Presence Of Disulfide Bonds Research Articles

Biophysical and structural studies of fibulin-2

Fibulin-2 is a multidomain, disulfide-rich, homodimeric protein which belongs to a broader extracellular matrix family. It plays an important role in the development of elastic fiber structures. Malfunction of fibulin due to mutation or poor expression can result in a variety of diseases including synpolydactyly, limb abnormalities, eye disorders leading to blindness, cardiovascular diseases and cancer. Traditionally, fibulins have either been produced in mammalian cell systems or were isolated from the extracellular matrix, a procedure that results in poor availability for structural and functional studies. Here, we produced seven fibulin-2 constructs covering 62% of the mature protein (749 out of 1195 residues) using a prokaryotic expression system. Biophysical studies confirm that the purified constructs are folded and that the presence of disulfide bonds within the constructs makes them extremely thermostable. In addition, we solved the first crystal structure for any fibulin isoform, a structure corresponding to the previously suggested three motifs related to anaphylatoxin. The structure reveals that the three anaphylatoxins moieties form a single-domain structure.

TXM peptides inhibit SARS-CoV-2 infection, syncytia formation, and lower inflammatory consequences

After three years of the SARS-CoV-2 pandemic, the search and availability of relatively low-cost benchtop therapeutics for people not at high risk for a severe disease are still ongoing. Although vaccines and new SARS-CoV-2 variants reduce the death toll, the long COVID-19 along with neurologic symptoms can develop and persist even after a mild initial infection. Reinfections, which further increase the risk of sequelae in multiple organ systems as well as the risk of death, continue to require caution. The spike protein of SARS-CoV-2 is an important target for both vaccines and therapeutics. The presence of disulfide bonds in the receptor binding domain (RBD) of the spike protein is essential for its binding to the human ACE2 receptor and cell entry. Here, we demonstrate that thiol-reducing peptides based on the active site of oxidoreductase thioredoxin 1, called thioredoxin mimetic (TXM) peptides, can prevent syncytia formation, SARS-CoV-2 entry into cells, and infection in a mouse model. We also show that TXM peptides inhibit the redox-sensitive HIV pseudotyped viral cell entry. These results support disulfide targeting as a common therapeutic strategy for treating infections caused by viruses using redox-sensitive fusion. Furthermore, TXM peptides exert anti-inflammatory properties by lowering the activation of NF-κB and IRF signaling pathways, mitogen-activated protein kinases (MAPKs) and lipopolysaccharide (LPS)-induced cytokines in mice. The antioxidant and anti-inflammatory effects of the TXM peptides, which also cross the blood-brain barrier, in combination with prevention of viral infections, may provide a beneficial clinical strategy to lower viral infections and mitigate severe consequences of COVID-19.

Itaconic acid-based sustainable polyurethane covalent adaptable networks with robust mechanical, reshaping and UV-resistant properties based on reversible disulfide bond

Thermoset polymer materials are widely used in various fields due to their excellent performance brought by their permanently cross-linked structures. However, their inability to be recycled also leads to a serious waste of resources. The emergence of covalent adaptable networks (CANs) has solved the problem perfectly that thermoset materials are difficult to recycle. Meanwhile, with the increasing concern for resources and the environment, bio-based materials have sparked a research boom. Itaconic acid is a biomass feedstock with great prospects for development. In this study, itaconic acid, 1,6-hexanediol, 6-mercapto-1-hexanol, isocyanate, and 4,4′-dithiodiphenylamine were used as raw materials. Itaconic acid-based polyols were first synthesized by thiol-ene click reaction, and a series of itaconic acid-based polyurethane (PU) materials containing dynamic covalent networks were synthesized by varying the ratios between the amino and hydroxyl groups. The obtained material has good mechanical properties with a maximum tensile strength of 31.5 MPa and a modulus of 717 MPa. The presence of disulfide bonds allows the material to be recycled by remodeling, and the recovery efficiency of tensile strength after three remodeling cycles is 85.1 %. The material also has excellent solvent resistance and UV-blocking resistance. In conclusion, this work provides possible ideas for the high-value utilization of itaconic acid in polyurethanes.

