disulfide bridge on the structure and the activity of GGN4 was investigated. The reduced form of GGN4 revealed a similar activity and conformation to native GGN4, suggesting that the disulfide bridge does not strongly affect the conformation and the antimicrobial activity of GGN4.