Β-sheet Content Research Articles

Co-delivery of vitamin C and β-carotene in W/O/W emulsions stabilized by modified aggregated insoluble soybean protein hydrolysate-xanthan gum complexes

Environmentally friendly emulsifiers safe for human consumption are urgently needed to stabilize emulsions for applications in the food industry. In this study, we prepared complexes combining modified aggregated insoluble soybean protein hydrolysate (AISPH) mixed with xanthan gum (XG) (0.05–0.3 %, w/v), and further to construct water-in-oil-in-water (W/O/W) emulsions to deliver vitamin C and β-carotene. We observed a decrease in the AISPH α-helix and β-sheet content, surface hydrophobicity, and fluorescence intensity all decreased after binding. In contrast, the particle size and absolute ξ-potential significantly increased, indicating that molecular non-covalent interactions occurred in the solution. The emulsification property of AISPH was also improved by adding XG, and the AISPH-XG-stabilized emulsion showed improved stability, encapsulation efficiency, and rheological properties. Among them, AISPH-XG-0.25-stabilized emulsion exhibited a smaller particle size (8.41 ± 0.49 μm) and the highest encapsulation efficiency for vitamin C (90.03 ± 0.23 %) and β-carotene (70.56 ± 0.06 %). Additionally, simulated gastric digestion indicated that vitamin C and β-carotene bioavailability increased by 3.6 and 5.8 times, respectively. Finally, the emulsion exhibited good pH, ionic, and thermal stability. In general, AISPH-XG-stabilized W/O/W emulsions showed good stability and carrying capacity, providing a theoretical basis for improving their application.

Radio frequency-assisted hot air drying of pacific white shrimp (Litopenaeus vannamei): drying kinetics, product quality and composition evaluation

ABSTRACT The influence of radio frequency-assisted hot air drying (RFHAD) on the drying kinetics, product quality and composition of Pacific white shrimp was evaluated and compared with radio frequency drying (RFD) and hot air drying (HAD). The results demonstrated that RFHAD showed faster drying rate and also ensured better product quality in terms of texture property, astaxanthin content, color quality, rehydration rate and microstructure of shrimp. Furthermore, RF wave could significantly reduce the formation of bitter amino acids (p < .05) and showed no significant difference in the content of umami amino acid. However, secondary structure changes were observed in RFHAD shrimp with a decrease in β-sheet and α-helix contents and increase in β-reverse fold, and RFHAD noticeably decreased the onset temperature, peak temperature and thermal enthalpy (ΔH) values of shrimp, which showed lower thermal stability. Therefore, the RFHAD method is a fast and efficient drying technology for shrimp with high-quality characteristic.

Regulation of ovalbumin allergenicity and structure-activity relationship analysis based on pulsed electric field technology

The study focused on the regulation of ovalbumin (OVA) allergenicity using pulsed electric field (PEF) technology and examined the structure-activity link. Following PEF treatment, the ability of OVA to bind to IgE and IgG1 at 6 kHz was inhibited by 30.41 %. According to the microstructure, PEF caused cracks on the OVA surface. Spectral analysis revealed a blue shift in the amide I band and a decrease in α-helix and β-sheet content indicating that the structure of OVA was unfolded. The disulfide bond conformation was transformed and the structure tended to be disordered. The increased fluorescence intensity indicated that tryptophan and tyrosine were exposed which led an increase in hydrophobicity. In addition, the results of molecular dynamics (MD) simulations confirmed that the stability of OVA was reduced after PEF, which was related to the reduction of hydrogen bonding and the sharp fluctuation of aspartic acid. Therefore, PEF treatment induced the exposure of hydrophobic amino acids and the transformation of disulfide bond configuration which in turn masked or destroyed allergenic epitopes, and ultimately inhibited OVA allergenicity. This study provided insightful information for the production of hypoallergenic eggs and promoted the use of PEF techniques in the food field.