CD-44 active cystamine-bridged hyaluronic acid-polydopamine nanoparticles for chemo-photothermal cancer therapy

The development of targeted drug delivery systems for cancer treatment has been a topic of great interest in recent years. We developed redox-responsive, NIR-activated, cystamine-bridged hyaluronan-polydopamine (Cys-HA-PDA) nanoparticles (NPs) loaded with doxorubicin (Dox) that bind to CD44 receptors on cancer cells and exhibit enhanced anticancer efficacy due to a chemo-photothermal effect under redox conditions. Synthesized Cys-HA-PDA NPs at 200 µg/mL concentration produced a temperature of 59.5 ℃ at 2 W/cm2, which destroyed tumor cells. The presence of disulfide bonds in Cys-HA-PDA NPs resulted in self-degradability in the cytosol of cancer cells. Furthermore, electrostatic interactions and pi-pi stacking allowed Dox to be successfully encapsulated in Cys-HA-PDA nanoparticles; calculated maximum drug loading and encapsulation efficiency were 21.6% and 88.3%, respectively. The anticancer activity of Dox-loaded NPs on HepG2 cancer cells was increased under NIR laser light (800 nm, 2 W/cm2). Furthermore, interaction with CD44 receptor enhanced the intracellular uptake of Dox-loaded Cys-HA-PDA NPs by HepG2 cells. Overall, the study shows that the use of redox-responsive nanoparticles for CD44-targeted drug delivery is a promising anticancer strategy.

Local Structural Analysis of Sulfide Polymer Electrolytes Prepared via I2-Induced Polymerization of Li3PS4

To avoid the volume change associated with the electrode active material during charge/discharge in all-solid-state batteries, an effective binder that improves the contact between the solid electrolyte and the electrode active material must be developed. To this aim, the polymerization of the sulfide solid electrolyte Li3PS4 offers a promising route. However, the local structure of the resulting solid electrolyte, i.e., a mixture of LiI crystals and glassy sulfide polymers, needs to be elucidated for further development of sulfide polymers as binders. Herein, we analyze the local structure of the isolated mixture via a differential pair distribution function analysis using X-ray total scattering. The presence of disulfide bonds between PS4 anions is confirmed in the glassy phase. The coordination number of the disulfide bonds is 0.65, and each PS4 tetrahedron is connected to two tetrahedrons to form a polymer chain structure of (−P–S–S−)n bonds containing Li ions. The coordination number of Li ions surrounding PS4 anions in the glassy phase is much smaller than that in the pure Li3PS4 glasses. This polymer may find application as a Li-ion conductive binder in sheet-type all-solid-state batteries having low resistance and high capacity retention.

Potential Development of Cow Hair Waste as Alternative Feed Protein Source for Poultry in South Sulawesi Province, Indonesia

Cow hair waste (CHW) is one of the wastes produced by the cowhide cracker processing industry. In South Sulawesi Province, every year, approximately 55.6 tons of CHW produced from this industry. The problem, the digestibility of CHW is very low so that the process requires certain technologies. The low of digestibility caused by the presence of disulfide bonds (S-S) in the protein component (keratin). The application of CHW as a protein source feed affects the productivity and quality of poultry meat. The study was aims to evaluate the productivity and quality of poultry meat (quail) which was added with CHW meal at various levels. A total of four levels of CHW meal (0% (control); 2%, 4% and 6%)(w/w) were applied to the composition of the quail (Coturnix-coturnix) feed rations that were kept in cages for 6 weeks. The results showed no significant effect (p>0,05) on the body weight (BW), meat shear force (MSF) and cooking loss (CL) with increasing levels of CHW meal administration. In general, it can be concluded that CHW can be applied in the composition of poultry feed at level of 0-6% (w/w).

Gastrointestinal fate of food allergens and its relationship with allergenicity.

Food allergens are closely related to their gastrointestinal digestion fate, but the changes in food allergens during digestion and related mechanisms are quite complicated. This review presents in detail digestion models for predicting allergenicity, the fates of food allergens in oral, gastric and duodenal digestion, and the applications of digestomics in mapping IgE-binding epitopes of digestion-resistant peptides. Moreover, this review highlights the structure-activity relationships of food allergens during gastrointestinal digestion. Digestion-labile allergens may share common structural characteristics, such as high flexibility, rendering them easier to be hydrolyzed into small fragments with decreased or eliminated allergenicity. In contrast, the presence of disulfide bonds, tightly wound α-helical structures, or hydrophobic domains in food allergens helps them resist gastrointestinal digestion, stabilizing IgE-binding epitopes, thus maintaining their sensitization. In rare cases, digestion leads to increased allergenicity due to exposure of new epitopes. Finally, the action of the food matrix and processing on the digestion and allergenicity of food allergens as well as the underlying mechanisms was overviewed. The food matrix can directly act on the allergen by forming complexes or new epitopes to affect its gastrointestinal digestibility and thereby alter its allergenicity or indirectly affect the allergenicity by competing for enzymatic cleavage or influencing gastrointestinal pH and microbial flora. Several processing techniques attenuate the allergenicity of food proteins by altering their conformation to improve susceptibility to degradation by digestive enzymes. Given the complexity of food components, the food itself rather than a single allergen should be used to obtain more accurate data for allergenicity assessment. PRACTICAL APPLICATION: The review article will help to understand the relationship between food protein digestion and allergenicity, and may provide fundamental information for evaluating and reducing the allergenicity of food proteins.

