Changes In Secondary Structure Research Articles

Natural compound plumbagin based inhibition of hIAPP revealed by Markov state models based on MD data along with experimental validations.

Human islet amyloid polypeptide (amylin or hIAPP) is a 37 residue hormone co-secreted with insulin from β cells of the pancreas. In patients suffering from type-2 diabetes, amylin self-assembles into amyloid fibrils, ultimately leading to the death of the pancreatic cells. However, a research gap exists in preventing and treating such amyloidosis. Plumbagin, a natural compound, has previously been demonstrated to have inhibitory potential against insulin amyloidosis. Our investigation unveils collapsible regions within hIAPP that, upon collapse, facilitates hydrophobic and pi-pi interactions, ultimately leading to aggregation. Intriguingly plumbagin exhibits the ability to bind these specific collapsible regions, thereby impeding the aforementioned interactions that would otherwise drive hIAPP aggregation. We have used atomistic molecular dynamics approach to determine secondary structural changes. MSM shows metastable states forming native like hIAPP structure in presence of PGN. Our in silico results concur with in vitro results. The ThT assay revealed a striking 50% decrease in fluorescence intensity at a 1:1 ratio of hIAPP to Plumbagin. This finding suggests a significant inhibition of amyloid fibril formation by plumbagin, as ThT fluorescence directly correlates with the presence of these fibrils. Further TEM images revealed disappearance of hIAPP fibrils in plumbagin pre-treated hIAPP samples. Also, we have shown that plumbagin disrupts the intermolecular hydrogen bonding in hIAPP fibrils leading to an increase in the average beta strand spacing, thereby causing disaggregation of pre-formed fibrils demonstrating overall disruption of the aggregation machinery of hIAPP. Our work is the first to report a detailed atomistic simulation of 22 μs for hIAPP. Overall, our studies put plumbagin as a potential candidate for both preventive and therapeutic candidate for hIAPP amyloidosis.

Magnetic nanoparticles immobilized thrombin ligand fishing to screen thrombin inhibitors in natural products

In this study, thrombin was immobilized with magnetic particles modified by glutaraldehyde. The changes in secondary structures of immobilized enzyme revealed an increment in conformational rigidity and stability, which can be reflected in temperature and pH stability as well as the tolerance of organic reagents. The optimal reutilization times of magnetic particle immobilized thrombin were 7 times, and the half-life of enzyme activity preserved at room temperature was 5 days, which was 2.5 times higher than that of free enzyme. Ligusticum chuanxiong and Anemarrhenae Rhizoma with high enzyme inhibitory activity were selected for primary screening, and six potential inhibitors of thrombin were identified by HPLC/MS. The results showed that three compounds in Anemarrhenae Rhizoma had better predictive thrombin inhibitory activity. Through the in vitro thrombin activity inhibition experiment, it was also verified that mangiferin and neo-mangiferin had an ideal thrombin activity inhibition effect, which was consistent with the results of molecular docking.

Human apo-metallothionein 1a is not a random coil: Evidence from guanidinium chloride, high temperature, and acidic pH unfolding studies

The structures of apo-metallothioneins (apo-MTs) have been relatively elusive due to their fluxional, disordered state which has been difficult to characterize. However, intrinsically disordered protein (IDP) structures are rather diverse, which raises questions about where the structure of apo-MTs fit into the protein structural spectrum. In this paper, the unfolding transitions of apo-MT1a are discussed with respect to the effect of the chemical denaturant GdmCl, temperature conditions, and pH environment. Cysteine modification in combination with electrospray ionization mass spectrometry was used to probe the unfolding transition of apo-MT1a in terms of cysteine exposure. Circular dichroism spectroscopy was also used to monitor the change in secondary structure as a function of GdmCl concentration. For both of these techniques, cooperative unfolding was observed, suggesting that apo-MT1a is not a random coil. More GdmCl was required to unfold the protein backbone than to expose the cysteines, indicating that cysteine exposure is likely an early step in the unfolding of apo-MT1a. MD simulations complement the experimental results, suggesting that apo-MT1a adopts a more compact structure than expected for a random coil. Overall, these results provide further insight into the intrinsically disordered structure of apo-MT.

