Alpha-helical Content Research Articles

Enhanced biochemical properties of soybean root nodule asparaginase through plant molecular farming compared to bacterial enzyme for cancer treatment

Asparaginase is a therapeutic enzyme used as an anticancer agent and is typically produced through microbial fermentation using organisms such as Escherichia coli and Erwinia chrysanthemi. However, this method faces challenges, including potential enzyme contamination during production, allergic reactions to the enzyme, and stability issues requiring stringent control measures. An innovative solution is the application of plant molecular farming, utilizing Rhizobium root symbiosis for asparaginase production. The objective is to optimize nodule development for asparaginase yield, characterize the enzyme's properties, and evaluate its anticancer efficacy against microbial enzyme. In our study, we established soybean root cultures and inoculated them with Bradyrhizobium japonicum to form root nodules. We evaluated eukaryotic asparaginase production at different incubation times. We purified asparaginase from the root nodule cultures and compared its physicochemical properties and anticancer activity with microbial asparaginase. Results showed that asparaginase reached maximum activity in root nodule cultures 10 days after rhizobium inoculation in the culture media. The root nodule asparaginase exhibited a high content of alpha helices and beta sheets and a low random coil. It demonstrated higher stability and activity across different pH levels and temperatures than Escherichia coli asparaginase. Additionally, root nodule asparaginase displayed better catalytic parameters and stability over time than E. coli asparaginase. Thus, root nodule asparaginase is superior to E. coli asparaginase as an anticancer agent. This ensures the root nodule asparaginase can effectively target cancer cells, enhancing the overall therapeutic outcome. This provides a renewable, cost-effective, and environmentally friendly alternative to traditional enzyme production methods.

Assembly of Hydrophobin class I from Agaricus bisporus produced different amyloid-like fibrils

This work studied the extraction, purification, characterization, and assembly of hydrophobin class I from Agaricus bisporus (ABH4). The highest soluble protein concentration was obtained from the pinhead, the extraction and purification were efficient for hydrophobin class I, obtaining a band of 12 kDa. The identified sequence of hydrophobin presented the eight cysteine residues; for the prediction of the structure, hydrophobin presented more alpha helix structures than beta sheets. It was observed that the hydrophobin managed to decrease and increase the contact angle in Teflon and glass, respectively, finding a micellar critical concentration of 221 μg mL−1. ThT experiments demonstrated that the production of fibrils decreased at basic pH, while acidic and neutral pH favoured the formation of fibrils. Likewise, the addition of colloidal Teflon affects the formation of fibrils. Circular dichroism spectra proved that hydrophobin class I undergo changes in its secondary structure, increasing its alpha helix and beta sheet content after vortexing. It was observed that the analysis by scanning electron microscopy and atomic force microscopy of the hydrophobin produced different amyloid-like structures in glass and mica.

Structural and retrospective bio-dosimetric study of gamma-irradiated human fingernails

Emergency involving ionising radiation is a rare occurrence, yet it can leave severe damage if a population is not equipped with necessary preparation. In such a situation, there is no viable way to know how much radiation dose is received by human beings. However, in present time only personnel of rescue teams have accessed to active dosimetry such as a personal dosimeter. Preliminary study has been made of human fingernails, a model in examining the potential of nails of the human fingers in retrospective and emergency bio-dosimetry applications, also offering effective atomic number near to that of soft human tissues at 7.4. Human fingernails were sampled from different age groups and exposed to 60Co gamma rays, delivering doses from 2 to 10 Gy. Structural alterations were observed, use being made of Raman spectroscopy and the crystalline structure of alpha keratin is studied using X-ray diffraction (XRD), while the elemental composition of the sample is determined by energy dispersive x-ray (EDX) analysis. Raman shows the ‘fingerprint’ of nail sample is identical in which the peak assignment concerns on the disulfide (S–S) bond and the alpha helix content at the amide 1 region. These are the few regions which displayed major Raman shift and intensity difference from a normal nail sample and an effected nail which have the same analogy as a non-irradiated nail and an irradiated nail sample. XRD shows the molecular structure of hard α-keratin from different age groups of fingernails, examining crystallite diameters (D) and crystallinity index (C.I.). The α-keratin characteristic bands can be seen in all groups of fingernails, albeit they become much weaker and shift to smaller angles with higher gamma doses. The results obtained by Raman spectroscopy and X-ray diffraction are complementary and suggest that the α-helix structure of keratin is partially destroyed by the presence of radiation.

