pH-dependent Interaction Research Articles

New Insights into pH-dependent Complex Formation between Lignosulfonates and Anthocyanins: Impact on Color and Oxidative Stability.

Anthocyanins have limited application as natural colorants and antioxidants due to their color loss and instability under certain conditions. This research explores the formation of a complex between lignosulfonates (LS) and cyanidin-3-O-glucoside (C3G) using a multitechnique approach as well as the effect on C3G's red color, oxidative stability, and antioxidant activity in acidic mediums. All data revealed pH-dependent LS-C3G interactions. The thermodynamic parameters showed weak noncovalent interactions, mainly electrostatic interactions, hydrogen bonds, and hydrophobic effect, with a higher association constant determined at pH 3. Fourier-transform infrared spectroscopy and Zeta-potential experiments further corroborate evidence of these LS-C3G interactions. Fluorescence quenching and lifetime experiments revealed static and dynamic quenching at pH 1 and 3, respectively. UV-visible spectroscopy demonstrated a bathochromic shift upon complex formation and a hyperchromic effect at pH 3 and 4, as a consequence of the improved red color of C3G. Electrochemical results suggested that at pH 3 the LS enhances C3G stability by protecting its oxidizable moieties over time, as well as improving the antioxidant activity of the anthocyanin in the complex.

Glu592 of the axon guidance receptor ROBO3 mediates a pH-dependent interaction with NELL2 ligand.

There are only a few studies on the function of neuronal axon guidance molecules during low brain pH conditions. We previously reported that roundabout (ROBO) 2, a receptor for the axon guidance molecule SLIT, can bind to the neural epidermal growth factor-like-like (NELL) ligands in acidic conditions by conformational change of its ectodomain. Here, we show that the ROBO3 receptor also exhibits a pH-dependent increase in binding to the NELL2 ligand. We found that the Glu592 residue of ROBO3 at the binding interface between NELL2 and ROBO3 is a pH sensor and that the formation of a new hydrogen bonding network, due to protonation of the Glu592, leads to increased binding in acidic conditions. These results suggest that NELL2-ROBO3 signaling could be regulated by extracellular pH.

Macroscopic Assembly of Materials with Engineered Bacterial Spores via Coiled-Coil Interaction.

Herein, we report macroscopic materials formed by the assembly of engineered bacterial spores. Spores were engineered by using a T7-driven expression system to display a high density of pH-responsive self-associating proteins on their surface. The engineered surface protein on the spore surface enabled pH-dependent binding at the protein level and enabled the assembly of granular materials. Mechanical properties remained largely constant with changing pH, but erosion stability was pH-dependent in a manner consistent with the pH-dependent interaction between the engineered surface proteins. Our finding utilizes synthetic biology for the design of macroscopic materials and illuminates the impact of coiled-coil interaction across various length scales.

Serotype-agnostic affinity purification of adeno-associated virus (AAV) via peptide-functionalized chromatographic resins

Adeno-associated viruses (AAVs) have emerged as a prominent family of vectors for gene delivery, providing therapeutic options to diseases once deemed incurable. At the same time, they necessitate efficient and affordable purification methods that can be platformed to serve all AAV serotypes. Current chromatographic tools, while affording high product purity, fail to bind certain serotypes, provide limited yield and lifetime, and impose harsh elution conditions that can compromise the vector's activity and safety. Addressing these challenges, this work demonstrates the application of new peptide ligands as the first serotype-agnostic technology for AAV purification by affinity chromatography. Our study reveals a pH-dependent affinity interaction: AAV2, AAV3, AAV6, AAV9, and AAVrh.10 are effectively captured at neutral pH, while binding AAV1, AAV5, AAV7, and AAV8 is stronger in a slightly acidic environment. The elution of bound AAVs was achieved using magnesium chloride at neutral pH for all serotypes, consistently affording capsid yields above 50% and genome yields above 80%, together with a >100-fold reduction in host cell proteins and nucleic acids. In particular, peptide ligand A10 exhibited remarkable binding capacity (> 1014 vp per mL of resin) and purification performance for all AAV serotypes, demonstrating broad applicability for gene therapy manufacturing. Finally, this work introduces novel alkaline-stable variants of A10 and demonstrates their use as the first affinity ligands capable of performing multiple cycles of AAV2, AAV8, and AAV9 purification with intermediate caustic cleaning without loss of capacity or product quality. Collectively, these results demonstrate the promise of this technology to further the impact and affordability of gene therapy.

