Changes In Secondary Structure Research Articles

Mapping the FF domain folding pathway via structures of transiently populated folding intermediates

Despite the tremendous accomplishments of AlpaFold2/3 in predicting biomolecular structure, the protein folding problem remains unsolved in the sense that accurate atomistic models of how protein molecules fold into their native conformations from an unfolded ensemble are still elusive. Here, using chemical exchange saturation transfer (CEST) NMR experiments and a comprehensive four-state kinetic model of the folding trajectory of a 71 residue four-helix bundle FF domain from human HYPA/FBP11 we present an atomic resolution structure of a transiently formed intermediate, I2, that along with the structure of a second intermediate, I1, provides a description of the FF domain folding trajectory. By recording CEST profiles as a function of urea concentration the extent of compaction along the folding pathway is evaluated. Our data establish that unlike the partially disordered I1 state, the I2 intermediate that is also formed before the rate-limiting folding barrier is well ordered and compact like the native conformer, while retaining nonnative interactions similar to those found in I1. The slow-interconversion from I2 to F, involving changes in secondary structure and the breaking of nonnative interactions, proceeds via a compact transition-state. Interestingly, the native state of the FF1 domain from human p190-A Rho GAP resembles the I2 conformation, suggesting that well-ordered folding intermediates can be repurposed by nature in structurally related proteins to assume functional roles. It is anticipated that the strategy for elucidation of sparsely populated and transiently formed structures of intermediates along kinetic pathways described here will be of use in other studies of protein dynamics.

Assessment of Punicalagin activity Against Influenza a Virus Proteins via In-silico Approaches

Background: To investigate the anti-influenza mechanism of punicalagin and its inhibitory effects on influenza virus proteins, we conducted molecular docking studies targeting 11 viral proteins. Additionally, molecular dynamics simulations were performed for the protein with the lowest free energy and the minimum concentration required for binding in a simulated environment. Methods: The molecular structure and data of punicalagin were obtained from the PubChem database and converted into a PDB file. FASTA sequences of viral proteins were retrieved from UniProt, and their PDB structures were predicted using the I-TASSER server. Molecular docking was performed using AutoDock 4.2 software, while molecular dynamics simulations were conducted with Gromacs 2022 software. Results: The PB1-F2 protein exhibited the best inhibitory performance, with a binding energy of -5.76 kcal/mol and the lowest inhibition constant (Ki). Docking of punicalagin to the PB1-F2 protein led to a significant decrease in the average total energy (TE), radius of gyration (Rg), and root mean square deviation (RMSD), as well as alterations in the secondary structure of the protein (P < 0.001). The most prominent secondary structural change was a reduction in coil structures and an increase in turn structures. Conclusions: Punicalagin displayed a strong binding affinity for the PB1-F2 protein compared to other influenza viral proteins. Considering the role of PB1-F2 in exacerbating inflammation caused by influenza, punicalagin may mitigate inflammation in patients by modulating the activity of this protein.

Probing the potential mechanism of permethrin exposure on Alzheimer's disease through enantiomer-specific network toxicology, multi-spectroscopic, and docking approaches

Latest observations indicated that exposure of organic environmental neurotoxins may increase the potential risk of Alzheimer's diseases (AD). As a suspected food-derived risk factor, permethrin, composed of cis-isomer and trans-isomer, is widely used as a broad-spectrum pyrethroid insecticide in agricultural crops for the arthropod pests controlling. Thus, evaluating the impact of permethrin exposure is of great importance to human health. In this study, we performed the toxicological network approach to decipher AD-related mechanisms of cis-permethrin and trans-permethrin. Based on the toxicological network construction and central network topological analysis, human serum albumin (HSA) was selected as the core targets in AD-related developing. From the analysis of the steady state and time-resolved fluorescence quenching of HSA in presence of permethrin mixture, it has been inferred that the nature of the quenching mainly originates from the dynamic modes. Experimentally, the thermodynamic parameters revealed hydrophobic interactions and van der Waals forces played a major role during quenching process. Tryptophan synchronous fluorescence spectra were blue shifted whereas the position of tyrosine synchronous spectra was red shifted during the complex formation. Three-dimensional fluorescence together with FT-IR experiment confirmed that permethrin caused the secondary structure changes in HSA. To better understand the binding patterns between HSA and cis/trans -permethrin, theoretical calculation and molecular docking were implemented. According to the electrostatic potential map, the electrophilic attack region corresponds for electron rich oxygen atoms, while the nucleophilic attack regions were mainly located at over the benzene rings and methyl on cyclopropane ring of permethrins. Docking results shown that cis-permethrin and trans-permethrin located in hydrophobic pocket nearby Domain IIA with the different binding affinity (−7.6 and −9.2 kcal/mol), which consistent with the competitive displacement experiment. All these findings generated in the present study facilitated the elucidation of the molecular mechanism details between permethrin mixture and HSA, which provided fresh insights into the links between environmental exposure and AD-related adverse health outcomes.