Oxidative Modification, Structural Conformation, and Gel Properties of Pork Paste Protein Mediated by Oxygen Concentration in Modified Atmosphere Packaging

The objective of this study was to investigate the effect of pork oxidation through modified atmosphere packaging (MAP) on gel characteristics of myofibrillar proteins (MP) during the heat-induced gelation process. The pork longissimus thoracis (LT) was treated by MAP at varying oxygen concentrations (0, 20, 40, 60, and 80% O2) with a 5-day storage at 4 °C for the detection of MP oxidation and gel properties. The findings showed the rise of O2 concentration resulted in a significant increase of carbonyl content, disulfide bond, and particle size, and a decrease of sulfhydryl content and MP solubility (p &lt; 0.05). The gel textural properties and water retention ability were significantly improved in MAP treatments of 0–60% O2 (p &lt; 0.05), but deteriorated at 80% O2 level. As the concentration of O2 increased, there was a marked decrease in the α-helix content within the gel, accompanied by a simultaneous increase in β-sheet content (p &lt; 0.05). Additionally, a judicious oxidation treatment (60% O2 in MAP) proved beneficial for crafting dense and uniform gel networks. Our data suggest that the oxidation treatment of pork mediated by O2 concentration in MAP is capable of reinforcing protein hydrophobic interaction and disulfide bond formation, thus contributing to the construction of superior gel structures and properties.

Fat analogue emulsions stabilized by peanut protein microgel particles: microscale and nanoscale structure and stabilization process analysis.

Biopolymer-based microgels are being regarded increasingly as promising building blocks in food applications. This study aimed to clarify the evolution process of the network for fat analogue emulsions stabilized by peanut protein isolate (PPI) microgel particles. It also investigated the interfacial structure and characteristics of emulsions (50% oil phase, w/w) stabilized by microgels under different pH conditions. There was an increasing interfacial adsorption capacity for PPI microgels over time (from 85.26% to the maximum of 89.78% at 24 h of storage) due to the aggregation of microgels around droplets and the development of cross-linking microgel chains between adjacent interfaces. The increased β-sheet content (from 35.51% to 41.12%) of adsorbed microgels indicated unfolding and the enhanced aggregation of microgels, which led to stronger droplet interaction. The network evolution observed with different microscopes clarified the transition to a self-supporting emulsion. The uneven adsorption of large microgel aggregates at the oil-water interface promoted larger and deformed droplets, so more fat-like medium internal phase emulsion stabilized by PPI microgel could be obtained by adjusting the microgel pH to 4.5. The interfacial membranes observed by scanning electron microscopy were thicker and coarser at pH 3.0 and 4.5 than those at pH 7.0 and 9.0. The adsorption of PPI microgel aggregates enhanced the structural strength and improved emulsion stability. This work could form a basis for further studies relating physical properties to the design of plant protein-based fat analogues. © 2024 Society of Chemical Industry.

Improved lipase performance by covalent immobilization of Candida antarctica lipase B on amino acid modified microcrystalline cellulose as green renewable support

Development of immobilized lipase with excellent catalytic performance and low cost is the major challenge for large-scale industrial applications. In this study, green renewable microcrystalline cellulose (MCC) that was hydrophobically modified with D-alanine (Ala) or L-lysine (Lys) was used for immobilizing Candida antarctica lipase B (CALB). The improved catalytic properties were investigated by experimental and computational methods. CALB immobilized on MCC-Ala with higher hydrophobicity showed better catalytic activity than CALB@MCC-Lys because the increased flexibility of the lid region of CALB@MCC-Ala favored the formation of open conformation. Additionally, the low root mean square deviation and the high β-sheet and α-helix contents of CALB@MCC-Ala indicated that the structure became more stable, leading to a significantly enhanced stability (54.80% and 90.90% relative activity at 70 °C and pH 9.0, respectively) and good reusability (48.92% activity after 5 cycles). This study provides a promising avenue to develop immobilized lipase with high catalytic properties for industry applications.

Inhibition of staphylococcal nuclease by benzimidazole-based Ligand: Implications in DNA-Mediated entrapment and uptake of MRSA by Macrophage-like cells

The staphylococcal nuclease also referred as micrococcal nuclease (MNase) is a key drug target as the enzyme degrades the neutrophil extracellular trap (NET) and empowers the pathogen to subvert the host innate immune system. To this end, the current study presents a critical evaluation of MNase inhibition rendered by benzimidazole-based ligands (C1 and C2) and probes its therapeutic implications. A nuclease assay indicated that MNase inhibition rendered by C1 and C2 was ∼ 55 % and ∼ 72 %, respectively, at the highest tested concentration of 10 µM. Studies on enzyme kinetics revealed that C2 rendered non-competitive inhibition and significantly reduced MNase turnover number (Kcat) and catalytic efficiency (Kcat/Km) with an IC50 value of ∼ 1122 nM. In CD spectroscopy, a notable perturbation in the β-sheet content of MNase was observed in presence of C2. Fluorescence-microscope analysis indicated that MNase inhibition by C2 could restore entrapment of methicillin-resistant Staphylococcus aureus (MRSA) in calf-thymus DNA (CT-DNA). Flow cytometry and confocal microscope analysis revealed that uptake of DNA-entrapped MRSA by activated THP-1 cells was reinstated by MNase inhibition rendered by C2. Inhibition of nuclease by the non-toxic ligand C2 holds therapeutic prospect as it has the potential to bolster the DNA-mediated entrapment machinery and mitigate MRSA infections.

