Protein Microenvironment Research Articles

Real-Time Visualization of Protein Microenvironment Changes with High Spatial Resolution in Live Cells via Site-Specific Incorporation of Rotor-Based Fluorescent Noncanonical Amino Acids.

Traditional methods, such as the use of fluorescent protein fusions and environment-sensitive fluorophores, have limitations when studying protein microenvironment changes at the finest spatial resolution. These techniques often rely on bulky proteins or tags restricted to the N- or C-terminus, which can disrupt the natural behavior of the target protein and dramatically limit the ability of their method to investigate noninvasively microenvironment effects. To overcome these challenges, we have developed an innovative strategy to visualize microenvironment changes of protein substructures in real-time by genetically incorporating environment-sensitive noncanonical amino acids (ncAAs) containing rotor-based fluorophores (RBFs) at specific positions within a protein of interest. Through computational redesign of aminoacyl-tRNA synthetase, we successfully incorporated these rotor-based ncAAs into various proteins in mammalian cells. By site-specifically placing these ncAAs in distinct regions of proteins, we detected microenvironmental changes of several different protein domains during events such as aggregation, clustering, aggregation disassembly, and cluster dissociation.

Synergistic Effects of 125I Seed Implantation Brachytherapy and Gemcitabine in Pancreatic Tumors.

Monotherapy consisting of radiotherapy or chemotherapy has limited efficacy in pancreatic tumors. This study aims to investigate whether the combination of 125I brachytherapy and gemcitabine (GEM) chemotherapy has a synergistic effect on pancreatic cancer (PC). In vitro, PANC-1 cells in the exponential phase were treated with 125I radioactive seeds (6 Gy) and GEM (30 nM). Cell proliferation, apoptosis, and mitochondrial membrane potential were measured using the Cell Counting Kit-8 (CCK-8) assay, Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, and flow cytometry, respectively. In vivo, we examined the inhibitory effect of three different treatment regimens on tumor growth in mice when combined with 125I brachytherapy and GEM. Next, we investigated the effects of the optimal scheme among the three on the tumor microenvironment, tumor tissue morphology, tumor cell apoptosis, systemic inflammatory response, and levels of apoptosis-related proteins in the tumor. Changes in the tumor microenvironment and levels of apoptosis-related proteins were measured by Western blot. The extent of damage to tumor tissue morphology was assessed by Hematoxylin and Eosin (HE) staining. Tumor cell apoptosis was measured by TUNEL staining. Changes in inflammation-related factors were determined by Enzyme-Linked Immunosorbent Assay (ELISA). The results of in vitro cell experiments demonstrated that the combination of 125I radioactive seeds (6 Gy) and GEM (30 nM) had a stronger inhibitory effect on PANC-1 cells than either alone (p < 0.05). In vivo, data showed that the GEM (after 3 d) + 125I treatment group had the strongest tumor inhibition effect on PC (p < 0.05). Western blot analysis showed that the combined treatment of 125I brachytherapy and GEM caused changes in the expression of collagen and connexin in the tumor microenvironment, promoted tumor cell apoptosis, upregulated the expression of pro-apoptotic proteins, and helped to restore pancreatic function (p < 0.01). Our research results suggest that the strategy of 125I seed implantation surgery in mice after 3 days of GEM treatment has a more pronounced synergistic effect on the treatment of PC.

Subpocket Similarity-Based Hit Identification for Challenging Targets: Application to the WDR Domain of LRRK2.

We herewith applied a priori a generic hit identification method (POEM) for difficult targets of known three-dimensional structure, relying on the simple knowledge of physicochemical and topological properties of a user-selected cavity. Searching for local similarity to a set of fragment-bound protein microenvironments of known structure, a point cloud registration algorithm is first applied to align known subpockets to the target cavity. The resulting alignment then permits us to directly pose the corresponding seed fragments in a target cavity space not typically amenable to classical docking approaches. Last, linking potentially connectable atoms by a deep generative linker enables full ligand enumeration. When applied to the WD40 repeat (WDR) central cavity of leucine-rich repeat kinase 2 (LRRK2), an unprecedented binding site, POEM was able to quickly propose 94 potential hits, five of which were subsequently confirmed to bind in vitro to LRRK2-WDR.