Protein Disulfide Bonds Detected by Tagging with High Molecular Weight Maleimide Derivative

Disulfide bridges are essential for maintaining the structure and function of proteins. Traditionally, studies of the disulfide bonds require expensive equipment and high purity of the protein sample, therefore, the development of simpler techniques is warranted. Here, were present a novel protocol for the detection of disulfide bonds in proteins, which is based on the labeling reduced disulfide bridges with a high molecular weight (HMW) maleimide derivative. After irreversible blocking of free thiol groups of proteins, the labeling of new thiols released from disulfide bridges with a high-molecular-weight (HMW) maleimide derivative is performed. To confirm localization of cysteines involved in the formation of disulfide bonds, cysteine mutagenesis was conducted. For validation, aquaporin 5 (AQP5) and transient receptor potential cation channel subfamily V member 4 (TRPV4) proteins were tagged with FLAG (DYKDDDDK) on N-termini. Increase in MW of the target proteins from immunoblot indicated the presence of disulfide bonds. No bands with increased MW were detected in AQP5, while TPRV4 cysteines at disulfide bridges-constituting positions 639, 645, 652, 660, 770 were detected and confirmed by cysteine mutagenesis. These data indicate that the proposed technique is feasible and effective for the detection of protein disulfide bonds.

Protein Disulfide Bonds Detected by Tagging with High Molecular Weight Maleimide Derivative

Disulfide bridges are essential for maintaining the structure and function of proteins. Traditionally, studies of the disulfide bonds require expensive equipment and high purity of the protein sample, therefore, the development of simpler techniques is warranted. Here, were present a novel protocol for the detection of disulfide bonds in proteins, which is based on the labeling reduced disulfide bridges with a high molecular weight (HMW) maleimide derivative. After irreversible blocking of free thiol groups of proteins, the labeling of new thiols released from disulfide bridges with a high-molecular-weight (HMW) maleimide derivative is performed. To confirm localization of cysteines involved in the formation of disulfide bonds, cysteine mutagenesis was conducted. For validation, aquaporin 5 (AQP5) and transient receptor potential cation channel subfamily V member 4 (TRPV4) proteins were tagged with FLAG (DYKDDDDK) on N-termini. Increase in MW of the target proteins from immunoblot indicated the presence of disulfide bonds. No bands with increased MW were detected in AQP5, while TPRV4 cysteines at disulfide bridges-constituting positions 639, 645, 652, 660, 770 were detected and confirmed by cysteine mutagenesis. These data indicate that the proposed technique is feasible and effective for the detection of protein disulfide bonds.

Redox-Responsive Dipeptide Nanostructures toward Targeted Cancer Therapy.

Materials that exhibit responsiveness toward biological signals are currently subjected to intense research in the field of drug delivery. In our study, we tried to develop cancer-targeted and redox-responsive nanoparticles (NPs) from disulfide-linked oxidized cysteine-phenylalanine (CFO). The NPs were conjugated with folic acid (FA) to specifically target cancer cells, and the presence of disulfide bonds would enabled the disintegration of the particles in the presence of elevated levels of glutathione (GSH) in cancer cells. Anticancer drug doxorubicin (Dox) was successfully loaded inside the disulfide-linked nanoparticles (CFO-Dox-NPs), which further demonstrated stimuli-responsive drug release in the presence of GSH. We have also demonstrated enhanced uptake of FA-derivatized NPs (FA-CFO-NPs) in cancerous cells (C6 glioma and B16F10 melanoma cells) than in normal cells (HEK293T cells) due to the overexpression of FA receptors on the surface of cancer cells. Cytotoxicity studies in C6 cells and B16F10 cells further revealed enhanced efficacy of Dox loaded (FA-CFO-Dox-NPs) as compared to the native drug. The findings of this study clearly demonstrated that the disulfide-linked nanoparticle system may provide a promising selective drug delivery platform in cancer cells.