Quality variation analysis and rehydration kinetics modeling of yuba subjecting to three different drying process

This study investigated the effects of hot air drying (HAD), microwave vacuum drying (MVD), and vacuum frying drying (VFD) on the quality of yuba and utilized mathematical models (Peleg model, Weibull equation, first-order kinetic model, and exponential association model) to explore the rehydration kinetics of yuba. The results indicated that, compared to HAD, MVD significantly increased yuba's L* value and sensory score, while VFD notably reduced the cooking loss rate and enhanced moisture retention. Significant changes were observed in the texture of yuba before and after cooking, with distinct differences noted under microscopic observation, and a pronounced change in protein secondary structure. The Peleg model can effectively fit the rehydration processes of HAD and VFD yuba, while the first-order kinetic model can effectively fit the rehydration processes of MVD yuba. This study provides a valuable reference for yuba's drying method and rehydration Kinetics model.

Exposure to Silicone Oil Endotamponades Induces VEGF Antibody Aggregation and Loss of Functionality.

To investigate the interaction of a common monoclonal antibody (bevacizumab) with silicone oils and vitreous substitute hydrogels. Protein content (spectrophotometry) and bioactivity (ELISA) of bevacizumab were assessed after direct contact to silicone oil and vitreous substitute hydrogels up to 24 hours. To detect antibody aggregation, particle size was determined by dynamic light scattering. Changes in secondary protein structure were assessed using circular dichroism. Experiments were translated to another antibody, trastuzumab, because its bioactivity can be additionally quantified by its binding to the membrane of HER2-positive cells. The functionality of bevacizumab gradually decreased during exposure to silicone oils with only 30% of activity remaining after 24 hours. Although circular dichroism did not reveal structural changes, the particle size increased drastically, indicating antibody aggregation for both antibodies. Exposure to silicone oil strongly reduced binding of trastuzumab to HER2-positive cells. The addition of polysorbate, a common stabilizer for antibody formulations, dose-dependently prevented aggregation, with 62% aggregation observed at 0.04% polysorbate compared to only 10% aggregation at 0.5% polysorbate. No aggregation occurred after exposure to vitreous body replacement hydrogels. Contact to silicone oils induces a major loss of functionality of bevacizumab and trastuzumab. This limits clinical use during silicone oil tamponade. The beneficial effect of adding polysorbate suggests that loss of solubilizer is a likely cause of aggregation. Hydrogels, alternatives for vitreous body replacement, do not impair antibody functionality.

DRama: Differential Ramachandran Plot as a Tool to Analyze Subtle Changes in Protein Secondary Structure.

Determination of the changes in protein structure is crucial for a better understanding of their function and properties, which is highly important in identifying the causes of the disease, new drug development, and clinical applications. The Ramachandran plot, displaying the set of torsional angles, phi (Φ) and psi (Ψ), of the protein backbone, serves as a popular and convenient tool for secondary structure analysis and interpretation. However, identifying subtle changes in protein structure is often hindered in traditional Ramachandran plot, especially with the large amount of data generated by molecular dynamics (MD) simulations. In this paper, we proposed a useful and efficient tool, that is, differential Ramachandran plot (dRama), which enables to compare protein structures and extract the differences, providing a highly readable graphical representation. dRama is available at: https://github.com/MaksWolf44/dRama.

Structural Evaluation of Interleukin-19 Cytokine and Interleukin-19-Bound Receptor Complex Using Computational Immuno-Engineering Approach