Replicating shear-mediated self-assembly of spider silk through microfluidics

The development of artificial spider silk with properties similar to native silk has been a challenging task in materials science. In this study, we use a microfluidic device to create continuous fibers based on recombinant MaSp2 spidroin. The strategy incorporates ion-induced liquid-liquid phase separation, pH-driven fibrillation, and shear-dependent induction of β-sheet formation. We find that a threshold shear stress of approximately 72 Pa is required for fiber formation, and that β-sheet formation is dependent on the presence of polyalanine blocks in the repetitive sequence. The MaSp2 fiber formed has a β-sheet content (29.2%) comparable to that of native dragline with a shear stress requirement of 111 Pa. Interestingly, the polyalanine blocks have limited influence on the occurrence of liquid-liquid phase separation and hierarchical structure. These results offer insights into the shear-induced crystallization and sequence-structure relationship of spider silk and have significant implications for the rational design of artificially spun fibers.

The extraction of buckwheat protein and its interaction with kappa-carrageenan: Textural, rheological, microstructural, and chemical properties

Current plant-based foods use plant proteins as a key structuring and texturing ingredient. The use of water for extraction can replace conventional protein extraction methods. Water extraction of protein is environmentally friendly and could prevent the loss of protein functionality due to extreme pH changes. This study demonstrates an aqueous extraction method, coupled with ultrasound as pre-treatment, to obtain buckwheat protein (BWPE) and assess its gelling property and composited gel with kappa-carrageenan (k-carr). Textural and rheological analyses showed that the hardness and storage modulus of the composited gel containing 1 % w/w BWPE and 1 % w/w k-carr was 4.2-fold and 100-fold, respectively, higher than k-carr gel at 1 % w/w. Light microscopy showed a mixed bi-continuous gel system, with k-carr reinforcing the protein gel network. Besides volume exclusion effects, chemical bond and FTIR analyses revealed that adding k-carr to BWPE altered the protein's secondary structure and mediated protein denaturation during heating. This results in greater β-sheet content, which creates a more organised gel structure. These results demonstrated that ultrasound-assisted water-extracted BWPE, together with varying concentrations of k-carr, can be used to develop composited gels of tailorable textural and rheological properties to suit different food applications.

Effect of pH on the Structure, Functional Properties and Rheological Properties of Collagen from Greenfin Horse-Faced Filefish (Thamnaconus septentrionalis) Skin.

Collagen is an important biopolymer widely used in food, cosmetics and biomedical applications. Understanding the effect of pH on the structure and properties of collagen is beneficial for its further processing and exploitation. In this study, greenfin horse-faced filefish skin collagen (GHSC) was prepared and identified as a type I collagen. We systematically investigated the effect of pH on the structural, functional and rheological properties of GHSC. Scanning electron microscopy showed that the collagen morphology changed from an ordered stacked sheet structure to a rough silk-like structure as pH increased. Gaussian-fitted Fourier infrared spectroscopy results of the collagen revealed that it unfolded with increasing pH. Moreover, the ordered structure was reduced, and random coils became the dominant conformation. Its β-sheet and random coil contents increased from 18.43 ± 0.08 and 33.62 ± 0.17 to 19.72 ± 0.02 and 39.53 ± 1.03%, respectively, with increasing pH. α-helices and β-turns decreased from 35.00 ± 0.26 and 12.95 ± 0.01 to 29.39 ± 0.92 and 11.36 ± 0.10%, respectively. The increase in β-sheets and random coils allowed the pI-treated collagen to exhibit maximum water contact angle. The emulsification and foaming properties decreased and then increased with increasing pH in a V-shape. The increased net surface charge and β-sheets in collagen benefited its emulsification and foaming properties. The rheological results showed that the protoprotein exhibited shear-thinning properties in all pH ranges. The collagen solutions showed liquid-like behaviour in low-pH (2, 4) solutions and solid-like behaviour in high-pH (6, 7.83 and 10) solutions. Moreover, the frequency-dependent properties of the storage modulus (G') and loss modulus (G″) of the collagen solutions weakened with increasing pH. Collagen has considerable frequency-dependent properties of G' and G″ at low pH (2, 4). Thus, the importance of collagen raw material preparation for subsequent processing was emphasised, which may provide new insights into applying collagen-based materials in food, biomaterials and tissue engineering.