Exploration of the pearl millet phospholipase gene family to identify potential candidates for grain quality traits

BackgroundPhospholipases constitute a diverse category of enzymes responsible for the breakdown of phospholipids. Their involvement in signal transduction with a pivotal role in plant development and stress responses is well documented.ResultsIn the present investigation, a thorough genome-wide analysis revealed that the pearl millet genome contains at least 44 phospholipase genes distributed across its 7 chromosomes, with chromosome one harbouring the highest number of these genes. The synteny analysis suggested a close genetic relationship of pearl millet phospholipases with that of foxtail millet and sorghum. All identified genes were examined to unravel their gene structures, protein attributes, cis-regulatory elements, and expression patterns in two pearl millet genotypes contrasting for rancidity. All the phospholipases have a high alpha-helix content and distorted regions within the predicted secondary structures. Moreover, many of these enzymes possess binding sites for both metal and non-metal ligands. Additionally, the putative promoter regions associated with these genes exhibit multiple copies of cis-elements specifically responsive to biotic and abiotic stress factors and signaling molecules. The transcriptional profiling of 44 phospholipase genes in two genotypes contrasting for rancidity across six key tissues during pearl millet growth revealed a predominant expression in grains, followed by seed coat and endosperm. Specifically, the genes PgPLD-alpha1-1, PgPLD-alpha1-5, PgPLD-delta1-7a, PgPLA1-II-1a, and PgPLD-delta1-2a exhibited notable expression in grains of both the genotypes while showing negligible expression in the other five tissues. The sequence alignment of putative promoters revealed several variations including SNPs and InDels. These variations resulted in modifications to the corresponding cis-acting elements, forming distinct transcription factor binding sites suggesting the transcriptional-level regulation for these five genes in pearl millet.ConclusionsThe current study utilized a genome-wide computational analysis to characterize the phospholipase gene family in pearl millet. A comprehensive expression profile of 44 phospholipases led to the identification of five grain-specific candidates. This underscores a potential role for at least these five genes in grain quality traits including the regulation of rancidity in pearl millet. Therefore, this study marks the first exploration highlighting the possible impact of phospholipases towards enhancing agronomic traits in pearl millet.

Ultrasound-assisted modification of soybean protein isolate with L-histidine: Relationship between structure and function

Herein, the effects of ultrasound-assisted L-histidine (L-His) on the physicochemical properties and conformation of soybean protein isolate (SPI) were investigated. Particle size, zeta potential, turbidity, and solubility were used to evaluate protein aggregation, and the relationship between structural and functional changes of the proteins was characterized using spectral analysis, surface hydrophobicity, emulsification, and antioxidant properties. After ultrasound-assisted L-His treatment, SPI exhibited a smaller particle size, higher solubility, and more homogeneous micromorphology owing to the decrease in alpha-helix content and subsequent increases in zeta potential and active sulfhydryl content. In addition, spectral analysis showed that L-His and SPI could form a complex, which changed the microenvironment of the amino acid residues in SPI, thus improving its emulsification and antioxidant properties. At the concentration of L-His was 0.3 % w/w, the nanocomplex had a smaller particle size (140.03 nm), higher ζ-potential (–23.63 mV), and higher emulsification stability (22.48 min).

Exploring the molecular basis of tucatinib interaction with human serum albumin: A spectroscopic and computational analysis

This study delves into the kinetics of interaction among anticancer drug tucatinib (TCT) and human serum albumin (HSA), a pivotal protein that transports substances in the bloodstream of humans. Employing biophysical techniques alongside in-silico approaches, the TCT-HSA complex formation is substantiated. This binding interaction is unequivocally confirmed by the inverse relationship between temperature and Stern-Volmer constant (KSV) and the hyperchromicity in UV–visible spectra. The binding constant (Kb) value (5.34 x 104 M−1) indicates a moderate affinity between TCT and HSA, with thermodynamic analysis pointing towards stabilization through hydrogen bonds and hydrophobic and van der Waals interactions. Moreover, despite causing alterations in the vicinity of Tyr and Trp amino acids, the introduction of TCT to HSA confers a substantial protective effect against the thermal denaturation of the protein. Circular dichroism unveils a decrease in the content of alpha helices upon TCT binding. At the same time, CD thermal analysis hints at a marginal increase in the melting temperature of HSA, indicating heightened stability at elevated temperatures. Molecular docking and competitive displacement assays confirm that the preferred binding site for TCT is subdomain IIA (Sudlow's site I) of HSA. Molecular dynamics simulations emphasize the stability of the TCT-HSA complex. This work sheds light on a detailed characterization of the HSA-TCT interaction, elucidating binding mechanisms and their consequential impact on HSA structure and stability.