Interleukin 19 (IL-19) is an anti-inflammatory cytokine that belongs to the IL-10 family, where IL-20 and IL-24 also exist. While IL-19 and IL-20 share some comparable structural folds, there are certain structural divergences in their N-terminal ends. To date, there are no reported IL-19 receptors; although, it has been suggested in the literature that IL-19 would bind to lL-20 receptor (IL-20R) and trigger the JAK-STAT signaling pathways. The present report examines the structure of the IL-19 cytokine and its receptor complex using a computational approach. Specifically, the postulated modes of interactions for IL-20R as an IL-19 receptor are examined on the basis of a set of computational findings. The author has used molecular docking and molecular dynamics simulation to generate a 3D model for the IL-19 complex with IL-20R. When a protein’s crystal structure is not available in the literature, predictive modeling is often employed to determine the protein’s 3D structure. The model assessment can be based on various factors, which include stability analysis using RMSD calculations, tracking changes in time-based secondary structures and the associated Gibbs energies, ΔG. Since one model complex (referred to as model A throughout this paper) can be used as a working hypothesis for future experiments, this structure has been explored here in detail to check its stability, subunit interfaces, and binding residues. The information gathered in this approach can potentially help to design specific experiments to test the validity of the model protein structure. Additionally, the results of this research should be relevant for understanding anti-inflammatory mechanisms and, eventually, could contribute to the efforts for therapeutic developments and targeted therapy.

His-tag based supramolecular biopolymerization

The term supramolecular polymer has been applied to polymeric materials in which the individual units, i.e., building blocks—are bound to each other via noncovalent interactions, including electrostatic or hydrogen bonding, as well as metal–ligand conjugation. The building blocks are generally low molecular weight amphiphiles. Methods for preparing biopolymers based on non-toxic, metal–ligand conjugation have been little studied; however, they offer significant potential for tuning the response of biologically relevant macromolecules. In this communication, we characterize the assembly and morphology of supramolecular biopolymers in which the building blocks are low- or medium-molecular weight globular proteins—ubiquitin and Cas9-interacting via metal–ligand conjugation. In each case, the protein gene was expressed in cell culture with the addition of hexa-His/linkers at both the N and C termini. Divalent cations investigated were Zn2+ and Ni2+. We observe in cryo-TEM imaging an absolute requirement for divalent cations for the formation of supramolecular biopolymers. In the presence of Ni2+, 1D assembled fibers are predominant, while with Zn2+, the more frequently detected structures are sheet-like. We use gel electrophoresis and CD spectroscopy to monitor possible secondary and tertiary structural changes in the protein building blocks during conjugation.

Changes in Secondary Structure Upon Pr to Pfr Transition in Cyanobacterial Phytochrome Cph1 Detected by DNP NMR.

Phytochromes perceive subtle changes in the light environment and convert them into biological signals by photoconversion between the red-light absorbing (Pr) and the far-red-absorbing (Pfr) states. In the primitive bacteriophytochromes this includes refolding of a tongue-like hairpin loop close to the chromophore, one strand of an antiparallel β-sheet being replaced by an α-helix. However, the strand sequence in the cyanobacterial phytochrome Cph1 is different from that of previously investigated bacteriophytochromes and has a higher β-sheet propensity. We confirm here the transition experimentally and estimate minimum helix length using dynamic nuclear polarisation (DNP) magic angle spinning NMR. Sample conditions were optimized for protein DNP NMR studies at high field, yielding Boltzmann enhancements ϵB of 19 at an NMR field of 18.801 T. Selective labelling of Trp, Ile, Arg, and Val residues with 13C and 15N enabled filtering for pairs of labelled amino acids by the 3D CANCOCA technique to identify signals of the motif 483Ile-Val-Arg485 (IVR) present in both sheet and helix. Those signals were assigned for the Pfr state of the protein. Based on the chemical shift pattern, we confirm for Cph1 the formation of a helix covering the IVR motif.

From Structure to Strength: Analyzing the Impact of Sulfuric Acid on Pig Bone Demineralization Through FTIR, LIBS, and AAS.

The present research aimed to investigate the demineralizing effects of sulfuric acid on pig bone. Alterations in collagen and phosphate contents and changes in the elemental composition of the bone during the 14-day-long immersion in sulfuric acid solutions of different concentrations were estimated using ATR-FTIR, LIBS, and AAS. FTIR spectra at amide I (1800-1600 cm-1) and phosphate ν1/ν3 (PO43-) (1300-900 cm-1) domains were scrutinized using the deconvolution method for monitoring changes in the protein secondary structure and mineral content. The results implicated sulfuric acid as a powerful demineralization agent and effective in targeting mineral components, such as hydroxyapatite, while leaving the collagen matrix relatively preserved with a complex secondary structure. Collagen maturity marker values gave valuable insights into the structural integrity of the bone. LIBS and AAS indicated changes in bone hardness; phosphorous-to-carbon ratio; and calcium, phosphorous, and magnesium content in the solutions left after the immersion period. The changes in the ratio of ionic-to-atomic calcium lines in the LIBS spectra indicated hardening of the bone, with increasing acid concentration and prolonged action, due to the deposition of calcium sulfate on the surface. The calcium concentration in the solutions decreased with increased acid concentration, while the change in phosphorus and magnesium concentrations was reversed.