Insight into the improvement mechanism of gel properties of pea protein isolate based on the synergistic effect of cellulose nanocrystals and calcium ions

Here, the influence and potential mechanism by which cellulose nanocrystals (CNC) collaborated with Ca2+ enhancing the heat-induced gelation of pea protein isolate (PPI) were investigated. It was found that the combination of 0.45% CNC and 15 mM Ca2+ synergistically increased the gel strength (from 14.18 to 65.42 g) and viscoelasticity of PPI while decreased the water holding capacity. The improved particle size, turbidity, and thermostability as well as the reduced solubility, crystallinity, and gel porosity were observed in CNC/CaCl2 composite system. CNC fragments bind to specific amino acids in 11S legumin and 7S vicilin mainly through hydrogen bonding and van der Waals forces. Moreover, changes in the protein secondary structure and enhancement of the molecular interaction induced by CNC and Ca2+ could favor the robust gel network. The results will provide a new perspective on the functional regulation of pea protein and the creation of pea protein gel-based food.

Interaction and complex formation of sonicated soluble lentil protein and Tremella fuciformis polysaccharide

Proteins’ physicochemical and structural properties critically affect their interactions with polysaccharides. The effect of ultrasound treatment (0–40 min) on the physicochemical properties of soluble lentil proteins (LPs) was investigated. The interaction between LPs (ultrasound-treated for different times, U0-LP, U5-LP, and U40-LP) and Tremella fuciformis polysaccharides (TPS) under different pHs, biopolymer mixing ratios, and total biopolymer concentrations was studied. With the sonication duration increasing, decreased intrinsic fluorescence, higher zeta potential values, and increased hydrophilicity were observed for the sonicated LP samples. The molecule weight of LPs remained unchanged, and their secondary structure changes were independent of the sonication duration. The soluble LPs:TPS complexes and insoluble complex coacervates formed at pH 6.5 (pHc) and pH 6.0 (pHɸ1), respectively, regardless of the mixing ratio and the LP type. The maximum formation of insoluble LPs:TPS complex coacervates (pHopt) occurred at the biopolymer mixing ratio of 4:1 at pH 3.0, 2:1 at pH 3.0, and 2:1 at pH 3.5 for the U0-LP:TPS, U5-LP:TPS, and U40-LP:TPS systems, respectively. The pHɸ1 and pHopt of all the three LPs:TPS systems shifted to higher pH values as the total biopolymer concentration increased. With the greatest dynamic quenching constants (Ksv) and binding site (n) values, U40-LP exhibited the strongest binding affinity with TPS. This was consistent with the highest viscosity of their mixture solution under pHs greater than pHopt. The overall results suggest that ultrasound-treated proteins showed a stronger electrostatic interaction with TPS compared to untreated proteins.

Study of Monoclonal Antibody Aggregation at the Air-Liquid Interface under Flow by ATR-FTIR Spectroscopic Imaging.

Throughout bioprocessing, transportation, and storage, therapeutic monoclonal antibodies (mAbs) experience stress conditions that may cause protein unfolding and/or chemical modifications. Such structural changes may lead to the formation of aggregates, which reduce mAb potency and may cause harmful immunogenic responses in patients. Therefore, aggregates need to be detected and removed or ideally prevented from forming. Air-liquid interfaces, which arise during various stages of bioprocessing, are one of the stress factors causing mAb aggregation. In this study, the behavior of an immunoglobulin G (IgG) at the air-liquid interface was investigated under flow using macro attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopic imaging. This chemically specific imaging technique allows observation of adsorption of IgG to the air-liquid interface and detection of associated secondary structural changes. Chemical images revealed that IgG rapidly accumulated around an injected air bubble under flow at 45 °C; however, no such increase was observed at 25 °C. Analysis of the second derivative spectra of IgG at the air-liquid interface revealed changes in the protein secondary structure associated with increased intermolecular β-sheet content, indicative of aggregated IgG. The addition of 0.01% w/v polysorbate 80 (PS80) reduced the amount of IgG at the air-liquid interface in a static setup at 30 °C; however, this protective effect was lost at 45 °C. These results suggest that the presence of air-liquid interfaces under flow may be detrimental to mAb stability at elevated temperatures and demonstrate the power of ATR-FTIR spectroscopic imaging for studying the structural integrity of mAbs under bioprocessing conditions.