Biophysical and functional characterization of N-terminal domain of Human Interferon Regulatory Factor 6.

Interferon regulatory factor 6 (IRF6) has a key function in palate fusion during palatogenesis during embryonic development, and mutations in IRF6 cause orofacial clefting disorders. The in silico analysis of IRF6 is done to obtain leads for the domain boundaries and subsequently the sub-cloning of the N-terminal domain of IRF6 into the pGEX-2TK expression vector and successfully optimized the overexpression and purification of recombinant glutathione S-transferase-fused NTD-IRF6 protein under native conditions. After cleavage of the GST tag, NTD-IRF6 was subjected to protein folding studies employing Circular Dichroism and Intrinsic fluorescence spectroscopy at variable pH, temperature, and denaturant. CD studies showed predominantly alpha-helical content and the highest stability of NTD-IRF6 at pH 9.0. A comparison of native and renatured protein depicts loss in the secondary structural content. Intrinsic fluorescence and quenching studies have identified that tryptophan residues are majorly present in the buried areas of the protein and a small fraction was on or near the protein surface. Upon the protein unfolding with a higher concentration of denaturant urea, the peak of fluorescence intensity decreased and red shifted, confirming that tryptophan residues are majorly present in a more polar environment. While regulating IFNβ gene expression during viral infection, the N-terminal domain binds to the promoter region of Virus Response Element-Interferon beta (VRE-IFNβ). Along with the protein folding analysis, this study also aimed to identify the DNA-binding activity and determine the binding affinities of NTD-IRF6 with the VRE-IFNβ promoter region. The protein-DNA interaction is specific as demonstrated by gel retardation assay and the kinetics of molecular interactions as quantified by Biolayer Interferometry showed a strong affinity with an affinity constant (KD) value of 7.96 × 10-10 M. NTD-IRF6 consists of a mix of α-helix and β-sheets that show temperature-dependent cooperative unfolding between 40 °C and 55 °C. Urea-induced unfolding shows moderate tolerance to urea as the mid-transition concentration of urea (Cm) is 3.2 M. The tryptophan residues are majorly buried as depicted by fluorescence quenching studies. NTD-IRF6 has a specific and high affinity toward the promoter region of VRE-IFNβ.

Biophysical and structural characterization of tetramethrin serum protein complex and its toxicological implications.

Tetramethrin (TMT) is a commonly used insecticide and has a carcinogenic and neurodegenerative effect on humans. The binding mechanism and toxicological implications of TMT to human serum albumin (HSA) were examined in this study employing a combination of biophysical and computational methods indicating moderate binding affinity and potential hepato and renal toxicity. Fluorescence quenching experiments showed that TMT binds to HSA with a moderate affinity, and the binding process was spontaneous and predominantly enthalpy-driven. Circular dichroism spectroscopy revealed that TMT binding did not induce any significant conformational changes in HSA, resulting in no changes in its alpha-helix content. The binding site and modalities of TMT interactions with HSA as computed by molecular docking and molecular dynamics simulations revealed that it binds to Sudlow site II of HSA via hydrophobic interactions through its dimethylcyclopropane carboxylate methyl propanyl group. The structural dynamics of TMT induce proper fit into the binding site creating increased and stabilizing interactions. Additionally, molecular mechanics-Poisson Boltzmann surface area calculations also indicated that non-polar and van der Waals were found to be the major contributors to the high binding free energy of the complex. Quantum mechanics (QM) revealed the conformational energies of the binding confirmation and the degree of deviation from the global minimum energy conformation of TMT. The results of this study provide a comprehensive understanding of the binding mechanism of TMT with HSA, which is important for evaluating the toxicity of this insecticide in humans.