Inhibiting insulin aggregation by chaperone-like green cholinium-based DESs as additives

Protein microenvironment is critical to study protein function and stability. In the current study, the ability of synthesized choline-based deep eutectic solvents (DESs) as additives to inhibit and disaggregate amyloid fibrils of human insulin was investigated. The DESs synthesized in this study were nontoxic and can be classified as green chemistry. Utilizing various biophysical methods indicated that choline-chloride glycine (Ch/Gly) and choline-chloride ascorbic acid (Ch/Asb) exhibited chaperone-like properties. This was attributed to their inhibitory effects on the lag phase by stabilizing insulin monomers and on the saturation phase by disaggregating mature insulin fibrils. In contrast, choline-chloride histidine (Ch/His) accelerated the aggregation of insulin. Our results suggest that antioxidant and hydrophobic properties, combined with lower surface activity and higher hydrogen bond formation ability, are the main features describing the inhibitory effect of DESs on protein aggregation.

Ionic Effect on the Microenvironment of Biomolecular Condensates.

Biomolecules such as proteins and RNA could organize to form condensates with distinct microenvironments through liquid-liquid phase separation (LLPS). Recent works have demonstrated that the microenvironment of biomolecular condensates plays a crucial role in mediating biological activities, such as the partition of biomolecules, and the subphase organization of the multiphasic condensates. Ions could influence the phase transition point of LLPS, following the Hofmeister series. However, the ion-specific effect on the microenvironment of biomolecular condensates remains unknown. In this study, we utilized fluorescence lifetime imaging microscopy (FLIM), fluorescence recovery after photobleaching (FRAP), and microrheology techniques to investigate the ion effect on the microenvironment of condensates. We found that ions significantly affect the microenvironment of biomolecular condensates: salting-in ions increase micropolarity and reduce the microviscosity of the condensate, while salting-out ions induce opposing effects. Furthermore, we manipulate the miscibility and multilayering behavior of condensates through ion-specific effects. In summary, our work provides the first quantitative survey of the microenvironment of protein condensates in the presence of ions from the Hofmeister series, demonstrating how ions impact micropolarity, microviscosity, and viscoelasticity of condensates. Our results bear implications on how membrane-less organelles would exhibit varying microenvironments in the presence of continuously changing cellular conditions.

Theoretical insights into the formation of oxygen activation sites with the affection of protein microenvironment in Flavin-Dependent monooxygenases MsuD

Flavin-dependent monooxygenases are very famous for their capability to catalyze the oxidation of various substrates using oxygen and reduced flavin mononucleotide (FMNH-) or reduced flavin adenine dinucleotide (FADH-). When investigating the catalytic mechanism of flavin-dependent monooxygenase systems, a fundamental problem remains unsolved is that the O2 binding scheme within the reaction cavity is unclear. By applying molecular dynamics simulations, MDpocket simulations, residue mutation molecular dynamics simulation and energy decomposition analyses, this work identified the O2 binding pockets and discovered a possible oxygen migration channel within MsuD enzyme. Molecular dynamics simulations after residue mutation proved that residues Asn104 plays an important role in maintaining the O2 position within the reaction cavity. By analyzing the interaction between O2 and the surrounding protein microenvironment, we demonstrated that N5 is a more favored reaction site and speculated a reasonable O2 activation process. This work recovers the formation of oxygen activation sites in MsuD enzyme, which is of great significance to further investigations of the catalytic reaction mechanism. This work provides a feasible method to study the O2 binding in proteins and the role of the protein microenvironment around the reaction cavity.