A pH-sensitive motif in an outer membrane protein activates bacterial membrane vesicle production

Outer membrane vesicles (OMVs) produced by Gram-negative bacteria have key roles in cell envelope homeostasis, secretion, interbacterial communication, and pathogenesis. The facultative intracellular pathogen Salmonella Typhimurium increases OMV production inside the acidic vacuoles of host cells by changing expression of its outer membrane proteins and modifying the composition of lipid A. However, the molecular mechanisms that translate pH changes into OMV production are not completely understood. Here, we show that the outer membrane protein PagC promotes OMV production through pH-dependent interactions between its extracellular loops and surrounding lipopolysaccharide (LPS). Structural comparisons and mutational studies indicate that a pH-responsive amino acid motif in PagC extracellular loops, containing PagC-specific histidine residues, is crucial for OMV formation. Molecular dynamics simulations suggest that protonation of histidine residues leads to changes in the structure and flexibility of PagC extracellular loops and their interactions with the surrounding LPS, altering membrane curvature. Consistent with that hypothesis, mimicking acidic pH by mutating those histidine residues to lysine increases OMV production. Thus, our findings reveal a mechanism for sensing and responding to environmental pH and for control of membrane dynamics by outer membrane proteins.

Environmentally dependent interactions shape patterns in gene content across natural microbiomes.

Sequencing surveys of microbial communities in hosts, oceans and soils have revealed ubiquitous patterns linking community composition to environmental conditions. While metabolic capabilities restrict the environments suitable for growth, the influence of ecological interactions on patterns observed in natural microbiomes remains uncertain. Here we use denitrification as a model system to demonstrate how metagenomic patterns in soil microbiomes can emerge from pH-dependent interactions. In an analysis of a global soil sequencing survey, we find that the abundances of two genotypes trade off with pH; nar gene abundances increase while nap abundances decrease with declining pH. We then show that in acidic conditions strains possessing nar fail to grow in isolation but are enriched in the community due to an ecological interaction with nap genotypes. Our study provides a road map for dissecting how associations between environmental variables and gene abundances arise from environmentally modulated community interactions.

A colloidal model for the equilibrium assembly and liquid-liquid phase separation of the reflectin A1 protein

Reflectin is an intrinsically disordered protein known for its ability to modulate the biophotonic camouflage of cephalopods based on its assembly-induced osmotic properties. Its reversible self-assembly into discrete, size-controlled clusters and condensed droplets are known to depend sensitively on the net protein charge, making reflectin stimuli-responsive to pH, phosphorylation, and electric fields. Despite considerable efforts to characterize this behavior, the detailed physical mechanisms of reflectin’s assembly are not yet fully understood. Here, we pursue a coarse-grained molecular understanding of reflectin assembly using a combination of experiments and simulations. We hypothesize that reflectin assembly and phase behavior can be explained from a remarkably simple colloidal model whereby individual protein monomers effectively interact via a short-range attractive and long-range repulsive (SA-LR) pair potential. We parameterize a coarse-grained SA-LR interaction potential for reflectin A1 from small-angle x-ray scattering measurements, and then extend it to a range of pH values using Gouy-Chapman theory to model monomer-monomer electrostatic interactions. The pH-dependent SA-LR interaction is then used in molecular dynamics simulations of reflectin assembly, which successfully capture a number of qualitative features of reflectin, including pH-dependent formation of discrete-sized nanoclusters and liquid-liquid phase separation at high pH, resulting in a putative phase diagram for reflectin. Importantly, we find that at low pH size-controlled reflectin clusters are equilibrium assemblies, which dynamically exchange protein monomers to maintain an equilibrium size distribution. These findings provide a mechanistic understanding of the equilibrium assembly of reflectin, and suggest that colloidal-scale models capture key driving forces and interactions to explain thermodynamic aspects of native reflectin behavior. Furthermore, the success of SA-LR interactions presented in this study demonstrates the potential of a colloidal interpretation of interactions and phenomena in a range of intrinsically disordered proteins.

