Complete Denaturation Research Articles

Model for electrical conductivity of muscle meat during Ohmic heating

A model is presented for predicting the electrical conductivity of muscle meat, which can be used for the evaluation of Ohmic heating. The model computes the conductivity as a function of composition, temperature and microstructure. The muscle meat is thought to be composed of protein, water, salt. Concerning the microstructure, the model takes into account the muscle fiber orientation with respect to the electric field, and the development of drip channels due to protein denaturation. The model includes a description of the protein denaturation kinetics. The model has been validated for different types of meat, varying in composition and heating rate. The submodel for protein denaturation is validated using independent DSC measurements. For meats heated faster than 20° per minute, the conductivity is a linear function of temperature - due to the absence of protein denaturation, and thus drip channel formation. If meat is heated slower than 10° per minute the conductivity is showing non-linear behaviour, with a significant decrease at temperatures above 70 degrees Celsius. This decrease is explained by the action of the complete protein denaturation. Our study shows that if Ohmic heating of meat is performed at fast rates, there is a large potential to retain most of its moisture during heating.

Changes of protein profiles in pork and beef meat caused by high hydrostatic pressure treatment

In the experiments pork loin and beef sirloin were treated by pressures of 100 to 600 MPa by 100 MPa steps for 5 min. Colour changes of samples and the changes of proteins were investigated. The latter were examined with isoelectric focusing and SDS polyacrylamide gel electrophoresis. We found that myoglobin behaved completely differently in case of the two different species. Myoglobin has mostly lost its native state at 300 MPa pressure in case of pork, but the beef myoglobin could remain native even up to 500 MPa. The treatment at 300 MPa or higher pressure values caused almost complete aggregation and denaturation in case of pork and beef proteins. The results of SDS-PAGE and the colour measurement confirmed this finding.

A novel protocol for enzymatic digestion based on covalent binding by protein immobilization.

The process of protein digestion is a critical step for successful protein identification in proteomic analysis. Many efforts have been dedicated to enhancing the digestion efficiency for sufficient digestion. Among these approaches, protein complete denaturation with denaturants is a common process for better digestion. However, the removal of denaturants was tedious or would cause protein loss and other problems. In this work, a feasible digestion approach, immobilized protein digestion (IPD), based on covalent binding has been developed. Proteins can be completely denatured and immobilized on the surface of functional materials by covalent binding to form a monolayer. Subsequently, varieties of denaturants or contaminants would be removed thoroughly by washing. To achieve fast immobilization and high digestion efficiency, different functional materials and denaturants were selected. Compared with traditional in-solution digestion, the method achieved a prominent increase in identified peptides numbers and sequence coverage of proteins. Data analysis also showed that covalent binding could evidently decrease enzymatic missed cleavage for various protein sequences. Furthermore, possible peptide losses due to covalent binding were also investigated. Also, it has been proved to be efficient for complex biological sample digestion. Graphical abstract Workflow of the IPD method, including protein denaturation, immobilization, digestion, and identification.

Cosolvent and Crowding Effects on the Temperature and Pressure Dependent Conformational Dynamics and Stability of Globular Actin.

Actin can be found in nearly all eukaryotic cells and is responsible for many different cellular functions. The polymerization process of actin has been found to be among the most pressure sensitive processes in vivo. In this study, we explored the effects of chaotropic and kosmotropic cosolvents, such as urea and the compatible osmolyte trimethylamine-N-oxide (TMAO), and, to mimic a more cell-like environment, crowding agents on the pressure and temperature stability of globular actin (G-actin). The temperature and pressure of unfolding as well as thermodynamic parameters upon unfolding, such as enthalpy and volume changes, have been determined by fluorescence spectroscopy over a wide range of temperatures and pressures, ranging from 10 to 80 °C and from 1 to 3000 bar, respectively. Complementary high-pressure NMR studies revealed additional information on the existence of native-like conformational substates of G-actin as well as a molten-globule-like state preceding the complete pressure denaturation. Different from the chaotropic agent urea, TMAO increases both the temperature and pressure stability for the protein most effectively. The Gibbs free energy differences of most of the native substates detected are not influenced significantly by TMAO. In mixtures of these osmolytes, urea counteracts the stabilizing effect of TMAO to some extent. Addition of the crowding agent Ficoll increases the temperature and pressure stability even further, thereby allowing sufficient stability of the protein at temperature and pressure conditions encountered under extreme environmental conditions on Earth.

Effects of extrusion cooking on the chemical composition and functional properties of dry common bean powders

The impact of extrusion cooking on the chemical composition and functional properties of bean powders from four common bean varieties was investigated. The raw bean powders were extruded under eight different conditions, and the extrudates were then dried and ground (particle size⩽0.5mm). Compared with corresponding non-extruded (raw) bean powders (particle size⩽0.5mm), the extrusion treatments did not substantially change the protein and starch contents of the bean powders and showed inconsistent effects on the sucrose, raffinose and stachyose contents. The extrusion cooking did cause complete starch gelatinization and protein denaturation of the bean powders and thus changed their pasting properties and solvent-retention capacities. The starch digestibilities of the cooked non-extruded and cooked extruded bean powders were comparable. The extruded bean powders displayed functional properties similar to those of two commercial bean powders.