Multiscale Modeling of Macromolecular Interactions between Tau-Amylin Oligomers and Asymmetric Lipid Nanodomains That Link Alzheimer's and Diabetic Diseases.

The molecular events of protein misfolding and self-aggregation of tau and amylin are associated with the progression of Alzheimer's and diabetes, respectively. Recent studies suggest that tau and amylin can form hetero-tau-amylin oligomers. Those hetero-oligomers are more neurotoxic than homo-tau oligomers. So far, the detailed interactions between the hetero-oligomers and the neuronal membrane are unknown. Using multiscale MD simulations, the lipid binding and protein folding behaviors of hetero-oligomers on asymmetric lipid nanodomains or raft membranes were examined. Our raft membranes contain phase-separated phosphatidylcholine (PC), cholesterol, and anionic phosphatidylserine (PS) or ganglioside (GM1) in one leaflet of the lipid bilayer. The hetero-oligomers bound more strongly to the PS and GM1 than other lipids via the hydrophobic and hydrophilic interactions, respectively, in the raft membranes. The hetero-tetramer disrupted the acyl chain orders of both PC and PS in the PS-containing raft membrane, but only the GM1 in the GM1-containing raft membrane as effectively as the homo-tau-tetramer. We discovered that the alpha-helical content in the heterodimer was greater than the sum of alpha-helical contents from isolated tau and amylin monomers on both raft membranes, indicative of a synergetic effect of tau-amylin interactions in surface-induced protein folding. Our results provide new molecular insights into understanding the cross-talk between Alzheimer's and diabetes.

Insights into the conformational, secondary structural, dynamical and hydration pattern changes of glucose mediated glycated HSA: a molecular dynamics approach

The robust structural nature of human serum albumin (HSA) is responsible for its multifarious functional property. The site specific glycation of HSA due to hyperglycaemia (excess glucose) causes structural changes which have an impact on the functioning of the protein. This work investigates the effects of glucose-mediated glycation in the altered inter-domain motion, distorted binding site conformation and modified hydration patterns, Trp214 orientation, and secondary structure transition using simulation approach. Here we have observed an increase of turns in the helices of glycated HSA, which modulates the open-close conformation of Sudlow I & II. The secondary structure changes of glycated HSA indicate plausible reduction in the alpha helical content in the helices which participates in ligand binding. It also affects geometrical features of drug binding sites (Sudlow I and II) such as volume and hydration. We found that glycation disturbs domain specific mobility patterns of HSA, a substantial feature for albumin drug binding ability which is also correlated with changes in the local environment of Trp214. Communicated by Ramaswamy H. Sarma

Effect of non-covalently bound polyphenols on the structural and functional properties of wheat germ protein

This study investigated the effects of epigallocatechin-3-gallate (EGCG), chlorogenic acid (CA), piceatannol (PIC) and ferulic acid (FA) on the conformational and functional properties of wheat germ protein (WGP). The WGP-polyphenol interaction was confirmed by native polyacrylamide gel electrophoresis and detection of polyphenol content in the complex. Among the four polyphenols, EGCG showed the highest binding affinity to WGP. Moreover, FT-IR analysis indicated that the polyphenols changed the secondary structure of WGP, inducing an enhancement in alpha-helix content and a reduction in β-sheet structure. Fluorescence spectrum analysis demonstrated the presence of polyphenols induced tertiary structure change of WGP, leading to the deconstruction and unfolding of protein. In addition, the main interaction force for WGP-EGCG was hydrogen bonding and van der Waals forces, and for WGP-CA complexes was electrostatic interaction, while interaction force for WGP-PIC and WGP-FA complexes were hydrophobic interaction. Moreover, the addition of polyphenols significantly increased the absolute value of zeta potential while decreased surface hydrophobicity and free sulfhydryl content of WGP. The solubility and functionality of WGP were mainly improved by CA and EGCG. The findings in this study suggest that WGP-polyphenol complexes have the potential to be applied as novel functional food ingredients.