PH-dependent interaction between acetaminophen and dioleoylphosphatidylcholine membranes

Abstract Acetaminophen may cause acute liver failure due to overdose. Understanding the processes of adsorption and incorporation of acetaminophen into cell membranes is crucial for elucidating the mechanisms of action and toxicity of acetaminophen. We investigated the interaction between acetaminophen and a model cell membrane (dioleoylphosphatidylcholine) using surface pressure–area isotherms. Acetaminophen was incorporated into the dioleoylphosphatidylcholine membrane under basic conditions, increasing the dioleoylphosphatidylcholine molecular area. The acetaminophen uptake was explained by the adsorption of hydroxide ions onto the lipid membrane and electrostatic interaction between acetaminophen and lipid molecules under basic conditions.

Impact of sulfur substitution on biotin binding affinity to streptavidin

In this study, we investigated how the replacement of the tetrahydrothiophene ring of biotin with either an oxolane or (methyl)pyrrolidine moiety may affect its molecular interactions, in an effort to identify alternative affinity ligands suitable for in vitro and in vivo applications in synthetic biology. Initial molecular dynamics (MD) simulations suggested the potential formation of a hydrogen bond between either the oxygen or nitrogen atom of the envisaged tetrahydroheteryl analogues and the Thr90 residue of streptavidin, mirroring the sulfur-centered hydrogen bond detected by the crystallographic analysis of the biotin-streptavidin interaction. Therefore, oxy-, aza-, and N-methylazabiotin were readily synthesized starting from chiral five- or six-carbon sugar precursors. Based on fluorescence-based titration experiments using the corresponding fluorescein conjugates, oxybiotin showed a binding behavior similar to biotin with streptavidin, while both amino analogues displayed lower binding capacities. Notably, azabiotin exhibited a pH-dependent interaction profile, demonstrating enhanced binding under acidic conditions but weaker binding under basic pH, which could be exploited for various purposes.

Cellular Neonatal Fc Receptor Recycling Efficiencies can Differentiate Target-Independent Clearance Mechanisms of Monoclonal Antibodies

In vivo clearance mechanisms of therapeutic monoclonal antibodies (mAbs) encompass both target-mediated and target-independent processes. Two distinct determinants of overall mAb clearance largely separate of target-mediated influences are non-specific cellular endocytosis and subsequent pH-dependent mAb recycling mediated by the neonatal Fc receptor (FcRn), where inter-mAb variability in the efficiency of both processes is observed. Here, we implemented a functional cell-based FcRn recycling assay via Madin-Darby canine kidney type II cells stably co-transfected with human FcRn and its light chain β2-microglobulin. Next, a series of pH-dependent internalization studies using a model antibody demonstrated proper function of the human FcRn complex. We then applied our cellular assays to assess the contribution of both FcRn and non-specific interactions in the cellular turnover for a panel of 8 clinically relevant mAbs exhibiting variable human pharmacokinetic behavior. Our results demonstrate that the interplay of non-specific endocytosis rates, pH-dependent non-specific interactions, and engagement with FcRn all contribute to the overall recycling efficiency of therapeutic monoclonal antibodies. The predictive capacity of our assay approach was highlighted by successful identification of all mAbs within our panel possessing clearance in humans greater than 5 mL/day/kg. These results demonstrate that a combination of cell-based in vitro assays can properly resolve individual mechanisms underlying the overall in vivo recycling efficiency and non-target mediated clearance of therapeutic mAbs.

PH-Dependent interactions between β-casein and blueberry anthocyanins based on typical thermal processing conditions: Stability, structural characterization, and binding mechanisms

The color and structural stability of blueberry anthocyanins (BAs) are significantly affected by pH changes during processing. In this study, we present the potential effects of the thermal stability, structural characterization, and binding mechanism of BAs with β-casein (β-CN) at pH 2, 4, and 6 for the first time. The pH was found significantly affect the ability of β-CN to protect BAs from thermal, vitamin C, and sucrose induced degradation. Scanning electron microscopy (SEM) resolved the microstructural differences of β-CN-BAs at varying pH values. Multispectral analysis showed that as pH increases, the electrostatic interaction between β-CN and the major monomer of anthocyanins Cyanidin-3-O-glucoside (C3G) are replaced by hydrogen bonding and van der Waals forces, and then electrostatic interactions are again the dominant force. In vitro gastrointestinal digestion confirmed that the protein stabilized C3G under varying pH conditions. Nuclear magnetic resonance (NMR) spectroscopy reveals the connection between the multiple equilibrium forms of C3G (hemiketal, cis-chalcone, and trans-chalcone) and β-CN on the formation of complexes between multiple equilibrium forms to specifically elucidate the mechanism of its stabilizing effect. Molecular docking simulation revealed the significance of electrostatic interaction and hydrogen bonding, where the highest binding match was achieved at pH 2, with a minimum binding energy. These interactions can effectively promote stable complexation of β-CN with C3G. These findings have practical implications for protein regulation of the relationship between the equilibrium forms of different anthocyanins and can guide the stabilization of anthocyanin-rich products.