Coherent Raman microscopy visualizes ongoing cellular senescence through amide I peak shifts originating from β sheets in disordered nucleolar proteins

Cellular senescence occurs through the accumulation of many kinds of stresses. Senescent cells in tissues also cause various age-related disorders. Therefore, detecting them without labeling is beneficial for medical research and developing diagnostic methods. However, existing biomarkers have limitations of requiring fixation and labeling, or their molecular backgrounds are uncertain. Coherent anti-Stokes Raman scattering (CARS) spectroscopic imaging is a novel option because it can assess and visualize molecular structures based on their molecular fingerprint. Here, we present a new label-free method to visualize cellular senescence using CARS imaging in nucleoli. We found the peak of the nucleolar amide I band shifted to a higher wavenumber in binuclear senescent cells, which reflects changes in the protein secondary structure from predominant α-helices to β-sheets originating from amyloid-like aggregates. Following this, we developed a procedure that can visualize the senescent cells by providing the ratios and subtractions of these two components. We also confirmed that the procedure can visualize nucleolar aggregates due to unfolded/misfolded proteins produced by proteasome inhibition. Finally, we found that this method can help visualize the nucleolar defects in naïve cells even before binucleation. Thus, our method is beneficial to evaluate ongoing cellular senescence through label-free imaging of nucleolar defects.

Influence of protein in low paste viscosities of Bambara groundnut flours from heat-treated Bambara groundnut seeds

Heat treatment of Bambara groundnut seeds has been reported to cause low paste viscosities in resulting flours. Structural changes in Bambara groundnut protein, due to heat treatment, causes the protein to encapsulate starch, making it unavailable to paste causing low paste viscosities. In this study, trypsin was used to hydrolyze proteins in the flour and the microstructure analysis confirmed the disappearance of aggregates. Flour microstructure analysis confirmed hydrolysis of protein from previously aggregated status and showed liberated individual starch granules. Following the treatment of flours by trypsin, Confocal laser scanning microscopy did not show a protein signal. Hydrolysing Bambara groundnut proteins significantly increased the flour paste viscosities (P < 0.05). The final viscosities for flours from 20 % moisture-conditioned and infrared heat-treated seeds for 5 min were 733.9 mPa s before protein hydrolysis and 2081.71 mPa s after protein hydrolysis. The gelatinization temperature (81 °C) did not show a significant change following protein hydrolysis. Sodium dodecyl-sulphate polyacrylamide gel electrophoresis band intensity increased indicative of disulfide bonding and protein polymerisation when microwave and infrared heat treatment were combined. There were changes in Bambara groundnut protein secondary structures such as an increase of 57 % in β-sheet along with a 60 % reduction in the α-helix as shown by the Fourier-transform infrared spectroscopy. The changes in secondary structure of Bambara groundnut protein were caused by microwave and infrared heating. Heat treatment of Bambara groundnut seeds is partly responsible for the reduction in paste viscosities of their flours.

Enhancing zein functionality through sequential limited Alcalase hydrolysis and transglutaminase treatment: Structural changes and functional properties

This study investigated the effects of sequential enzymatic hydrolysis using Alcalase, followed by transglutaminase conjugation on the secondary and tertiary structures, hydrophobicity, free amine content, protein-protein interactions, and functional properties of zein. Fourier-transform infrared spectroscopy showed that the most significant secondary structural changes, characterized by a decrease in α-helix content and an increase in β-turns, occurred at a higher degree of hydrolysis. At a 2 % degree of hydrolysis, it revealed notable emulsifying activity (65.96 m2/g), while at 5 % hydrolysis, it achieved the highest solubility (75.06 %). Additionally, the zein hydrolysate with a 7 % hydrolysis degree, treated with transglutaminase, demonstrated improved H0 values (2992.33), enhanced foam capacity (65.95 %), and increased solubilized protein content in a dithiothreitol extractant (31.35 %). Meanwhile, native zein treated with transglutaminase showed the highest water holding capacity (4.47 g/g). Overall, the combined enzymatic approach modified zein structure and properties, suggesting potential for improving functionality in plant-based food applications.