Studies on <i>in vitro</i> binding of parecoxib to p38MAPK using spectroscopic methods

Purpose: To investigate the interaction between parecoxib and p38MAPK under simulated physiological conditions.Methods: The interaction between parecoxib and p38MAPK was studied under simulated physiological conditions using spectroscopy-based methods. The effect of parecoxib on the microenvironment and conformation of p38MAPK chromophore was studied by synchronous fluorescence spectroscopy, three-dimensional fluorescence, time-resolved fluorescence spectroscopy, circular dichroism spectroscopy, and ultraviolet-visible absorption spectroscopy.Results: Synchronous fluorescence spectroscopy showed that addition of parecoxib changed the structure of p38MAPK and destroyed the original stable structure. Three-dimensional fluorescence spectroscopy showed that the hydrophilicity of the microenvironment in which the fluorescent chromophore is located was enhanced, and the polarity increased such that the serum protein macromolecules tend to be unfolded, and the alpha-helix content reduced. Time-resolved fluorescence spectroscopy showed that the presence of parecoxib hardly affected the fluorescence quenching of p38MAPK, and the combination of parecoxib and p38MAPK forms a stable complex (static quenching). Circular dichroism spectroscopy revealed the combined parecoxib change, the secondary structure of p38MAPK and reduced the alpha-helix content. Ultraviolet-visible absorption spectroscopy revealed changes in the microenvironment of the three amino acid residues as well as the tertiary structure of the protein.Conclusion: The results shows that parecoxib has a significant effect on the structure of p38MAPK. In addition to explicitly inhibiting COX-2 and blocking arachidonic acid synthesis of prostaglandins, it inhibits the pathway involved in p38MAPK.

Conformational variation of site specific glycated albumin: A Molecular dynamics approach

Human serum albumin (HSA) is a major cargo protein, which undergoes glycation in hyperglycaemic conditions and results in impaired function. In physiological conditions, HSA plays a crucial role in pharmacological activities such as drug transport or delivery through its binding capacity and also by its enzymatic activity, which enables the translation of pro-drugs into active drugs. In this study, the impact of the methylglyoxal-mediated glycation on dynamic behaviour of inter-domain motion, Cys34 reactivity, binding site residual interaction and secondary structure transition were investigated through molecular dynamics simulation. The alteration in inter-domain motion reflects the effect of glycation-mediated changes on the structural conformation of albumin. The binding site residue interactions and volume analysis revealed the impact of glycation on the geometry of the binding site. We also found the correlation of Cys34 reactivity with increase of turns in the region between Ia-h4 and Ia-h5. The rise in turn formation in that region keeps Tyr84 farther away from Cys34 which could lead to higher Cys34 reactivity. In parallel, significant alterations in alpha helical content of helices in the binding sites were observed. These structural and conformational changes in glycated albumin could be the causative agents for functional impairment which leads to diabetic complications.

Reverse-QTY code design of active human serum albumin self-assembled amphiphilic nanoparticles for effective anti-tumor drug doxorubicin release in mice

Human serum albumin (HSA) is a highly water-soluble protein with 67% alpha-helix content and three distinct domains (I, II, and III). HSA offers a great promise in drug delivery with enhanced permeability and retention effect. But it is hindered by protein denaturation during drug entrapment or conjugation that result in distinct cellular transport pathways and reduction of biological activities. Here we report using a protein design approach named reverse-QTY (rQTY) code to convert specific hydrophilic alpha-helices to hydrophobic to alpha-helices. The designed HSA undergo self-assembly of well-ordered nanoparticles with highly biological actives. The hydrophilic amino acids, asparagine (N), glutamine (Q), threonine (T), and tyrosine (Y) in the helical B-subdomains of HSA were systematically replaced by hydrophobic leucine (L), valine (V), and phenylalanine (F). HSArQTY nanoparticles exhibited efficient cellular internalization through the cell membrane albumin binding protein GP60, or SPARC (secreted protein, acidic and rich in cysteine)-mediated pathways. The designed HSArQTY variants displayed superior biological activities including: i) encapsulation of drug doxorubicin, ii) receptor-mediated cellular transport, iii) tumor cell targeting, and iv) antitumor efficiency compare to denatured HSA nanoparticles. HSArQTY nanoparticles provided superior tumor targeting and antitumor therapeutic effects compared to the albumin nanoparticles fabricated by antisolvent precipitation method. We believe that the rQTY code is a robust platform for specific hydrophobic modification of functional hydrophilic proteins with clear-defined binding interfaces.