Modification of structure, epitope and allergenicity on heat-stressed ovalbumin by resveratrol

Allergen modification is a novel strategy for preventing and treating food allergy. Resveratrol (RES) can be used as a modifying ligand for allergens, but the interaction mechanism between RES and allergens is still not detailed, and the ramifications of this interaction on the structure, epitope and allergenicity of allergens are also lacking a clear explanation. In addition, the application of heat can promote protein structure to unfold and expose more binding sites to facilitate interaction. Therefore, in this study, ovalbumin (OVA) was selected as the allergen model and was heated to expose more binding sites. The effects of the interaction between RES and heat-stressed ovalbumin (HOVA) on allergen structure, epitope and allergenicity were investigated by combining routine experimental analysis and computer simulation. Results showed heat stress at 323 K for 15 min could better unfold the molecular structure of OVA. On this basis, the interaction between RES and OVA was strengthened, which showed a static quenching of the ground state complex with binding affinity of 1.25 × 106 L/mol. The binding of RES induced the variation of protein microenvironment, and the overall structure tended to disordered direction. Computer simulation had shown RES could directly act on the IgE epitope region (AA251-260) of the OVA. The allergenicity evaluation experiment further indicated the combination of RES alleviated the IgE binding capacity of HOVA. These findings are helpful to further study the mechanism of RES ligand modification to change food allergy.

Charged Amino Acid Substitutions Affect Conformation of Neuroglobin and Cytochrome c Heme Groups.

Neuroglobin (Ngb) is a cytosolic heme protein that plays an important role in protecting cells from apoptosis through interaction with oxidized cytochrome c (Cyt c) released from mitochondria. The interaction of reduced Ngb and oxidized Cyt c is accompanied by electron transfer between them and the reduction in Cyt c. Despite the growing number of studies on Ngb, the mechanism of interaction between Ngb and Cyt c is still unclear. Using Raman spectroscopy, we studied the effect of charged amino acid substitutions in Ngb and Cyt c on the conformation of their hemes. It has been shown that Ngb mutants E60K, K67E, K95E and E60K/E87K demonstrate changed heme conformations with the lower probability of the heme planar conformation compared to wild-type Ngb. Moreover, oxidized Cyt c mutants K25E, K72E and K25E/K72E demonstrate the decrease in the probability of methyl-radicals vibrations, indicating the higher rigidity of the protein microenvironment. It is possible that these changes can affect electron transfer between Ngb and Cyt c.

Succinic Acid Ameliorates Concanavalin A-Induced Hepatitis by Altering the Inflammatory Microenvironment and Expression of BCL-2 Family Proteins.

Autoimmune hepatitis (AIH) is a severe immune-mediated inflammatory liver disease that currently lacks feasible drug treatment methods. Our study aimed to evaluate the protective effect of succinic acid against AIH and provide a reliable method for the clinical treatment of AIH. We performed an in vivo study of the effects of succinic acid on concanavalin A (ConA)-induced liver injury in mice. We examined liver transaminase levels, performed hematoxylin and eosin (HE) staining, and observed apoptotic phenotypes in mice. We performed flow cytometry to detect changes in the number of neutrophils and monocytes, and used liposomes to eliminate the liver Kupffer cells and evaluate their role. We performed bioinformatics analysis, reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and western blotting to detect mitochondrial apoptosis-induced changes in proteins from the B-cell lymphoma 2(Bcl-2) family. Succinic acid ameliorated ConA-induced AIH in a concentration-dependent manner, as reflected in the survival curve. HE and TUNEL staining and terminal deoxynucleotidyl transferase dUTP nick end labeling revealed decreased alanine transaminase and aspartate aminotransferase levels, and reduced liver inflammation and apoptosis. RT-qPCR and enzyme-linked immunosorbent assay revealed that succinic acid significantly reduced liver pro-inflammatory cytokine levels. Flow cytometry revealed significantly decreased levels of liver neutrophils. Moreover, the protective effect of succinic acid disappeared after the Kupffer cells were eliminated, confirming their important role in the effect. Bioinformatics analysis, RT-qPCR, and western blotting showed that succinic acid-induced changes in proteins from the Bcl-2 family involved mitochondrial apoptosis, indicating the molecular mechanism underlying the protective effect of succinic acid. Succinic acid ameliorated ConA-induced liver injury by regulating immune balance, inhibiting pro-inflammatory factors, and promoting anti-apoptotic proteins in the liver. This study provides novel insights into the biological functions and therapeutic potential of succinic acid in the treatment of autoimmune liver injury.