PSSbond-PseAAC: Prediction of disulfide bonding sites by integration of PseAAC and statistical moments

The structure of protein gains additional stability against various detrimental effects by the presence of disulfide bonds. The formation of correct disulfide bonds between cysteine residues ensures proper in vivo and in vitro folding of the protein. Many cysteine residues can be present in the polypeptide chain of a protein, however, not all cysteine residues are involved in the formation of a disulfide bond, and therefore, accurate prediction of these bonds is crucial for identifying biophysical characteristics of a protein. In the present study, a novel method is proposed for the prediction of intramolecular disulfide bonds accurately using statistical moments and PseAAC. The pSSbond-PseAAC uses PseAAC along with position and composition relative features to calculate statistical moments. Statistical moments are important as they are very sensitive regarding the position of data sequences and for prediction of intramolecular disulfide bonds, moments are combined together to train neural networks. The overall accuracy of the pSSbond-PseAAC is 98.97% to sensitivity value 98.92%, specificity 98.99% and 0.98 MCC; and it outperforms various previously reported studies.

Redox-responsive polymeric micelles formed by conjugating gambogic acid with bioreducible poly(amido amine)s for the co-delivery of docetaxel and MMP-9 shRNA

A novel redox-sensitive system for co-delivering hydrophobic drugs and hydrophilic siRNA or shRNA was developed by conjugating gambogic acid (GA) with poly(amido amine)s (PAAs) through amide bonds, which is called GA-conjugated PAAs (PAG). PAG can self-assemble into micelles as amphiphilic block copolymers, which exhibits an excellent loading ability for the co-delivery of docetaxel (DTX) and MMP-9 shRNA with adjustable dosing ratios. In addition, confocal microscopy, flow cytometry and in vitro transfection analyses demonstrated more efficient cellular internalization of DTX and MMP-9 shRNA after incubation with PAG/DTX- MMP-9 shRNA micelles (PAG/DTX-shRNA) than with free drugs. Unlike traditional amphiphilic copolymer micelles, GA conjugated in PAG possesses an intrinsic anticancer efficacy. The presence of disulfide bonds in PAAs enables rapid disassembly of PAG micelles in response to reducing agents, inducing the release of loaded drugs (DTX, GA and MMP-9 shRNA). In vitro cellular assays revealed that PAG/DTX-shRNA micelles inhibited MCF-7 cell proliferation more efficiently than the single drug or single drug-loaded micelles. In vivo biodistribution and anti-tumor effect studies using an MCF-7 breast cancer xenograft mouse model have indicated that PAG/DTX-shRNA micelles can enhance drug accumulation compared with the free drug, thereby sustaining the therapeutic effect on tumors. Additionally, PAG/DTX-shRNA micelles displayed a greater anti-tumor efficacy than Taxotere® and PAG-shRNA micelles. These results suggest that the redox-sensitive PAG platform is a promising co-delivery system for combining drugs and gene therapy for the treatment of cancer. Statement of SignificanceThe PAG micelles were designed by conjugating gambogic acid (GA) with poly(amido amine)s (PAAs), which would serve dual purposes as both gene and drugs co-delivery carrier and an anti-tumor prodrug. Unlike traditional amphiphilic micelles, GA conjugated in PAG could exert its intrinsic efficacy and provide synergistic antiproliferative effects with docetaxel (DTX) on MCF-7 cells. Disulfide bonds in PAG enables a rapid disassembly of PAG micelles in response to reducing agents and to release all loaded drugs (DTX, GA and MMP-9 shRNA) at tumor sites. PAG/DTX-shRNA micelles displayed greater anti-tumor efficacy than that of Taxotere®, indicating the design concept for PAG works well. And the strategy for PAG could be used to develop a series of similar co-delivery systems through conjugations of other small-molecule drugs with PAAs, such as doxorubicin, methotrexate and other drugs with carboxy groups in their structure.

