Wheat germ agglutinin (WGA) demonstrates potential as an oral delivery agent owing to its selective binding to carbohydrates and its capacity to traverse biological membranes. In this study, we employed differential scanning calorimetry and molecular dynamics simulations to comprehensively characterize the thermal unfolding process of both the complete lectin and its four isolated domains. Furthermore, we present the nuclear magnetic resonance structures of three domains that were previously lacking experimental structures in their isolated forms. Our results provide a collective understanding of the energetic and structural factors governing the intricate unfolding mechanism of the complete agglutinin, shedding light on the specific role played by each domain in this process. The analysis revealed negligible interdomain cooperativity, highlighting instead significant coupling between dimer dissociation and the unfolding of the more labile domains. By comparing the dominant interactions, we rationalized the stability differences among the domains. Understanding the structural stability of WGA opens avenues for enhanced drug delivery strategies, underscoring its potential as a promising carrier throughout the gastrointestinal environment.

A major concern for wineries is haze formation in white wines due to protein instability. Despite its prevalent use, the conventional bentonite method has shortcomings, including potential alteration of color and aroma, slow processing times, and notable wine wastage. Zirconium oxide (ZrO2) effectively removes proteins without affecting wine characteristics. However, producing cost-effective ZrO2 materials with efficient protein removal capabilities poses a significant challenge. This research aims to assess the viability of designing a porous material impregnated with zirconia to remove turbidity-causing proteins effectively. For this purpose, the support material alone (Al2O3) and the zirconia-impregnated support (ZrO2/Al2O3) were subjected to different calcination temperatures. It was observed that high-temperature treatments (750 °C) enhanced wine stability and protein adsorption capacity. The optimal adsorbent achieved a notable reduction in turbidity, decreasing the ΔNTU from 42 to 18, alongside a significant 44 % reduction in the total protein content, particularly affecting proteins in the molecular weight range of 10 to 70 kDa. This result is attributed to modifying the textural properties of ZrO2/Al2O3, characterized by the reduction of acidic sites, augmented pore diameters from 4.81 to 7.74 nm, and the emergence of zirconia clusters across the surface of the porous support. In summary, this study presents the first application of zirconia on the alumina support surface for protein stabilization in white wine. Combining ZrO2/Al2O3 and a high-temperature treatment emerges as a promising, cost-efficient, and environmentally sustainable strategy for protein removal in white wine.

The fibrillation of therapeutic peptides can present significant quality concerns and poses challenges for manufacturing and storage. A fundamental understanding of the mechanisms of fibrillation is critical for the rational design of fibrillation-resistant peptide drugs and can accelerate product development by guiding the selection of solution-stable candidates and formulations. The studies reported here investigated the effects of structural modifications on the fibrillation of a 29-residue peptide (PepA) and two sequence modified variants (PepB, PepC). The C-terminus of PepA was amidated, whereas both PepB and PepC retained the carboxylate, and Ser16 in PepA and PepB was substituted with a helix-stabilizing residue, α-aminoisobutyric acid (Aib), in PepC. In thermal denaturation studies by far-UV CD spectroscopy and fibrillation kinetic studies by fluorescence and turbidity measurements, PepA and PepB showed heat-induced conformational changes and were found to form fibrils, whereas PepC did not fibrillate and showed only minor changes in the CD signal. Pulsed hydrogen-deuterium exchange mass spectrometry (HDX-MS) showed a high degree of protection from HD exchange in mature PepA fibrils and its proteolytic fragments, indicating that most of the sequence had been incorporated into the fibril structure and occurred nearly simultaneously throughout the sequence. The effects of the net peptide charge and formulation pH on fibrillation kinetics were investigated. In real-time stability studies of two formulations of PepA at pH\'s 7.4 and 8.0, analytical methods detected significant changes in the stability of the formulations at different time points during the study, which were not observed during accelerated studies. Additionally, PepA samples were withdrawn from real-time stability and subjected to additional stress (40 °C, continuous shaking) to induce fibrillation; an approach that successfully amplified oligomers or prefibrillar species previously undetected in a thioflavin T assay. Taken together, these studies present an approach to differentiate and characterize fibrillation risk in structurally related peptides under accelerated and real-time conditions, providing a model for rapid, iterative structural design to optimize the stability of therapeutic peptides.

This study focuses on the modulation of protein aggregation and immunogenicity. As a starting point for investigating long-range interactions within a non-native protein, the effects of perturbing denatured protein states on their aggregation, including the formation of amyloid fibrils, were evaluated. The effects of adducts, sugar modifications, and stabilization on protein aggregation were then examined. We also investigated how protein immunogenicity was affected by enhancing protein conformational stability and other factors.