Deciphering the interaction mechanism between soy protein isolate and fat-soluble anthocyanin on experiments and molecular simulations

In this work, the acylated anthocyanin (Ca-An) was prepared by enzymatic modification of black rice anthocyanin with caffeic acid, and the binding mechanism of Ca-An to soybean protein isolate (SPI) was investigated by experiments and computer simulation to expand the potential application of anthocyanin in food industry. Multi-spectroscopic studies revealed that the stable binding of Ca-An to SPI induced the folding of protein polypeptide chain, which transformed the secondary structure of SPI trended to be flexible. The microenvironment of protein was transformed from hydrophobic to hydrophilic, while tyrosine played dominant role in quenching process. The binding sites and forces of the complexes were determined by computer simulation for further explored. The protein conformation of the 7S and 11S binding regions to Ca-An changed, and the amino acid microenvironment shifted to hydrophilic after binding. The results showed that more non-polar amino acids existed in the binding sites, while in binding process van der Waals forces and hydrogen bonding played a major role hydrophobicity played a minor role. Based on MM-PBSA analysis, the binding constants of 7S-Ca-An and 11S-Ca-An were 0.518 × 106 mol−1 and 5.437 × 10−3 mol−1, respectively. This information provides theoretical guidance for further studying the interaction between modified anthocyanins and biomacromolecules.

Osmolyte-specific counteraction of ethanol-induced aggregation and structure-function distortion of catalase

Catalase, an industrially important enzyme, was found to unfold and aggregate in presence of ethanol. Aggregation of catalase was observed in 20% and higher ethanol concentrations as evident by typical light scattering aggregation profile with complete absence of lag phase, enhanced Thioflavin T fluorescence, increased 8-anilinonaphthalene-1-sulfonic acid binding depicting enhanced hydrophobic exposure, unfolded tertiary structure with an altered Fourier transform infrared and soret spectra. To prevent ethanol-induced aggregation of catalase, osmolytes were screened for their ability to counteract the ethanol effects on catalase. Among all the osmolytes tested, only three i.e., betaine, trehalose and sucrose counteracted the ethanol-induced aggregation of catalase by about 30–50%. Results infer that addition of osmolytes to ethanol-treated catalase persuaded changes in solvent polarity and protein microenvironment leading to overall counteraction of ethanol-induced structural changes in catalase responsible for its aggregation. Fluorescence microscopy also confirmed the counteraction of ethanol-induced aggregation by osmolytes as evident from the decreased presence of aggregates in osmolyte-incubated ethanol-treated catalase. Overall, the osmolyte-induced counteraction of ethanol effects on catalase is biologically significant as it provides significant insights regarding the prevention of organic solvent induced aggregation of industrially important enzymes, being otherwise vulnerable to the solvent-induced distortion of their structure-function integrity.

Capture of endogenous lipids in peptidiscs and effect on protein stability and activity

Compared to protein-protein and protein-nucleic acid interactions, our knowledge of protein-lipid interactions remains limited. This is primarily due to the inherent insolubility of membrane proteins (MPs) in aqueous solution. The traditional use of detergents to overcome the solubility barrier destabilizes MPs and strips away certain lipids that are increasingly recognized as crucial for protein function. Recently, membrane mimetics have been developed to circumvent the limitations. In this study, using the peptidisc, we find that MPs in different lipid states can be isolated based on protein purification and reconstitution methods, leading to observable effects on MP activity and stability. Peptidisc also enables re-incorporating specific lipids to fine-tune the protein microenvironment and assess the impact on downstream protein associations. This study offers a first look at the illusive protein-lipid interaction specificity, laying the path for a systematic evaluation of lipid identity and contributions to membrane protein function.