Deciphering the binding behavior and interaction mechanism of apigenin and α-glucosidase based on multi-spectroscopic and molecular simulation studies

This study investigated the molecular mechanism underlying the binding interaction between apigenin (API) and α-glucosidase (α-glu) by a combination of experimental techniques and computational simulation strategies. The spontaneously formation of stable API-α-glu complex was mainly driven by hydrogen bonds and hydrophobic forces, leading to a static fluorescence quenching of α-glu. The binding of API induced secondary structure and conformation changes of α-glu, decreasing the surface hydrophobicity of protein. Computational simulation results demonstrated that API could bind into the active cavity of α-glu via its interaction with active residues at the binding site. The important roles of key residues responsible for the binding stability and affinity between API and α-glu were further revealed by MM/PBSA results. In addition, it can be found that the entrance of active site tended to close after API binding as a result of its interaction with gate keeping residues. Furthermore, the structural basis for the binding interaction behavior of API was revealed and visualized by weak interaction analysis. The findings of our study revealed atomic-level mechanism of the interaction between API, which might shed light on the development of better inhibitors.

Infrared Spectroscopy for Tracking Changes in Proteins Secondary Structure and Lipids During Wound Healing Process of Diabetic Mice After Treated by a Cold Atmospheric Plasma Jet

Infrared Spectroscopy for Tracking Changes in Proteins Secondary Structure and Lipids During Wound Healing Process of Diabetic Mice After Treated by a Cold Atmospheric Plasma Jet

Site-directed immobilization of enzymes on nanoparticles using self-assembly systems

Enzyme immobilization is an effective method for improving the stability and reusability. However, linking at random sites on the enzyme results in low catalytic efficiency due to blockage of the active site or conformational changes. Therefore, controlling the orientation of enzymes on the carrier has been developed. Here, the site-specific mutation and the SpyTag/SpyCatcher systems were used to prepare a site-directed immobilized enzyme. The thermal stability of the immobilized enzyme was better than that of the free enzyme, and ≥80 % of the catalytic activity was retained after 30 days of storage. Furthermore, the Michaelis constant (Km) and the turnover number (kcat) of the immobilized enzyme were 5.23-fold lower and 6.11-fold higher than those of the free enzyme, respectively, which appeared to be related to changes in secondary structure after immobilization. These findings provide a new and effective option for enzyme-directed immobilization.

Computational insights into dynamics and conformational stability of N-acetylmannosamine kinase mutations

The activity of UDP-GlcNAc 2-epimerase/ManNAc kinase (GNE) is essential for the biosynthesis of sialic acid, which is involved in cellular processes in health and diseases. GNE contains an N-terminal epimerase domain and a C-terminal kinase domain (N-acetylmannosamine kinase, MNK). Mutations of the GNE protein led to hypoactivity of the enzyme and cause sialurea or autosomal recessive inclusion body myopathy/Nonaka myopathy. Here, we used all-atom molecular dynamics (MD) simulations to comprehend the folding, dynamics and conformational stability of MNK variants, including the wild type (WT) and three mutants (H677R, V696M and H677R/V696M). The deleterious and destabilizing nature of MNK mutants were predicted using different prediction tools. Results predicted that mutations modulate the stability, flexibility and function of MNK. The effect of mutations on the conformational stability and dynamics of MNK was next studied through the free-energy landscape (FEL), hydrogen-bonds and secondary structure changes. The FEL results show that the mutations interfere with various conformational transitions in both WT and mutants, exposing the structural underpinnings of protein destabilization and unfolding brought on by mutation. We discover that, when compared to the other two mutations, V696M and H677R/V696M, H677R has the most harmful effects. These findings have a strong correlation with published experimental studies that demonstrate how these mutations disrupt MNK activity. Hence, this computational study describes the structural details to unravel the mutant effects at the atomistic resolution and has implications for understanding the GNE's physiological and pathological role. Communicated by Ramaswamy H. Sarma