Orientational Pathways during Protein Translocation through Polymer-Modified Nanopores.

Protein translocation through nanopores holds significant promise for applications in biotechnology, biomolecular analysis, and medicine. However, the interpretation of signals generated by the translocation of the protein remains challenging. In this way, it is crucial to gain a comprehensive understanding on how macromolecules translocate through a nanopore and to identify what are the critical parameters that govern the process. In this study, we investigate the interplay between protein charge regulation, orientation, and nanopore surface modifications using a theoretical framework that allows us to explicitly take into account the acid-base reactions of the titrable amino acids in the proteins and in the polyelectrolytes grafted to the nanopore surface. Our goal is to thoroughly characterize the translocation process of different proteins (GFP, β-lactoglobulin, lysozyme, and RNase) through nanopores modified with weak polyacids. Our calculations show that the charge regulation mechanism exerts a profound effect on the translocation process. The pH-dependent interactions between proteins and charged polymers within the nanopore lead to diverse free energy landscapes with barriers, wells, and flat regions dictating translocation efficiency. Comparison of different proteins allows us to identify the significance of protein isoelectric point, size, and morphology in the translocation behavior. Taking advantage of these insights, we propose pH-responsive nanopores that can load proteins at one pH and release them at another, offering opportunities for controlled protein delivery, separation, and sensing applications.

Engineering fibronectin-templated multi-component fibrillar extracellular matrices to modulate tissue-specific cell response

Cells assemble fibronectin, the major extracellular matrix (ECM) protein, into fibrillar matrices, which serve as 3D architectural scaffolds to provide, together with other ECM proteins tissue-specific environments. Although recent approaches enable to bioengineer 3D fibrillar fibronectin matrices in vitro, it remains elusive how fibronectin can be co-assembled with other ECM proteins into complex 3D fibrillar matrices that recapitulate tissue-specific compositions and cellular responses. Here, we introduce the engineering of fibrillar fibronectin-templated 3D matrices that can be complemented with other ECM proteins, including vitronectin, collagen, and laminin to resemble ECM architectures observed in vivo. For the co-assembly of different ECM proteins, we employed their innate fibrillogenic mechanisms including shear forces, pH-dependent electrostatic interactions, or specific binding domains. Through recapitulating various tissue-specific ECM compositions and morphologies, the large scale multi-composite 3D fibrillar ECM matrices can guide fibroblast adhesion, 3D fibroblast tissue formation, or tissue morphogenesis of epithelial cells. In other examples, we customize multi-composite 3D fibrillar matrices to support the growth of signal propagating neuronal networks and of human brain organoids. We envision that these 3D fibrillar ECM matrices can be tailored in scale and composition to modulate tissue-specific responses across various biological length scales and systems, and thus to advance manyfold studies of cell biological systems.

Multi-spectroscopic and molecular docking studies for the pH-dependent interaction of β-lactoglobulin with (−)-epicatechin gallate and/or piceatannol: Influence on antioxidant activity and stability

(−)-Epicatechin gallate (ECG) and piceatannol (PIC) are commonly polyphenols with excellent biological activities. β-Lactoglobulin (BLG) is a food-grade globule protein and its morphologies are sensitive to pH. This study used experimental and computational methods to determine the interaction of single or combined ECG and PIC with BLG at different pHs. The static quenching process was determined through fluorescence and ultraviolet–visible spectroscopy. Compared with ECG, PIC could significantly bind to BLG with higher affinity. Their binding affinity for BLG with different morphologies followed the tendency of monomer > dimer > tetramer. The negative contribution of van der Waals forces, electrostatic interactions, and hydrogen bonds to ΔHo exceeded the positive contribution of hydrophobic interactions in the spontaneous and exothermic process. The reduced binding affinity in the ternary systems demonstrated the competitive binding between ECG and PIC on BLG, and the hinder effect of ECG or PIC was enhanced with increasing pH. Molecular docking studies revealed the same binding sites of ECG and PIC on various conformations of BLG and identical driven forces as thermodynamic results. Tryptophan and tyrosine were the main participators in the BLG + ECG and BLG + PIC systems, respectively. The conformational changes in the binary and ternary systems could be ascertained through synchronous fluorescence, circular dichroism, and dynamic light scattering. Furthermore, the effects of pH and BLG encapsulation on the antioxidant capacity and stability of ECG or PIC were also implemented. ECG or PIC was the most stable in the (BLG + PIC) + ECG system at pH 6.0. This study could clarify the interaction mechanism between ECG/PIC and BLG and elucidate the pH effect on their binding information. The results will provide basic support for their usage in food processing and applications.