Biophysical insight into the binding mechanism of 1,3-benzodioxole-based lidocaine tagged ionic liquids with BSA: An in-silico, and spectroscopic studies

The interaction between bio-macromolecules and drugs has attracted significant interest for several decades due to their pharmacokinetics and pharmacological implications. Ionic liquids have a major importance in the development of effective drugs that can majorly target and bind to the proteins present in the body. A slight disturbance in the micro-environment of protein induced by a drug can alter the conformations of proteins to lead them to work differently. The interaction of ionic liquids with proteins present in blood such as HSA (Human Serum Albumin) and BSA (Bovine Serum Albumin) provides useful evidence for synthesizing API-based liquid drug and their delivery. In this study, the interaction of BSA with LPAc & LPOT ILs (LPAc: N-(benzo[d][1,3]dioxol-5-ylmethyl)-2-((2,6-dimethylphenyl)amino)-N,N-diethyl-2-oxoethan-1-aminium acetate and LPOT: N-(benzo[d][1,3]dioxol-5-ylmethyl)-2-((2,6-dimethylphenyl)amino)-N,N-diethyl-2-oxoethan-1-aminiumtrifluoromethane sulfonate) using spectroscopic methods like UV–visible, fluorescence, CD spectroscopy, computational studies (molecular docking, MD simulation studies, and ADMET studies). UV–vis spectra showed a slight rise in absorbance intensity with the addition of ionic liquid. A static quenching of BSA with blue shift was observed according to fluorescence spectra on the addition of ionic liquids (LPAc & LPOT) and conformational alterations of BSA were observed by analyzing CD spectra. LPAc and LPOT showed strong binding with BSA with molecular docking scores of −330.2 and −365.2 kcal/mol respectively. The molecular dynamic simulation and in-silico pharmacological and pharmacokinetics studies support the spectroscopic study and molecular docking results inferring that LPOT ionic liquid showed strong binding interactions with BSA. In-vitro studies against A549 cell lines showed that both LAPc and LPOT ionic liquids had antiproliferative properties and LPOT has better anticancer activity as inferred by IC50 value: 38.83 and may be further useful for drug design as cancer therapeutic.

Learning the shape of protein microenvironments with a holographic convolutional neural network

Proteins play a central role in biology from immune recognition to brain activity. While major advances in machine learning have improved our ability to predict protein structure from sequence, determining protein function from its sequence or structure remains a major challenge. Here, we introduce holographic convolutional neural network (H-CNN) for proteins, which is a physically motivated machine learning approach to model amino acid preferences in protein structures. H-CNN reflects physical interactions in a protein structure and recapitulates the functional information stored in evolutionary data. H-CNN accurately predicts the impact of mutations on protein stability and binding of protein complexes. Our interpretable computational model for protein structure-function maps could guide design of novel proteins with desired function.

NIR-I emissive cyanine derived molecular probe for selective monitoring of hepatic albumin levels during hyperglycemia.