Nanogel delivery systems for cationic peptides: More than a ‘One Size Fits All’ solution

Self-assembled hyaluronic acid-based nanogels are versatile drug carriers due to their biodegradable nature and gentle preparation conditions, making them particularly interesting for delivery of peptide therapeutics. This study aims to elucidate the relation between peptide structure and encapsulation in a nanogel. Key peptide properties that affect encapsulation in octenyl succinic anhydride-modified hyaluronic acid nanogels were identified as we explored the effect on nanogel characteristics using 12 peptides with varying charge and hydrophobicity. The size and surface properties of the microfluidics-assembled peptide-loaded nanogels were evaluated using dynamic light scattering, laser Doppler electrophoresis, and small angle neutron scattering. Additionally, the change in peptide secondary structure upon encapsulation in nanogels, their release from the nanogels, and the in vitro antimicrobial activity were assessed. In conclusion, the more hydrophobic peptides showed stronger binding to the nanogel carrier and localized internally rather than on the surface of the nanogel, resulting in more spherical nanogels with smoother surfaces and slower release profiles. In contrast, cationic and hydrophilic peptides localized at the nanogel surface resulting in fluffier nanogel structures and quick and more complete release in biorelevant medium. These findings emphasize that the advantages of nanogel delivery systems for different applications depend on the therapeutic peptide properties.

Investigating the influence of protein secondary structure on the dissolution behavior of β-lactoglobulin-based amorphous solid dispersions

Proteins acting as carriers in amorphous solid dispersions (ASDs) demonstrate a notable sensitivity to the spray drying process, potentially leading to changes in their conformation. The main aim of this study was to investigate the dissolution performance of ASDs based on proteins with different content of secondary structures, specifically β-sheet and α-helix structures. We prepared β-sheet-rich and α-helix-rich β-lactoglobulin (BLG), along with corresponding ASDs containing 10 wt% and 30 wt% drug loadings, through spray drying using celecoxib as the model drug. Circular dichroism and Fourier Transform Infrared Spectroscopy results revealed that even though changes in secondary structure were obtained in the spray-dried powders, the BLGs exhibited reversibility upon re-dissolving in phosphate buffer with varying pH levels. Both β-sheet-rich BLG and α-helix-rich BLG exhibited enhanced dissolution rates and higher solubility in the media with pH values far from the isoelectric point (pI) of BLG (pH 2, 7, 8, and 9) compared to the pH closer to the pI (pH 3, 4, 5, and 6). Notably, the release rate and solubility of the drug and BLG from both types of BLG-based ASDs at 10 wt% drug loading were largely dependent on the solubility of pure SD-BLGs. α-helix-rich BLG-ASDs consistently exhibited equivalent or superior performance to β-sheet-rich BLG-ASDs in terms of drug release rate and solubility, regardless of drug loading. Moreover, both types of BLG-based ASDs at 10 wt% drug loading exhibited faster release rates and higher solubility, for both the drug and BLG, compared to the ASDs at 30 wt% drug loading in pHs 2, 7, and 9 media.

Effect of glycation-induced concentration-dependent change in albumin structure and alteration in its binding capacity

Reducing sugars causes confirmatory alterations in albumin structure by the nonenzymatic glycation of the amino group of serum albumin. In this study, glucose and its hazardous metabolic products like glyoxal and methylglyoxal were incubated with bovine serum albumin (BSA). The confirmational changes in BSA molecule’s structure by glycating substances was investigated using a variety of spectroscopic methods, including deconvolutionFourier Transform Infra-red (FT-IR) spectroscopy, fluorescence spectroscopy, UV spectroscopy and circular dichroism (CD) spectroscopy. Dynamic fluorescence quenching was observed in the case of glucose, while static quenching was observed in the case of methyl glyoxal and glyoxal. Similarly, employing deconvolution FT-IR spectroscopy and CD spectroscopy for determination of change in secondary structures in terms signature of α-helix, β-turn, β-sheet and random coil modifications. Destabilization or unfolding of the albumin structure, due to the disruption of the hydrogen bonding pattern that stabilizes the albumin manifold, causes a 25-50% reduction in α-helix and a 2-fold increase in β-sheet and turns in glycated BSA. The competitive displacement interaction studies with warfarin were performed using the ultrafiltration technique and quantitative determination of free drug in ultrafiltrate using LC-MS/MS. The binding of carbamazepine (CBZ) or its active metabolite to proteins was unaffected by the glycation of BSA with glucose and methyl glyoxal. Nevertheless, with glyoxal-modified BSA, it changed the binding of selected analytes significantly. Based on in vitro observations and results, it could be anticipated that the serum CBZ concentration variation may be worsened in uncontrolled diabetes circumstances, with an overall variance of 30–40% possible. Communicated by Ramaswamy H. Sarma