Understanding the Inhibition Mechanism of Lignin Adsorption to Cellulase in Terms of Changes in Composition and Conformation of Free Enzymes

The adsorption of lignin to cellulase is the major obstacle in the sugar-platform conversion of lignocellulosic bioresources. In this study, the adsorption behavior of untreated and pretreated lignin samples from corn stover to cellulase was investigated, in particular the effects of lignin adsorption on the composition and spatial conformation of free enzymes were explored. The results showed that pretreatments decreased the hydrophobic groups contents of lignin, i.e., aromatic ring, ether and carbonyl, as well as the content of ionizable group, i.e., carboxyl, which reduced its hydrophobicity and negative charge density, thus weakening the adsorption ability of lignin to cellulase. The lignin samples mainly adsorbed the CBHII component of cellulase to inhibit the synergistic effect of free enzymes. Lignin adsorption altered the spatial position of tryptophan residues in free enzymes, exposing them to the protein surface. In addition, the secondary structure of free enzymes was altered, with a decrease in the alpha-helix content and an increase in the random coil content, thus loosening the spatial conformation of free enzymes. The change degree in the spatial structure of free enzymes correlated with the adsorption capacity of the lignin, i.e., lignin with low adsorption capacity caused the least damage to free enzyme, with NaOH pretreated lignin being the best. It appears that appropriate pretreatment and chemical modification of enzymes to resist lignin adsorption is a promising long-term pathway to overcome the lignin inhibition during sugar-platform conversion of lignocellulosic bioresources.

Investigation of the interaction between the functionalized mesoporous silica nanocarriers and bovine serum albumin via multi-spectroscopy

It is well known that the physicochemical properties of nanocarriers, which are closely related to the surface modification of nanoparticles, have crucial impacts on their biological effects. Herein, the interaction between functionalized degradable dendritic mesoporous silica nanoparticles (DDMSNs) and bovine serum albumin (BSA) was investigated for probing into the nanocarriers’ potential toxicity using multi-spectroscopy such as ultraviolet/visible (UV/Vis), synchronous fluorescence, Raman and circular dichroism (CD) spectroscopy. BSA, owing to its structural homology and high sequence similarity with HSA, was employed as the model protein to study the interactions with DDMSNs, amino-modified DDMSNs (DDMSNs-NH2) and hyaluronic acid (HA) coated nanoparticles (DDMSNs-NH2-HA). It was found that the static quenching behavior of DDMSNs-NH2-HA to BSA was accompanied by an endothermic and hydrophobic force-driven thermodynamic process, which was confirmed by fluorescence quenching spectroscopic studies and thermodynamic analysis. Furthermore, the conformational variations of BSA upon interaction with nanocarriers were observed by combination of UV/Vis, synchronous fluorescence, Raman and CD spectroscopy. The microstructure of amino residues in BSA changed due to the existence of nanoparticles, for example, the amino residues and hydrophobic groups exposed to microenvironment and the alpha helix (α-helix) content of BSA decreased. Specially, through thermodynamic analysis, the diverse binding modes and driving forces between nanoparticles and BSA were discovered because of different surface modifications on DDMSNs, DDMSNs-NH2 and DDMSNs-NH2-HA. We believe that this work can promote the interpretation of mutual impact between nanoparticles and biomolecules, which will be in favor of predicting the biological toxicity of nano-DDS and engineering functionalized nanocarriers.

Molecular interactions between soybean glycinin (11S) and genistein using spectroscopic and in silico analyses

Interactions and binding mechanisms between soybean glycinin (11S) and genistein were investigated at near neutral pH (7.4) and ambient temperature using various spectroscopic techniques and molecular docking. Ultra-violet visible spectroscopy revealed non-covalent bonds were responsible for the stabilisation of the complex. Following complexation, minor reductions in the alpha helix and beta sheet content of the protein were confirmed by circular dichroism (CD) and infrared spectroscopy (FTIR). Fluorescence quenching and molecular docking predicted a 11S trimer and genistein complex with a 1:1 stoichiometric ratio, with the binding location predominantly on the interior portion of the protein, stabilised in majority by hydrogen bonds. Although binding was determined to be of moderate strength, secondary structure characterisation suggested that the structural components of the protein remained largely unchanged. This outcome indicates that the ligand can be accommodated in the interior of the 11S trimer, forming a stable complex within the present conditions of neutral pH and ambient temperature.