In this study, we report a small molecule optical marker BI-CyG derived from the structural engineering of a cyanine scaffold. The developed probe offers suitable advantages over existing cyanine-based albumin specific probes in terms of its excitation and emission wavelengths, which are 760 and 830-832 nm, respectively. Structural tuning of the cyanine architecture leading to extended π-conjugation and resulting in a suitable bathochromic shift in the emission wavelength of the probe is represented in this study. The probe besides emitting in the NIR region, also possesses the desirable characteristics of being a potential target selective optical marker, as established from various biophysical studies. Molecular modelling and simulation studies provided critical insights into the binding of the probe in the protein microenvironment, which was further supported by experimental studies. The probe displayed intracellular albumin selectivity and was utilized for demonstrating alteration in albumin levels in pathological states such as hyperglycemia in hepatic cells. The present study also sheds some light on using BI-CyG as an imaging probe and on the role of metformin as a suitable drug for balancing hyperglycemia-induced reduced intra-hepatic albumin levels. The study, thus, attempts to highlight the structural derivatization of cyanine to afford a potential probe for serum albumin and its deployment to image altering albumin levels in an induced pathological condition, hyperglycemia.

A Live Tracker of Dormant Disseminated Tumor Cells.

The high prevalence of dormant disseminated tumor cells (DTCs) persisting systemically in patients with metastatic cancer is a major threat to long-lasting cure (Aguirre-Ghiso, Nat Rev Cancer 7:834-846, 2007; Klein, Nat Rev Cancer 20(11):681-694, 2020; Lyden et al. Cancer Cell 40:787-791, 2022). Despite its clinical significance, the study of what drives DTCs in and out of dormancy while they linger in distant sites has been challenged by the lack of tools to find and follow dormant DTCs inside a living organism. Here, leveraging the fact that dormant DTCs are mostly quiescent, we describe a live cell reporter to distinguish dormant from cycling DTCs (Correia, Nat Rev Cancer 22(7):379, 2022; Correia et al. Nature 594(7864):566-571, 2021). Cancer cell lines are engineered to coexpress a luciferase-tdTomato reporter and a fluorescent fusion protein of mVenus with a mutant form of the cell cycle inhibitor p27 (mVenus-p27K-) that identifies quiescent cells. When implanted in animal models or assembled in cocultures in vitro, labeled cells can be imaged longitudinally over time or retrieved alive alongside their surrounding microenvironment for downstream gene, protein, and metabolite profiling, allowing the mapping of tissue-specific determinants of cancer dormancy and metastasis.

An injectable photocuring silk fibroin-based hydrogel for constructing an antioxidant microenvironment for skin repair.

Skin has a protein microenvironment dominated by functional collagen fibers, while oxidative stress caused by injury can greatly slow down the progress of wound healing. Here, methacrylated dopamine was incorporated into methacrylated silk fibroin molecule chains to develop an injectable hydrogel with photocuring properties for constructing an antioxidant skin protein microenvironment. This silk fibroin-based hydrogel (SF-g-SDA) showed good tensile and adhesion properties for adapting to the wound shape and skin movement, exhibited stable mechanical properties, good biodegradability and cytocompatibility, and promoted cell adhesion and vascularization in vitro. In addition, its phenolic hydroxyl-mediated antioxidant properties effectively protected cells from damage caused by oxidative stress and supported normal cellular life activities. In animal experiments, SF-g-SDA achieved better skin repair effects in comparison to commercial Tegaderm™ in vivo, showing its ability to accelerate wound healing, improve collagen deposition and alignment in newly fabricated tissues, and promote neovascularization and hair follicle formation. These experimental results indicated that the SF-g-SDA hydrogel is a promising wound dressing.

Interaction and binding mechanism of ovalbumin with cereal phenolic acids: improved structure, antioxidant activity, emulsifying and digestion properties for potential targeted delivery systems