The L84S mutation has been observed frequently in the ORF8 protein of SARS-CoV-2, which is an accessory protein involved in various important functions such as virus propagation, pathogenesis, and evading the immune response. However, the specific effects of this mutation on the dimeric structure of ORF8 and its impacts on interactions with host components and immune responses are not well understood. In this study, we performed one microsecond molecular dynamics (MD) simulation and analyzed the dimeric behavior of the L84S and L84A mutants in comparison to the native protein. The MD simulations revealed that both mutations caused changes in the conformation of the ORF8 dimer, influenced protein folding mechanisms, and affected the overall structural stability. In particular, the 73YIDI76 motif has found to be significantly affected by the L84S mutation, leading to structural flexibility in the region connecting the C-terminal β4 and β5 strands. This flexibility might be responsible for virus immune modulation.  The free energy landscape (FEL) and principle component analysis (PCA) have also supported our investigation. Overall, the L84S and L84A mutations affect the ORF8 dimeric interfaces by reducing the frequency of protein-protein interacting residues (Arg52, Lys53, Arg98, Ile104, Arg115, Val117, Asp119, Phe120, and Ile121) in the ORF8 dimer.  Our findings provide detail insights for further research in designing structure-based therapeutics against the SARS-CoV-2.Communicated by Ramaswamy H. Sarma.

The eye is a vital organ in the visual system, which is composed of transparent vascular tissue. αB-crystallin, a significant protein found in the lens, plays a crucial role in our understanding of lens diseases. Mutations in the αB-crystallin protein can cause lens diseases, such as cataracts and myopathy. However, the molecular mechanism underlying the R120G mutation is not fully understood. In this study, we utilized molecular dynamics simulations to illustrate, in atomic detail, how the R120G mutation leads to the aggregation of αB-crystallin and scattering of light in the lens. Our findings show that the R120G mutation alters the dynamic and structural properties of the αB-crystallin protein. Specifically, this mutation causes the angle of the hairpin at the C-terminal to increase from 80° to 150°, while reducing the distance between the hydrophobic patches around residues 10 and 44-55 from 1.5 nm to 1 nm. In addition, our results showed that the mutation could disrupt the IPI motif - β4/β8 interaction. The disruption of this interaction could affect the αB-crystallin oligomerization and the chaperone activity of αB-crystallin protein. The exposed hydrophobic area at the IPI motif - β4/β8 could become the primary site for interprotein interactions, which are responsible for large-scale aggregation. We have demonstrated that, in wild-type αB-crystallin protein, salt bridges R120 and D109, R107 and D80 are formed. However, in the case of the R120G mutation, the salt bridges R120 and R109 are disrupted, and a new salt bridge with a different pattern is formed. In our study, it has been found that all of the changes associated with the R120G mutation are located at the interface of chains A and B, which could impact the multimerization of the αB-crystallin. Previous research on the K92-E99 residue has shown that a salt bridge in the dimer I can reduce the chaperone activity of the protein. Furthermore, the salt bridges R120 and D109, as well as R107 and D80 in dimer II, induce changes in the hydrophobic envelope of β-sheets in the α-crystallin domain (ACD). These changes could have an impact on the multimerization of the αB-crystallin, leading to disruption of the oligomer structure and aggregation. Moreover, the changes in the αB-crystallin resulting from the R120G mutation can lead to faulty interactions with other proteins, which can cause the aggregation of αB-crystallin with other proteins, such as desmin. These findings may provide new insights into the development of treatments for lens diseases.Communicated by Ramaswamy H. Sarma.

In the bloodstream and in local tissues, most IGF molecules are associated with the one of the members of the IGF-binding protein (IGFBP) family, which are divided into six distinct types. IGF-binding proteins have been demonstrated to either decrease or increase the growth-promoting effects of IGFs on cell culture, by extending their half-life. They alter how IGFs interact with the receptors on their cell surfaces. IGFBP6 gene is associated with disease in-situ carcinoma. Upregulation or downregulation of IGFBP6 gene has been implicated in different types of cancer in humans. Nonsynonymous SNPs changes have the potential to affect the protein\'s structure and function. Potential functional SNPs can be assessed before undertaking studies in larger populations because validation of these functional SNPs can be a crucial problem. So, in this in-silico investigation, we used a variety of sequence- and structure-based bioinformatics methods to separate the potential nsSNPs of the IGFBP6 gene from the neutral ones. In total of 216 nsSNPs, 5 were found to have potential effects using 5 prediction tools. From which, 2 nsSNPs (R128G and R164H) were selected as potentially damaging due to their presence in highly conserved region and ability to decrease protein stability. Among these 2 nsSNPs, only R164H was found to be associated with Uterine corpus endometrial carcinoma. It was also found that both, upregulation or downregulation of IGFBP6 gene can lead to the different types of cancers. The findings of the present study will certainly be valuable in the future large population-based investigations as well as drug discovery, especially developing personalized medicine.Communicated by Ramaswamy H. Sarma.