Mechanochemical Evolution of the Giant Muscle Protein Titin as Inferred from Ancient Proteins

We use phylogenetic analysis and single-molecule atomic force spectroscopy (AFS) to investigate the nanomechanical evolution of titin, a giant protein responsible for the elasticity, integrity and signal transduction of muscle filaments. We bring back to life an eight-domain fragment of titin (I65-I72) from different extinct species ranging from 179 to 356 Mya that have been compared with their modern descendants. Using AFS techniques we have observed that the mechanical stability of titin domains and the presence of disulfide bonds are key elements in the evolution of titin. Our experiments together with structure prediction analysis and biochemical assays confirm that ancient titin domains were richer in disulfide bonds and have higher mechanical stability. While titin domains from birds and reptiles have kept similar mechanochemical features than those of the ancient ones, titin from mammals show lower mechanical stability and lower number of disulfide bonds. Our findings show a paleomechanical trend that suggests that disulfide bonds are mechanical regulators in titin that have disappeared over the course of evolution in mammals generating more flexible and extensible titin domains. We hypothesize that this substitution derived in physiological changes that allowed the muscular development and morphological diversity of modern vertebrates. A comparative analysis between animal physiological properties and titin mechanochemical properties supports our hypothesis and also allows us inferring some physiological properties of ancient species.

Charge site assignment in native proteins by ultraviolet photodissociation (UVPD) mass spectrometry.

Characterization of all gas-phase charge sites of natively sprayed proteins and peptides is demonstrated using 193 nm UVPD. The high sequence coverage offered by UVPD is exploited for the accurate determination of charge sites in protein systems up to 18 kDa, allowing charge site to be studied as a function of protein conformation and the presence of disulfide bonds. Charging protons are found on both basic sidechains and on the amide backbone of less basic amino acids such as serine, glutamine, and proline. UVPD analysis was performed on the 3+ charge state of melittin, the 5+ to 8+ charge states of ubiquitin, and the 8+ charge state of reduced and oxidized β-lactoglobulin. The location of charges in gas-phase proteins is known to impact structure; molecular modeling of different charge site motifs of 3+ melittin demonstrates how placement of protons in simulations can dramatically impact the predicted structure of the molecule. The location of positive charge sites in ubiquitin and β-lactoglobulin are additionally found to depend on the presence or absence of salt-bridges, columbic repulsion across the length of the peptide, and protein conformation. Charge site isomers are demonstrated for ubiquitin and β-lactoglobulin but found to be much less numerous than previously predicted.

Dynamics and thermodynamic properties of CXCL7 chemokine.

Chemokines form a family of signaling proteins mainly responsible for directing the traffic of leukocytes, where their biological activity can be modulated by their oligomerization state. We characterize the dynamics and thermodynamic stability of monomer and homodimer structures of CXCL7, one of the most abundant platelet chemokines, using experimental methods that include circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy, and computational methods that include the anisotropic network model (ANM), molecular dynamics (MD) simulations and the distance constraint model (DCM). A consistent picture emerges for the effects of dimerization and Cys5-Cys31 and Cys7-Cys47 disulfide bonds formation. The presence of disulfide bonds is not critical for maintaining structural stability in the monomer or dimer, but the monomer is destabilized more than the dimer upon removal of disulfide bonds. Disulfide bonds play a key role in shaping the characteristics of native state dynamics. The combined analysis shows that upon dimerization flexibly correlated motions are induced between the 30s and 50s loop within each monomer and across the dimer interface. Interestingly, the greatest gain in flexibility upon dimerization occurs when both disulfide bonds are present, and the homodimer is least stable relative to its two monomers. These results suggest that the highly conserved disulfide bonds in chemokines facilitate a structural mechanism that is tuned to optimally distinguish functional characteristics between monomer and dimer.

CoagVDb: a comprehensive database for coagulation factors and their associated SAPs.

The current state of the art in medical genetics is to identify and classify the functional (deleterious) or non-functional (neutral) single amino acid substitutions (SAPs), also known as non-synonymous SNPs (nsSNPs). The primary goal is to elucidate the mechanisms through which functional SAPs exert their effects, and ultimately interrogating this information for association with complex phenotypes. This work focuses on coagulation factors involved in the coagulation cascade pathway which plays a vital role in the maintenance of homeostasis in the human system. We developed an integrated coagulation variation database, CoagVDb, which makes use of the biological information from various public databases such as NCBI, OMIM, UniProt, PDB and SAPs (rsIDs/variant). CoagVDb enriched with computational prediction scores classify SAPs as either deleterious or tolerated. Also, various other properties are incorporated such as amino acid composition, secondary structure elements, solvent accessibility, ordered/disordered regions, conservation, and the presence of disulfide bonds. This specialized database provides integration of various prediction scores from different computational methods along with gene, protein, and disease information. We hope our database will act as a useful reference resource for hematologists to reveal protein structure–function relationship and disease genotype–phenotype correlation.Electronic supplementary materialThe online version of this article (doi:10.1186/s40659-015-0028-5) contains supplementary material, which is available to authorized users.