Relationship between the baking quality of wheat (Triticum aestivum L.) and the protein composition and structure after shading

Although wheat (Triticum aestivum L.) grain protein content is increased by shade stress, the relationship between the baking quality of wheat flour and protein composition and structure remains unclear. Here, we investigated the effects of shade stress on wheat flour protein composition and structure. The contents of the flour protein, α/β-gliadins and disulfide and hydrogen bonds were significantly increased by shade stress. Glutenins, UPP%, and β-sheet contents also increased, whereas that of α-helices decreased. Spearman correlations revealed that the flour protein content, Glu:Gli ratio, and disulfide, hydrogen, and ionic bonds can predict the specific volume and number of crumb cells in bread, whereas α/β-gliadins content can predict the crumb cell wall thickness and diameter of bread. Under shade stress, variations in protein composition and structure help increase the specific volume and crumb cells number and decrease crumb cell wall thickness and diameter of bread, ultimately leading to improved baking quality.

High moisture extrusion of soybean protein isolate: Effect of β-glucan on physicochemical properties of extrudates

This study is focused on exploring the effect of twin-screw high-moisture extrusion technology on the physico-chemical properties of β-glucan-soybean protein isolate (SPI-BG) extrudates. Different proportions (0 %, 1 %, 2 %, 3 %, 4 %, 5 %) of oat β-glucan (BG) were added with soybean protein isolate (SPI) to prepare SPI-BG extrudates. Results showed that the addition of a high quantity of β-glucan (BG) decreased the elastic properties of soy protein isolate (SPI) extrudates and then increased. The strengthening of S1-S2-S3 interactions (hydrogen bonds, hydrophobic interactions, and disulfide bonds) was primarily responsible for this trend. Fourier transform infrared (FTIR) spectral analysis was conducted which revealed that BG did not significant affect random coil content of SPI. However, it was observed that α-helix content was increased significantly and the β-sheet content was decreased. An observation was noted in the value of enthalpy (ΔH) change that increased from 115.80 J/g to 159.68 J/g.

Study on the anti-retrogradation of wheat amylopectin by addition of alkali-soluble glutenin

The retrogradation of wheat amylopectin during cold storage is the main reason for the increasing hardness of flour products such as steamed bread, bread and pastries, etc. Addition of gluten protein components is a green, safe, cheap and efficient method to inhibit the retrogradation of wheat amylopectin. In this paper, as being stored at 4 °C for 7 d, retrogradation rate of wheat amylopectin decreased from 55.02 % to 14.37 % after it was mixed with 20 % alkali-soluble glutenin (ASG) at 30 °C for 90 min, a 73.8 % reduction. The infrared results showed that the intensity of bending vibration of water molecules and intra-molecular β-sheet content of ASG decreased during the interaction between amylopectin and ASG. Meanwhile, intermolecular β-sheet and random coil contents of ASG increased. The results of 13C Solid-state NMR indicated that Qβ, Pγ and Lγ of ASG involved in interaction of wheat amylopectin, ASG and molecule of water. Under the optimal conditions, the interaction of wheat amylopectin and ASG began to form spheres containing disulfide bonds, resulting in the attenuation or disappearance of the diffraction peak at 2θ 19.7°, which may be marked as the criterion for the best mixing time of wheat amylopectin and ASG. The retrogradation kinetic index (n) of wheat amylopectin decreased significantly with the addition of ASG and formation of disulfide bond was the key factor. ASG could be potentially used as an anti-retrogradation agent for amylopectin.

Characterization of ultrasound-assisted covalent binding interaction between β-lactoglobulin and dicaffeoylquinic acid: Great potential for the curcumin delivery

The low bioavailability and poor gastrointestinal instability of curcumin hampers its application in pharmaceutical and food industries. Thus, it is essential to explore efficient carrier (e.g. a combination of polyphenols and proteins) for food systems. In this study, covalent β-lactoglobulin (LG)-dicaffeoylquinic acids (DCQAs) complexes were prepared by combining ultrasound and free radical induction methods. Covalent interactions between LG and DCQAs were confirmed by analyzing reactive groups. Variations in secondary or tertiary structure and potential binding sites of covalent complexes were explored using Fourier transform infrared spectroscopy and circular dichroism. Results showed that the β-sheet content decreased and the unordered content increased significantly (P < 0.05). The embedding rate of curcumin in prepared LG-DCQAs complexes using ultrasound could reach 49 % − 62 %, proving that complexes could embed curcumin effectively. This study highlights the benefit of ultrasound application in fabrication of protein–polyphenol complexes for delivering curcumin.