Detergents have been major contributors to membrane-protein structural study for decades. However, membrane proteins solubilized in conventional detergents tend to aggregate or denature over time. Stability of large eukaryotic membrane proteins with complex structures tends to be particularly poor, necessitating development of novel detergents with improved properties. Here, we prepared a novel class of detergents, designated 3,4-bis(hydroxymethyl)hexane-1,6-diol-based maltosides (HDMs). When tested on three membrane proteins, including two G-protein-coupled receptors (GPCRs), the new detergents displayed significantly better behaviors compared with DDM. Moreover, the HDMs were superior or comparable to LMNG, an amphiphile widely used for GPCR structural study. An optimal balance of detergent rigidity vs. flexibility of the HDMs is likely responsible for their favorable behaviors toward membrane-protein stability. Thus, the current study not only introduces the HDMs, with significant potential for membrane-protein structural study, but also suggests a useful guideline for designing novel detergents for membrane-protein research.

Although bulk biotherapeutics are often frozen during fill finish and shipping to improve their stability, they can undergo degradation leading to losses in biological activity during sub-optimal freeze-thaw (F/T) process. Except for a few small-scale studies, the relative contribution of various F/T stresses to the instability of proteins has not been addressed. Thus, the objective of this study was to determine the individual contributions of freeze-concentration, ice surface area, and processing time to protein destabilization at a practical manufacturing-scale. Lactate dehydrogenase (LDH) in histidine buffer solutions were frozen in 1L containers. The frozen solutions were sliced into representative samples and assessed for the ice specific surface area (SSA) and extent of solutes freeze-concentration. For the first time to our knowledge, ice SSA was measured in dried samples from large-volume protein solutions using volumetric nitrogen adsorption isotherms. SSA measurements of the freeze-dried cakes showed that the ice surface area increased with an increase in the freezing rate. The ice SSA was also impacted by the position of the sample within the container: samples closer to the active cooled surface of the container exhibited smaller ice surface area compared to ice-cored samples from the center of the bottle. The freeze-concentrate composition was determined by measuring LDH concentration in the ice-cored samples. The protein distributed more evenly throughout the frozen solution after fast freezing which also correlated with enhanced protein stability compared to slow freezing conditions. Overall, better protein stability parameters correlated with higher ice SSA and lower freeze-concentration extent which was achieved at a faster freezing rate. Thus, extended residence time of the protein at the freeze-concentrated microenvironment is the critical destabilizing factor during freezing of LDH in bulk histidine buffer system. This study expands the understanding of the relative contributions of freezing stresses which, coupled with the knowledge of cryoprotection mechanisms, is imperative to the development of optimized processes and formulations aiming stable frozen protein solutions.

Human serum retinol-binding protein (RBP) is a plasma transport protein for vitamin A. RBP is a prime subclass of lipocalins, which bind nonpolar ligands within a β-barrel. To understand the role of Trp 24, one of the highly conserved residues in RBP, free energy simulations have been carried out to understand the effects of the mutations from Trp at position 24 to Leu, Phe, and Tyr in the apo-RBP on its thermal stability. We examine various unfolded systems to study the dependence of the free energy differences on the denatured structure. Our calculated free energy difference values for the three mutations are in excellent agreement with the experimental values when the initial coordinates of the seven-residue peptide segments truncated from the crystal structure are used for the denatured systems. Our free energy change differences for the Trp→Leu, Trp→Phe, and Trp→Tyr mutations are 2.50 ± 0.69, 2.58 ± 0.50, and 2.49 ± 0.48 kcal/mol, respectively, when the native-like seven-residue peptides are used as models for the denatured systems. The main contributions to the free energy change differences for the Trp24→Leu and Trp24→Phe mutations are mainly from van der Waals and covalent interactions, respectively. Electrostatic, van der Waals and covalent terms equally contribute to the free energy change difference for the Trp24→Tyr mutation. The free energy simulation helps understand the detailed microscopic mechanism of the stability of the RBP mutants relative to the wild type and the role of the highly conserved residue, Trp24, of the human RBP.Communicated by Ramaswamy H. Sarma.