Confinement and Polarity Effects on the Peptide Packing Density on Mesoporous Silica Nanoparticles.

The adsorption of cationic peptide JM21 onto different mesoporous silica nanoparticles (MSNs) from an aqueous solution was studied as a function of pH. In agreement with the literature, the highest loading degrees could be achieved at pH close to the isoelectric point of the peptide where the peptide-peptide repulsion is minimum. However, mesopore size, mesopore geometry, and surface polarity all had an influence on the peptide adsorption in terms of both affinity and maximum loading at a given pH. This adsorption behavior could largely be explained by a combination of pH-dependent electrostatic interactions and confinement effects. It is demonstrated that hydrophobic interactions enhance the degree of peptide adsorption under pH conditions where the electrostatic attraction was absent in the case of mesoporous organosilica nanoparticles (MONs). The lower surface concentration of silanol groups for MON led to a lower level of peptide adsorption under optimum pH conditions compared to all-silica particles. Finally, the study confirmed the protective role of MSNs in preserving the biological activity of JM#21 against enzymatic degradation, even for large-pore MSNs, emphasizing their potential as nanocarriers for therapeutic peptides. By integrating experimental findings with theoretical modeling, this research elucidates the complex interplay of factors that influence peptide-silica interactions, providing vital insights for optimizing peptide loading and stabilization in biomedical applications.

Unravelling pH/pKa influence on pH-responsive drug carriers: Insights from ibuprofen-silica interactions and comparative analysis with carbon nanotubes, sulfasalazine, and alendronate

This study employs density functional theory to explore the interaction between ibuprofen (IBU) and silica, emphasizing the influence of the trimethylsilyl (TMS) functional group for designing pH-responsive drug carriers. The surface (S) and drug (D) molecules' neutral (0) or deprotonated (−1) states were taken into consideration during the investigation. The likelihood of these states was determined based on the pKa values and the desired pH conditions. To calculate the pH-dependent interaction energy (EintpH), four different situations have been identified: S0D0, S0D−1, S−1D0, and S−1D−1.The electrostatic component of interaction energy aligns favorably with its theoretical value in both the Debye-Hückel and Grahame models. The investigation has gathered first-hand experimental data on the drug loading and release of pH-responsive mesoporous silica nanoparticles. Effective drug loading was observed in the acidic environment of the stomach (pH 2–5), followed by a release in the slightly basic to neutral pH of the small intestine (pH 7.4), These findings align with existing literature. The results revealed horizontal drug adherence on silica surfaces, improving binding capabilities. Comparisons were made with combinations involving carboxylated carbon nanotubes and ibuprofen, silica, and sulfasalazine, and silica and alendronate, exploring drug loading/release dynamics associated with positive/negative interaction energies. The investigation, supported by experimental data, contributes valuable insights into pH-responsive mesoporous silica nanoparticles, offering new design possibilities for drug carriers.

Insights into the pH-dependent interactions of sulfadiazine antibiotic with soil particle models

Sulfonamide antibiotics (SAs) are widely used as a broad-spectrum antibiotic, leading to global concerns due to their potential soil accumulation and subsequent effects on ecosystems. SAs often exhibit remarkable environmental persistence, necessitating further investigation to uncover the ultimate destiny of these molecules. In this work, molecular dynamics simulations combined with complementary quantum chemistry calculations were employed to investigate the influence of pH on the behavior of sulfadiazine (SDZ, a typical SAs) in soil particle models (silica, one of the main components of soil). Meanwhile, the quantification of SDZ molecules aggregation potential onto silica was further extended. SDZ molecules tend to form a monolayer on the soil surface under acidic conditions while forming aggregated adsorption on the surface under neutral conditions. Due to the hydrophilicity of the silica, multiple hydration layers would form on its surface, hindering the further adsorption of SDZ molecules on its surface. The calculated soil-water partition coefficient (Psoil/water) of SDZ+ and SDZ were 9.01 and 7.02, respectively. The adsorption evaluation and mechanisms are useful in controlling the migration and transformation of SAs in the soil environment. These findings provide valuable insights into the interactions between SDZ and soil components, shedding light on its fate and transport in the environment.