Edible insect protein concentrates: Optimized salt-assisted extraction methods evaluation

This study explores the extraction of proteins from edible insects such as Tenebrio molitor, Acheta domesticus, and Locusta migratoria using alkaline extraction and acid precipitation (AEAP) as a control method and evaluates the influence of salting-in (NaCl-assisted), salting-out ((NH₄)₂SO₄), and combined salting-in/out techniques on the resulting protein quality and functional properties. We hypothesized that salt-assisted methods would enhance protein extraction efficiency and functionality compared to AEAP. Molecular size distribution analysis confirmed that the salting-out method preferentially aggregated smaller proteins (<30 kDa). FT-IR spectroscopy revealed notable changes in protein secondary structure across extraction methods, while amino acid profiling identified 17 amino acids, with essential amino acids (EAAs) comprising 37.8–44.2 % of total amino acids. Salt-assisted methods significantly increased the zeta potential (up to −62.67 mV) and modulated particle size distribution (180–492 nm) compared to the control samples. Rheological properties varied with extraction techniques, with potential fluid-type transitions. Proteins extracted via salt-assisted methods demonstrated high purity (>70 %), enhanced solubility (>60 % at pH 7.4), improved oil- and water-holding capacities (1.40–8.09 g/g, 2.41–4.4 g/g), and superior emulsifying properties (EAI >47 m2 g−1, ESI >50 %). These findings highlight that salt-assisted extraction methods can improve the quality and functionality of insect protein concentrates, supporting their potential for food-grade applications.

Inhibitory mechanism of apigenin, quercetin, and phloretin on α-glucosidase

Flavonoids present promising potential as α-glucosidase inhibitors, with potential benefits in lowering blood glucose levels. In our previous study, apigenin, quercetin, and phloretin obtained from whole-grain highland barley demonstrated notable efficacy as α-glucosidase inhibitors. Therefore, in this study, we used enzyme kinetics, fluorescence spectroscopy, circular dichroism, and molecular docking to explore the interactions between apigenin, quercetin, and phloretin, and α-glucosidase. With the addition of three flavonoids, the inhibitory effect of all the flavonoids on α-glucosidase activity was reversible and exhibited a mixed-type pattern. Molecular docking analysis revealed that the three flavonoids predominantly bind to yeast-derived α-glucosidase mainly through hydrophobic interactions and van der Waals forces, whereas the interaction with human-derived α-glucosidase involved hydrophobic interactions. Further experiments revealed that the flavonoids quenched the fluorescence of α-glucosidase due to the complex formation between the flavonoids and α-glucosidase, leading to changes in the protein secondary structure and conformational changes, which was further supported by molecular dynamics simulations. These results provided insights into the mechanism of the flavonoid inhibition of α-glucosidase. These findings could promote the consumption of whole-grain highland barley and the development of hypoglycemic foods rich in flavonoids.

Synthesis of sucrose 6-acetate by immobilized aspergillus Niger lipase imprinted with oleic acid and sorbitol

Biological and interfacial imprinting both were effective methods to improve the catalytic performance of lipase in organic solvent. Bioimprinting combined with interfacial activation of lipase was investigated for obtaining imprinted lipase with excellent catalytic performance. Enzymatic synthesis of sucrose-6-acetate in organic solvents to test the performance of imprinted lipase immobilized by adsorbing on macroporous resin. Lipase imprinted by substrate analog (oleic acid) then immobilization catalyzed the synthesis of sucrose-6-acetate with a 1.7 times increase in sucrose esterification rate compared to unimprinted immobilized lipase and a selectivity toward sucrose-6-acetate of 91.7 %. The sucrose esterification rate of reaction catalyzed by immobilized lipase was increased to 2.0 times with addition of sorbitol after imprinting by oleic acid. Imprinting by oleic acid and interfacial effect generated by oleic acid with sorbitol both induced zymoprotein secondary structure change that were conducive to enhance catalytic activity and stability of lipase in organic solvent.

Interaction of 4-ethyl phenyl sulfate with bovine serum albumin: Experimental and molecular docking studies.

4-ethyl phenyl sulfate (EPS), a protein-bound uremic toxin found in serum of patients suffering from autism spectrum disorders (ASD) and chronic kidney disease (CKD). As per recent advances in the field, gut metabolites after their formation goes to blood stream crosses blood brain barrier and causes neuro related problems. Increased levels of 4-EPS in human body causes anxiety in patients and its role remains elusive. 4-EPS interacts with serum albumin in human body and thus, a model study of interaction of BSA with 4-EPS is presented in support of it. Absorption spectroscopy result demonstrated decrease in bovine serum albumin (BSA) absorption upon interaction with increasing concentration of EPS in a range from 2 μM to 100 μM. Moreover, this interaction was confirmed by the fluorescence quenching in presence of metabolite. The change in secondary structure was demonstrated by circular dichroism, synchronous fluorescence and Fourier transform infra-red spectroscopy. Docking studies reveals binding score of -5.28 Kcal mol-1, demarking that 4-EPS is involved in interaction with BSA via amino acid residues, forming the stable complex. This interaction study may be helpful in devising strategies for the treatment of chronic kidney disease and other neuro related diseases, by producing synthetic compound that competes with albumin binding sites to allow 4-EPS clearance from the body.