Preferential Binding of Polyphenols in Blackcurrant Extracts with Milk Proteins and the Effects on the Bioaccessibility and Antioxidant Activity of Polyphenols.

Milk proteins are well-known delivery agents; however, there is no clear understanding of the competitive interactions of milk proteins with polyphenols in mixed complex systems. Here, we investigate the preferential competitive interactions of different polyphenols present in blackcurrant extract with milk proteins by quantifying the protein-bound polyphenols and comparing the factors affecting these interactions. In addition, bioaccessibility and antioxidant activity were studied after in vitro gastric digestion. Our results indicated that polyphenols from blackcurrant extracts were preferentially bound to caseins more than whey proteins, with noncovalent interactions causing secondary structural changes in the protein. The hydrophobicity and the charge of the polyphenols were negatively and positively related to the number of polyphenols bound to casein and whey proteins, respectively. Moreover, the bioaccessibility and antioxidant activity of polyphenols were enhanced in the presence of milk proteins in milk-based blackcurrant samples when compared to polyphenol and protein-alone samples in the in vitro gastric phase. These findings underscore the critical role of milk proteins in encapsulating or delivering polyphenols. This will pave the way for boosting the bioavailability of polyphenols by complexing them with milk proteins and formulating functional dairy foods, integrating the beneficial effects of these compounds.

Effect of different oligomerization assemblies of γ-conglutin on its interaction behavior with vitexin.

Several different factors underlie the molecular mechanisms of phenolic compound-protein interactions. They include the environmental conditions. In the case of γ-conglutin, pH conditions translate directly into the adoption of two distinct oligomeric assemblies, i.e. hexameric (pH 7.5) or monomeric (pH 4.5). This paper reports research on the pH-dependent oligomerization of γ-conglutin in terms of its ability to form complexes with a model flavonoid (vitexin). Fluorescence-quenching thermodynamic measurements indicate that hydrogen bonds, electrostatic forces, and van der Waals interactions are the main driving forces involved in the complex formation. The interaction turned out to be a spontaneous and exothermic process. Assessment of structural composition (secondary structure changes and arrangement/dynamics of aromatic amino acids), molecular size, and the thermal stability of the different oligomeric forms showed that γ-conglutin in a monomeric state was less affected by vitexin during the interaction. The data show precisely how environmental conditions might influence phenolic compound-protein complex formation directly. This knowledge is essential for the preparation of food products containing γ-conglutin. The results can contribute to a better understanding of the detailed fate of this unique health-promoting lupin seed protein after its intake. © 2023 Society of Chemical Industry.

Tomato (Solanum lycopersicum) leaf juice induced whey protein gelling: Unveiling the potential of endogenous proteases in novel applications