Ovalbumin (OVA) has been considered as a nutrient carrier for bioactive, which has high nutrition value and multiple properties. Recently, proteins-phenolic acids composite delivery systems have received widespread attention. Therefore, this research aimed to investigate the interaction between OVA and cereal phenolic acids (CPA) to establish delivery systems for bioactive. Spectroscopy results have found that CPA generated complexes with OVA, causing the microenvironment changes of OVA. Ferulic acid (FA), p-coumaric acid (CA), vanillic acid (VA), syringic acid (SY), sinapic acid (SI), and protocatechuic acid (PA) not only quenched the intrinsic fluorescence of OVA, but also altered protein microenvironment. Further investigation showed these complexes were formed by static quenching mode, while hydrogen bond and hydrophobic interaction were dominant binding forces. Meanwhile, the interaction decreased α-helix contents and increased β-sheet contents, leading to conformational changes in OVA. Besides, OVA/CPA complexes displayed an increase in hydrophobicity with a reduce in free-SH. After combination with FA, SY, CA, VA, SI, PA, it was found that all formed complexes had superior solubility, emulsifying and antioxidant activities than native OVA. Among them, OVA-PA exhibited the highest emulsifying activity index and emulsion stability index values (36.4 ± 0.39 m2/g and 60.4 ± 0.94 min) and stronger antioxidant activities. Finally, the combination with phenolic acids further improved the digestion efficiency in vitro of OVA. The OVA-CPA complexes showed improved properties for excellent delivery systems. Overall, OVA-CPA complexes could be a good carrier for bioactive, which provided valuable avenues in target delivery system application.

Deciphering the binding mode and structural perturbations in floxuridine-human serum albumin complexation with spectroscopic, microscopic, and computational techniques

The binding mode of antineoplastic antimetabolite, floxuridine (FUDR), with human serum albumin (HSA), the leading carrier in blood circulation, was ascertained using multi-spectroscopic, microscopic, and computational techniques. A static fluorescence quenching was established due to decreased Ksv values with rising temperatures, suggesting FUDR-HSA complexation. UV–vis absorption spectral results also supported this conclusion. The binding constant, Ka values, were found within 9.7–7.9 × 103 M−1 at 290, 300, and 310 K, demonstrating a moderate binding affinity for the FUDR-HSA system. Thermodynamic data (ΔS = +46.35 J.mol−1.K−1 and ΔH = −8.77 kJ.mol−1) predicted the nature of stabilizing forces (hydrogen-bonds, hydrophobic, and van der Waals interactions) for the FUDR-HSA complex. Circular dichroism spectra displayed a minor disruption in the protein’s 2° and 3° structures. At the same time, atomic force microscopy images proved variations in the FUDR-HSA surface morphology, confirming its complex formation. The protein's microenvironment around Trp/Tyr residues was also modified, as judged by 3-D fluorescence spectra. FUDR-bound HSA showed better resistance against thermal stress. As disclosed from ligand displacement studies, the FUDR binding site was placed in subdomain IIA (Site I). Further, the molecular docking analysis corroborated the competing displacement studies. Molecular dynamics evaluations revealed that the complex achieved equilibrium during simulations, confirming the FUDR–HSA complex's stability.

Contrasting effects of molecular crowding on the membrane-perturbing and chaperone-like activities of major bovine seminal plasma protein, PDC-109

Crowded environments inside cells and biological fluids greatly affect protein stability and activity. PDC-109, a polydisperse oligomeric protein of the bovine seminal plasma selectively binds choline phospholipids on the sperm cell surface and causes membrane destabilization and lipid efflux, leading to acrosome reaction. PDC-109 also exhibits chaperone-like activity (CLA) and protects client proteins against various kinds of stress, such as high temperature and low pH. In the present work, we have investigated the effect of molecular crowding on these two different activities of PDC-109 employing Dextran 70 (D70) – a widely used polymeric dextran – as the crowding agent. The results obtained show that presence of D70 markedly increases membrane destabilization by PDC-109. Isothermal titration calorimetric studies revealed that under crowded condition the binding affinity of PDC-109 for choline phospholipids increases approximately 3-fold, which could in turn facilitate membrane destabilization. In contrast, under identical conditions, its CLA was reduced significantly. The decreased CLA could be correlated to reduced surface hydrophobicity, which was due to stabilization of the protein oligomers. These results establish that molecular crowding exhibits contrasting effects on two different functional activities of PDC-109 and highlight the importance of microenvironment of proteins in modulating their functional activities.