Study of the interfacial viscoelasticity of human serum albumin microcapsules using a viscoelasto-electrohydrodynamic technique.

Crosslinked proteins are widely used as the encapsulating membranes in microcapsules for many biomedical and food industries. The interfacial rheological properties of these capsules are due to the complex microstructure of cross-linked globular proteins owing to structural changes at quaternary, tertiary and secondary levels. These changes in structure can be induced by high protein concentration, hydrophobic-hydrophillic interfaces, and pH. In this work, the interfacial viscoelastic rheological properties of human serum albumin (HSA) microcapsules are estimated using a novel electrodeformation technique exhibiting creep and oscillatory responses. Insights into the microstructure-rheology relationship are obtained using FTIR and SEM studies. The results show a complex dependence of the interfacial properties on the size, concentration and pH of the capsules. An interplay of inter-molecular interactions, adsorption and multilayer formation, accessibility to reactive functional groups, and dependence on the relative content of alpha helix, beta sheet and beta turn is observed. The interfacial rheological properties are estimated using the Burger model and creep is found to sensitively affect the rheological properties due to irreversible changes in microstructure. Furthermore, the electrodeformation technique allows analysis of interfacial rheology at high frequencies, 10 Hz to 1 kHz, which is otherwise not easily possible with conventional rheometers.

Formation of Fibrils by the Periplasmic Molecular Chaperone HdeB from Escherichia coli.

The molecular chaperones HdeA and HdeB of the Escherichia coli (E. coli) periplasm protect client proteins from acid denaturation through a unique mechanism that utilizes their acid denatured states to bind clients. We previously demonstrated that the active, acid-denatured form of HdeA is also prone to forming inactive, amyloid fibril-like aggregates in a pH-dependent, reversible manner. In this study, we report that HdeB also displays a similar tendency to form fibrils at low pH. HdeB fibrils were observed at pH < 3 in the presence of NaCl. Similar to HdeA, HdeB fibrils could be resolubilized by a simple shift to neutral pH. In the case of HdeB, however, we found that after extended incubation at low pH, HdeB fibrils were converted into a form that could not resolubilize at pH 7. Fresh fibrils seeded from these “transformed” fibrils were also incapable of resolubilizing at pH 7, suggesting that the transition from reversible to irreversible fibrils involved a specific conformational change that was transmissible through fibril seeds. Analyses of fibril secondary structure indicated that HdeB fibrils retained significant alpha helical content regardless of the conditions under which fibrils were formed. Fibrils that were formed from HdeB that had been treated to remove its intrinsic disulfide bond also were incapable of resolubilizing at pH 7, suggesting that certain residual structures that are retained in acid-denatured HdeB are important for this protein to recover its soluble state from the fibril form.

Tailoring dexamethasone loaded albumin nanoparticles: A full factorial design with enhanced anti-inflammatory activity In vivo

Protein-based nanoparticles (NPs) are biodegradable, biocompatible, and easily amenable to chemical modifications to allow for the incorporation of bioactive compounds. In the current study, we adopted a full factorial design to optimize dexamethasone disodium phosphate (Dex) encapsulation within NPs formed with bovine serum albumin (BSA) and stabilized using polyethylenimine (PEI). The optimized NP size (<200 nm dia.), zeta potential (−23.3 mV), and entrapment efficiency (67.4%) were occurred at 0.652 mg, 0.04, 5%, and 8.3 for solution concentration of Dex, PEI/BSA molar ratio, BSA solution concentration, and solution pH, respectively. Incorporation of Dex resulted in a decrease in alpha helix content of BSA indicating a change in its secondary structure. Dex loaded NPs yielded a bimodal release of Dex over an extended period of time via a quasi-Fickian diffusion mechanism. The in vivo anti-inflammatory activity of BSA/PEI Dex NPs surpassed that of free Dex in carrageenan-induced hind paw edema in rats as evidenced by enhanced suppression of oxidative stress, abrogation of NF-κB-p65 expression, as well as reduced myositis and inflammatory cell infiltration. The extended-release profile of BSA/PEI Dex NPs is crucial for achieving a significantly significant anti-inflammatory activity.