Tomato leaves are by-products of tomato production and a potential source of proteins. Part of these proteins are proteases that carry out important functions for the plants. These endogenous proteases can bring challenges in downstream protein extraction, and can alter protein functionality. The aim of this study was to characterize the endogenous proteases in tomato leaf juice and to investigate the hydrolysis by these proteases on both endogenous leaf proteins and WPI. In addition, we characterized the gelation behavior, rheological properties and structural and thermal properties change of WPI by the juice proteases. 38 different proteases were identified in the juice, along with 4 protease inhibitors. The proteases hydrolyzed both endogenous leaf proteins and WPI, with predominantly an endopeptidase manner for the latter. The protein hydrolysis was positively affected by the incubation time and temperature. The protease activity in juice could not be fully eliminated by heat deactivation or the addition of protease inhibitors. The protein hydrolysis however remained limited (max. 43 %) under all conditions below 60 °C. This hydrolysis resulted in small changes in both secondary structure and thermal properties of WPI. Limited hydrolysis led to WPI gelation at lower protein concentration. Gel formed with WPI after limited hydrolysis had higher gel strength and more brittle structure than the gel with intact proteins. To conclude, the complex and active nature of proteases in tomato leaves poses challenge for downstream protein extraction. Alternatively, juice from tomato leaves could be used as natural gelling agent for WPI.

Monitoring the interactions between POPG phospholipid bilayer and amyloid-forming protein human cystatin C. Does the bilayer influence the oligomeric state and structure of the protein?

A biological membrane is a structure characteristic for various cells and organelles present in almost all living organisms. Even though, it is one of the most common structures in organisms, where it serves crucial functions, a phospholipid bilayer may also take part in pathological processes leading to severe diseases. Research indicates that biological membranes have a profound impact on the pathological processes of oligomerization of amyloid-forming proteins. These processes are a hallmark of amyloid diseases, a group of pathological states involving, e.g., Parkinson's or Alzheimer's disease. Even though amyloidogenic diseases reap the harvest in modern societies, especially in elderly patients, the mechanisms governing the amyloid deposition are not clearly described. Therefore, the presented study focuses on the description of interactions between a model biological membrane (POPG) and one of amyloid forming proteins – human cystatin C. For the purpose of the study molecular dynamics simulations were applied to confirm interactions between the protein and POPG membrane. Next the NMR techniques were used to verify how the data obtained in solution compared to MD simulations and determine fragments of the protein responsible for interactions with POPG. Finally, circular dichroism was used to monitor the changes in secondary structure of the protein and size exclusion chromatography was used to monitor its oligomerization process. Obtained data indicates that the protein interacts with POPG submerging itself into the bilayer with the AS region. However, the presence of POPG bilayer does not significantly affect the structure or oligomerization process of human cystatin C.

Changes in Secondary Structure and Properties of Bovine Serum Albumin as a Result of Interactions with a Gold Surface.

The front cover artwork is provided by Prof. Barbara Jachimska's group at the Jerzy Haber Institute of Catalysis and Surface Chemistry, PAS. The image represents the process of changes in the secondary structure of Bovine Serum Albumin (BSA) as a result of its interactions with a gold surface. Read the full text of the Research Article at 10.1002/cphc.202300505.

Front Cover: Changes in Secondary Structure and Properties of Bovine Serum Albumin as a Result of Interactions with Gold Surface (ChemPhysChem 2/2024)

Front Cover: Changes in Secondary Structure and Properties of Bovine Serum Albumin as a Result of Interactions with Gold Surface (ChemPhysChem 2/2024)

Lowering added sugar and increasing fibre in croissants using short-chain fructooligosaccharides (sc-FOS)

This work explores the potential of short-chain fructooligosaccharides (sc-FOS) as a sugar replacer in bakery products, particularly croissants. The effects of sucrose replacement with sc-FOS on croissant dough properties, as well as the quality of baked croissants, are studied. It was found that the changes in croissant on inclusion of sc-FOS are primarily related to changes in water redistribution and the binding state of water in the dough, with minimal role of change in secondary structure of proteins. Rheological properties of sugar-reduced croissant dough were characterized. The use of sc-FOS as a sucrose replacement increased storage and loss moduli, indicating enhanced interactions and a firmer dough. Creep compliance revealed less deformation and improved elastic recovery, suggesting strengthened bonding, especially in the gluten network. The upper crust of croissants showed no significant differences in colour, but the lower crust darkened slightly with sc-FOS. Texture analysis revealed a firmer texture with complete sucrose replacement, while the porosity remained consistent. Sensory evaluation indicated changes in hardness and overall acceptability with sc-FOS, emphasizing the need for reformulation and consideration of the impacts on starch gelatinization.