Co-stabilization effects of gluten/carrageenan to the over-heated myofibrillar protein: Inhibit the undesirable gel weakening and protein over-aggregations

High-temperature (120 °C) sterilization is an indispensable process for manufacturing ready-to-eat surimi products, yet risking the denaturation of their myofibrillar proteins (MP), thus significantly reducing the gelling properties. To resolve this problem, herein, a synergistic co-strengthening strategy was designed. The negatively charged polysaccharide carrageenan (CG) was introduced into MP simultaneously with wheat gluten, followed by 120 °C thermal treatment for 30 min. A substantial enhancement in mechanical strength, up to four times greater (from 9.86 to 42.38 g·cm), was observed for MP gels, which even surpassed that subjected to conventional gelation processes at 90 °C (36.53 g·cm). Gels that were concurrently added with gluten and CG exhibited porous networks, uniform water distribution, and improved water holding capacity. Accordingly, over-aggregation behaviors of MP were restricted, as evidenced by their reduced particle sizes and polymer dispersity index. Other heat-induced protein deteriorations at 120 °C, i.e., changes of secondary structures and disulfide bonding conformations, were also alleviated. By varying the CG types, it was shown that the κ-CG/gluten-added MP achieved highest gel strength, while the ι-CG/gluten combination may better stabilize the moisture in gel networks. This study introduces a co-reinforcement paradigm and scientific insights to the quality improvement of ready-to-eat meat products.

Mapping the sclerostin-LRP4 binding interface identifies critical interaction hotspots in loops 1 and 3 of sclerostin.

The interaction of sclerostin (Scl) with the low-density lipoprotein receptor-related protein 4 (LRP4) leads to a marked reduction in bone formation by inhibiting the Wnt/β-catenin pathway. To characterize the Scl-LRP4 binding interface, we sorted a combinatorial library of Scl variants and isolated variants with reduced affinity to LRP4. We identified Scl single-mutation variants enriched during the sorting process and verified their reduction in affinity toward LRP4-a reduction that was not a result of changes in the variants' secondary structure or stability. We found that Scl positions K75 (loop 1) and V136 (loop 3) are critical hotspots for binding to LRP4. Our findings establish the foundation for targeting these hotspots for developing novel therapeutic strategies to promote bone formation.

Novel insights into fouling mechanisms in anaerobic acidification membrane bioreactor: The leading role of pH value

Anaerobic acidification membrane bioreactors (AAMBRs) have recently emerged as effective systems for recovering volatile fatty acids (VFAs) from municipal wastewater. However, membrane fouling remains a critical challenge, with its underlying mechanisms not yet fully understood. Given its importance in inhibiting methanogenic bacteria and stabilizing VFAs production, this study explores the direct and indirect effects of pH on membrane fouling mechanisms in AAMBRs. The results demonstrated that compared to pH 7, the fouling potential of anaerobic sludge was significantly higher at pH levels of 5 and 10 owing to a decrease of sludge floc size and an increase of soluble microbial products (SMP) especially proteins. Analytical techniques such as confocal laser scanning microscopy (CLSM), fourier transform infrared spectroscopy (FTIR), and fluorescence excitation-emission matrix (F-EEM) analyses revealed a substantial increase in organic foulants particularly proteins on the membrane surface at pH levels of 5 and 10. Circular dichroism (CD) spectroscopy further indicated a significant change in protein secondary structure, specifically an increase in α-helix content under extreme pH conditions. Furthermore, the extended Derjaguin-Landau-Verwey-Overbeek (XDLVO) theory indicated that elevated protein concentrations enhance the adhesion of sludge and SMP to membrane surfaces. Insights from the Flory-Huggins theory demonstrated that variations in protein structure significantly contribute to increased filtration resistance within the gel layer. Both experimental data and theoretical models underscore the dual role of pH in optimizing VFAs production and managing membrane fouling, offering insights for enhancing AAMBR performance and mitigating fouling issues.