Approach to Characterization of the Higher Order Structure of Disulfide-Containing Proteins Using Hydrogen/Deuterium Exchange and Top-Down Mass Spectrometry

Top-down hydrogen/deuterium exchange (HDX) with mass spectrometric (MS) detection has recently matured to become a potent biophysical tool capable of providing valuable information on higher order structure and conformational dynamics of proteins at an unprecedented level of structural detail. However, the scope of the proteins amenable to the analysis by top-down HDX MS still remains limited, with the protein size and the presence of disulfide bonds being the two most important limiting factors. While the limitations imposed by the physical size of the proteins gradually become more relaxed as the sensitivity, resolution and dynamic range of modern MS instrumentation continue to improve at an ever accelerating pace, the presence of the disulfide linkages remains a much less forgiving limitation even for the proteins of relatively modest size. To circumvent this problem, we introduce an online chemical reduction step following completion and quenching of the HDX reactions and prior to the top-down MS measurements of deuterium occupancy of individual backbone amides. Application of the new methodology to the top-down HDX MS characterization of a small (99 residue long) disulfide-containing protein β2-microglobulin allowed the backbone amide protection to be probed with nearly a single-residue resolution across the entire sequence. The high-resolution backbone protection pattern deduced from the top-down HDX MS measurements carried out under native conditions is in excellent agreement with the crystal structure of the protein and high-resolution NMR data, suggesting that introduction of the chemical reduction step to the top-down routine does not trigger hydrogen scrambling either during the electrospray ionization process or in the gas phase prior to the protein ion dissociation.

Plant antimicrobial peptides

Plant antimicrobial peptides (AMPs) are a component of barrier defense system of plants. They have been isolated from roots, seeds, flowers, stems, and leaves of a wide variety of species and have activities towards phytopathogens, as well as against bacteria pathogenic to humans. Thus, plant AMPs are considered as promising antibiotic compounds with important biotechnological applications. Plant AMPs are grouped into several families and share general features such as positive charge, the presence of disulfide bonds (which stabilize the structure), and the mechanism of action targeting outer membrane structures.

Cyclam-Based Polymeric Copper Chelators for Gene Delivery and Potential PET Imaging

A series of reducible polycationic copper chelators (RPCs) based on 1,4,8,11-tetraazacyclotetradecane (cyclam) were synthesized by Michael addition. Molecular weight of the polycations was controlled by reaction stoichiometry and reaction conditions, resulting in polymers with molecular weights ranging from 4400 to 13 800. The cyclam moieties in the polycations retained their ability to form complexes with Cu(II). The presence of disulfide bonds in the polycations resulted in substantially lower cytotoxicity than control 25 kDa poly(ethyleneimine). RPC as well as their complexes with Cu(II) exhibited high transfection activity in vitro. The reported polycationic Cu(II) chelates represent promising nucleic acid delivery vectors with potential for future theranostic applications.

Molecular Dynamics Simulations on Pars Intercerebralis Major Peptide-C (PMP-C) Reveal the Role of Glycosylation and Disulfide Bonds in its Enhanced Structural Stability and Function

Fucosylation of Thr 9 in pars intercerebralis major peptide-C (PMP-C) enhances its structural stability and functional ability as a serine protease inhibitor. In order to understand the role of disulfide bonds and glycosylation on the structure and function of PMP-C, we have carried out multiple explicit solvent molecular dynamics (MD) simulations on fucosylated and non-fucosylated forms of PMP-C, both in the presence and absence of the disulfide bonds. Our simulations revealed that there were no significant structural changes in the native disulfide bonded forms of PMP-C due to fucosylation. On the other hand, the non-fucosylated form of PMP-C without disulfide bonds showed larger deviations from the starting structure than the fucosylated form. However, the structural deviations were restricted to the terminal regions while core β-sheet retained its hydrogen bonded structure even in absence of disulfide bonds as well as fucosylation. Interestingly, fucosylation of disulfide bonded native PMP-C led to a decreased thermal flexibility in the residue stretch 29–32 which is known to interact with the active site of the target proteases. Our analysis revealed that disulfide bonds covalently connect the residue stretch 29–32 to the central β-sheet of PMP-C and using a novel network of side chain interactions and disulfide bonds fucosylation at Thr 9 is altering the flexibility of the stretch 29–32 located at a distal site. Thus, our simulations explain for the first time, how presence of disulfide bonds between conserved cysteines and fucosylation enhance the function of PMP-C as a protease inhibitor.