Effect of high-molecular-weight glutenin subunits silencing on dough aggregation characteristics

The qualities of wheat dough are influenced by the high-molecular-weight glutenin subunits (HMW-GS), a critical component of wheat gluten protein. However, it is still unknown how HMW-GS silencing affects the aggregation characteristics of dough. Two groups of near-isogenic wheat were used to study the effects of HMW-GS silencing on dough aggregation characteristics, dough texture characteristics, and dough microstructure. It was observed that the content of gliadin in LH-11 strain significantly increased compared to the wild-type (WT). Additionally, the amount of glutenin macropolymer and the glutenin/gliadin both decreased. The aggregation characteristics and rheological characteristics of the dough in LH-11 strain were significantly reduced, and the content of β-sheet in the dough was significantly reduced. The HMW-GS silencing resulted in a reduction in the aggregation of the gluten network in the dough, which related to the alteration of the secondary and microstructure of the gluten.

Tuning the viscoelastic properties of peptide coacervates by single amino acid mutations and salt kosmotropicity

Coacervation, or liquid-liquid phase separation (LLPS) of biomacromolecules, is increasingly recognized to play an important role both intracellularly and in the extracellular space. Central questions that remain to be addressed are the links between the material properties of coacervates (condensates) and both the primary and the secondary structures of their constitutive building blocks. Short LLPS-prone peptides, such as GY23 variants explored in this study, are ideal model systems to investigate these links because simple sequence modifications and the chemical environment strongly affect the viscoelastic properties of coacervates. Herein, a systematic investigation of the structure/property relationships of peptide coacervates was conducted using GY23 variants, combining biophysical characterization (plate rheology and surface force apparatus, SFA) with secondary structure investigations by infrared (IR) and circular dichroism (CD) spectroscopy. Mutating specific residues into either more hydrophobic or more hydrophilic residues strongly regulates the viscoelastic properties of GY23 coacervates. Furthermore, the ionic strength and kosmotropic characteristics (Hofmeister series) of the buffer in which LLPS is induced also significantly impact the properties of formed coacervates. Structural investigations by CD and IR indicate a direct correlation between variations in properties induced by endogenous (peptide sequence) or exogenous (ionic strength, kosmotropic characteristics, aging) factors and the β-sheet content within coacervates. These findings provide valuable insights to rationally design short peptide coacervates with programmable materials properties that are increasingly used in biomedical applications.

Effect of dynamic high-pressure microfluidization treatment on soybean protein isolate–rutin non-covalent complexes

In this investigation, soybean protein isolate–rutin (SPI–RT) complexes were treated using dynamic high-pressure microfluidization (DHPM). The effects of this process on the physicochemical and thermodynamic properties of SPI were investigated at different pressures. Fourier-transform infrared spectroscopy and fluorescence spectroscopy provided evidence that the SPI structure had been altered. The binding of SPI to RT resulted in a decrease in the percentage of α-helices and random curls as well as an increase in the percentage of β-sheets. In particular, the α-helix content decreased from 29.84 % to 26.46 %, the random curl content decreased from 17.45 % to 15.57 %, and the β-sheet content increased from 25.37 % to 26.53 %. Moreover, fluorescence intensity decreased, and the emission peak of the complex was red-shifted by 6 nm, exposing the internal groups. Based on fluorescence quenching analysis, optimal SPI–RT complexation was achieved after 120-MPa DHPM treatment, and molecular docking analysis verified the interaction between SPI and RT. The minimum particle size, maximum absolute potential, and total phenolic content of the complexes were 78.06 nm, 21.4 mV and 74.35 nmol/mg protein, respectively. Furthermore, laser confocal microscopy revealed that the complex particles had the best microstructure. Non-covalent interactions between the two were confirmed using sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Moreover, the hydrophobicity of the complex particle's surface increased to 16,045 after 120-MPa DHPM treatment. The results of this study suggest that DHPM strongly promotes the improvement of the physicochemical properties of SPI, and provide a theoretical groundwork for further research.