PH-dependent electrostatic interactions between enzymes and nanoparticles in Pickering emulsions – Influence on activity and droplet size

Pickering emulsions (PE) are liquid-liquid systems that are stabilized by solid (nano)particles at the fluid interface. They offer higher stability, easier separation and lower toxicity compared to classical emulsions stabilized by surfactants. Common applications range from food science to drug delivery. In the last decade they have become of more interest in the field of multiphasic biocatalysis. First, this study aims to present the influence of pH, salt strength and particle charge on enzyme activity. The different behavior of two lipases (CaLA and CRL) is shown. While the activity optimum of CaLA changed from pH 6.5 to pH 5 by applying particles with negative instead of positive surface charge, the CRL activity optimum stayed at pH 5–5.5. This enables particle charge as an additional parameter to optimize biocatalytic reactions in PEs. Second, the resulting drop sizes were measured to elucidate further interactions between the enzymes and particles in PEs. Drop sizes in PEs prepared with CaLA were not influenced by pH, but increased for positively and decreased for negatively charged particles upon the addition of CaLA. Electrostatic attraction between particles and CRL increased the droplet diameter from 10 μm up to 30 μm and therefore destabilized the PE for both particle types.

PH-dependent interactions of coacervate-forming histidine-rich peptide with model lipid membranes

Peptide-based liquid droplets (coacervates) produced by spontaneous liquid-liquid phase separation (LLPS), have emerged as a promising class of drug delivery systems due to their high entrapping efficiency and the simplicity of their formulation. However, the detailed mechanisms governing their interaction with cell membranes and cellular uptake remain poorly understood. In this study, we investigated the interactions of peptide coacervates composed of HBpep—peptide derived from the histidine-rich beak proteins (HBPs) of the Humboldt squid—with model cellular membranes in the form of supported lipid bilayers (SLBs). We employed quartz crystal microbalance with dissipation monitoring (QCM-D), neutron reflectometry (NR) and atomistic molecular dynamics (MD) simulations to reveal the nature of these interactions in the absence of fluorescent labels or tags. HBpep forms small oligomers at pH 6 whereas it forms µm-sized coacervates at physiological pH. Our findings reveal that both HBpep oligomers and HBpep-coacervates adsorb onto SLBs at pH 6 and 7.4, respectively. At pH 6, when the peptide carries a net positive charge, HBpep oligomers insert into the SLB, facilitated by the peptide’s interactions with the charged lipids and cholesterol. Importantly, however, HBpep coacervate adsorption at physiological pH, when it is largely uncharged, is fully reversible, suggesting no significant lipid bilayer rearrangement. HBpep coacervates, previously identified as efficient drug delivery vehicles, do not interact with the lipid membrane in the same manner as traditional cationic drug delivery systems or cell-penetrating peptides. Based on our findings, HBpep coacervates at physiological pH cannot cross the cell membrane by a simple passive mechanism and are thus likely to adopt a non-canonical cell entry pathway.

PH-dependent interactions of biologically important metal ions with hen egg white lysozyme based on its hydration properties: Thermodynamic and mechanistic insights

Importance of metal ion selectivity in biomolecules and their key role in proteins are widely explored. However, understanding the thermodynamics of how hydrated metal ions alter the protein hydration and their conformation is also important. In this study, the interaction of some biologically important Ca2+, Mn2+, Co2+, Cu2+, and Zn2+ ions with hen egg white lysozyme at pH 2.1, 3.0, 4.5 and 7.4 has been investigated. Intrinsic fluorescence studies have been employed for metal ion-induced protein conformational changes analysis. Thermostability based on protein hydration has been investigated using differential scanning calorimetry (DSC). Thermodynamic parameters emphasizing on metal ion-protein binding mechanistic insights have been well discussed using isothermal titration calorimetry (ITC). Overall, these experiments have reported that their interactions are pH-dependent and entropically driven. This research also reports the strongly hydrated metal ions as water structure breaker unlike osmolytes based on DSC studies. These experimental results have highlighted higher concentrations of different metal ions effect on the protein hydration and thermostability which might be helpful in understanding their interactions in aqueous solutions.