Study of the molecular structure of proteins in fermented Maize-Soybean meal-based rations based on FTIR spectroscopy

This research investigates the dynamic alterations that occur in protein molecular structure during the fermentation process of feed. Fourier transform infrared spectroscopy (FTIR), coupled with deconvolution, second derivative and curve-fitting methodologies, was employed to comparatively analyse the protein molecular structures in fermented feed. At the 48-h fermentation mark, the α-helix and β-sheet contents reached their peaks, while the random coil and β-turn contents were at their lowest. Simultaneously, the β-sheet/α-helix ratio was minimized. FTIR spectroscopy emerged as a comprehensive tool, revealing the nuanced changes in molecular structure throughout the fermentation process of corn-soybean meal feed. When integrated with spectral quantitative analysis, it provides a novel perspective for evaluating the nutritional value of fermented feed.

Insights into the baicalein-induced destabilization of LS-shaped Aβ42 protofibrils using computer simulations.

Amyloid-β (Aβ) peptides aggregate spontaneously into various aggregating species comprising oligomers, protofibrils, and mature fibrils in Alzheimer's disease (AD). Disrupting β-sheet rich neurotoxic smaller soluble Aβ42 oligomers formed at early stages is considered a potent strategy to interfere with AD pathology. Previous experiments have demonstrated the inhibition of the early stages of Aβ aggregation by baicalein; however, the molecular mechanism behind inhibition remains largely unknown. Thus, in this work, molecular dynamics (MD) simulations have been employed to illuminate the molecular mechanism of baicalein-induced destabilization of preformed Aβ42 protofibrils. Baicalein binds to chain A of the Aβ42 protofibril through hydrogen bonds, π-π interactions, and hydrophobic contacts with the central hydrophobic core (CHC) residues of the Aβ42 protofibril. The binding of baicalein to the CHC region of the Aβ42 protofibril resulted in the elongation of the kink angle and disruption of K28-A42 salt bridges, which resulted in the distortion of the protofibril structure. Importantly, the β-sheet content was notably reduced in Aβ42 protofibrils upon incorporation of baicalein with a concomitant increase in the coil content, which is consistent with ThT fluorescence and AFM images depicting disaggregation of pre-existing Aβ42 fibrils on the incorporation of baicalein. Remarkably, the interchain binding affinity in Aβ42 protofibrils was notably reduced in the presence of baicalein leading to distortion in the overall structure, which agrees with the structural stability analyses and conformational snapshots. This work sheds light on the molecular mechanism of baicalein in disrupting the Aβ42 protofibril structure, which will be beneficial to the design of therapeutic candidates against disrupting β-sheet rich neurotoxic Aβ42 oligomers in AD.

Biophysical characterization and insights into the oligomeric nature of CD2-associated protein.

Glomerular podocytes are specialized epithelial cells localized to the blood-urine interface of the kidney. Podocyte slit-diaphragm (SD), a size-and-charge-selective junction, is instrumental in blood ultrafiltration and the formation of protein-free urine. The SD consists of macromolecular complexes of several proteins, such as nephrin, podocin, and CD2-associated protein (CD2AP). CD2AP is an adapter protein and is considered to be crucial for the integrity of SD. Mutations in the SD proteins cause nephrotic syndrome (NS), characterized by proteinuria. SD proteins' structural features must be elucidated to understand the mechanism of proteinuria in NS. In this study, we expressed, purified, and biophysically characterized heterologously expressed human CD2AP. Codon-optimized human CD2AP was expressed in E. coli Rosetta cells. The recombinant protein was induced with 1 mM IPTG and purified by Ni-NTA affinity chromatography. Analytical size-exclusion chromatography, blue native-PAGE, circular dichroism, and fluorescence spectroscopy were performed to decipher the oligomeric nature, secondary structural content, and tertiary packing of CD2AP. Our analysis revealed that CD2AP adopts a predominantly disordered secondary structure despite exhibiting moderate tertiary packing, characterized by low helical and β-sheet content. CD2AP readily assembles into homo-oligomers, with octamers and tetramers constituting the primary population. Interestingly, the inherent flexibility of CD2AP's secondary structural elements appears resistant to thermal denaturation. Frameshift mutation (p.K579Efs*7) that leads to loss of the coiled-coil domain promotes aberrant oligomerization of CD2AP through SH3 domains. We successfully expressed full-length human CD2AP in a heterologous system, wherein the secondary structure of CD2AP is predominantly disordered. CD2AP can form higher-order oligomers, and the significance of these oligomers and the impact of mutations in the context of size-selective permeability of SD needs further investigation.