Physico-Chemical Characterization of the Interaction of Red Fluorescent Protein-DsRed With Thin Silica Layers.

The Discosoma recombinant red fluorescent (DsRed) protein is the latest member of the family of fluorescent proteins. It holds great promise for applications in biotechnology and cell biology. However, before being used for rational engineering, knowledge on the behavior of DsRed and the underlying mechanisms relating its structural stability and adsorption properties on solid surfaces is highly demanded. The physico-chemical analysis performed in this study reveals that the interaction of DsRed with SiO2 surfaces does not lead to complete protein denaturation after adsorption and dehydration. Nevertheless, the photoluminescence emission of dehydrated DsRed small droplets was found to be slightly red-shifted, peaking at 590 nm. The measured contact angles of droplets containing different concentration of DsRed proteins determine the interaction as hydrophilic one, however with larger contact angles for larger DsRed concentrations. The DsRed protein behavior is not pH-dependent with respect of the contact angle measurements, in agreement with previously reported studies.

The thermal and storage stability of bovine haemoglobin by ultraviolet–visible and circular dichroism spectroscopies

The effects of temperature, pH and long-term storage on the secondary structure and conformation changes of bovine haemoglobin (bHb) were studied using circular dichroism (CD) and ultraviolet--visible (UV–vis) spectroscopies. Neural network software was used to deconvolute the CD data to obtain the fractional content of the five secondary structures. The storage stability of bHb solutions in pH 6, 7 and 8 buffers was significantly higher at 4°C than at 23°C for the first 3 days. A complete denaturation of bHb was observed after 40 days irrespective of storage temperature or pH. The bHb solutions were also exposed to heating and cooling cycles between 25 and 65°C and structural changes were followed by UV–vis and CD spectroscopies. These experiments demonstrated that α-helix content of bHb decreased steadily with the increasing temperature above 35°C at all pH values. The loss in α-helicity and gain in random coil conformations was pH-dependent and the greatest under alkaline conditions. Furthermore, there was minimal recovery of the secondary structure content upon cooling to 25°C. The use of bHb as a model drug is very common and this study elucidates the significance of storage and processing conditions on its stability.

When Enzymes and Green Surfactants Meet

Surfactants and proteins coexist in many different industries, ranging from detergents to pharmaceutics. Surfactant-protein interactions are multi-faceted and can range from limited interactions, as typically observed with nonionic surfactants, to complete denaturation of the protein, more typical of anionic surfactants such as sodium dodecyl sulphate (SDS). Denaturation of proteins and enzymes can be driven by both monomeric and micellar surfactant interactions [1].Biosurfactants are produced by microorganisms and have promising properties. They have strong interfacial properties [2] that are promising for industrial use. Despite growing interest, their impact on protein and enzyme stability remains at an early stage of investigation. Studies with model proteins suggest a relatively mild interaction with slow inactivation kinetics [3]. Membrane proteins can be refolded in their presence [4].Here we report interactions between industrial/detergent enzymes and two glycolipid-biosurfactants: Anionic rhamnolipid (RL) produced in Pseudomonas aeruginosa and mixed anionic/nonionic sophorolipid (SL) produced in Candida bombicola. RL and SL both act as mild biosurfactants that despite their anionic charge are compatible with industrial enzymes. The lipase from Thermomyces lanuginosus (TlL) shows pH dependent interactions. At pH 8 we do not detect interactions between TlL and RL or SL, with only minor perturbations to stability and activity. However, lowering pH from 8 to 6 favor stronger interactions between TlL and RL/SL that strongly stabilizes TlL and opens the active site with a 3- to 6-fold boost to activity. We use SAXS to qualitatively determine formation of complexes between TlL and SL and RL micelles at pH 6.1.Otzen, D.E., P. Sehgal, and P. Westh. J Colloid Interface Sci, 2009.329(2): p.273-83.2.Nguyen, T.T. and D.A. Sabatini. Int J Mol Sci, 2011.12(2): p.1232-44.3.Andersen, K.K. and D.E. Otzen. Biochim Biophys Acta, 2014.1844: p.2338-2345.4.Andersen, K.K. and D.E. Otzen. FEBS Lett, 2014.588: p.1955-60.

Modeling and simulation of protein-surface interactions: achievements and challenges.

Understanding protein-inorganic surface interactions is central to the rational design of new tools in biomaterial sciences, nanobiotechnology and nanomedicine. Although a significant amount of experimental research on protein adsorption onto solid substrates has been reported, many aspects of the recognition and interaction mechanisms of biomolecules and inorganic surfaces are still unclear. Theoretical modeling and simulations provide complementary approaches for experimental studies, and they have been applied for exploring protein-surface binding mechanisms, the determinants of binding specificity towards different surfaces, as well as the thermodynamics and kinetics of adsorption. Although the general computational approaches employed to study the dynamics of proteins and materials are similar, the models and force-fields (FFs) used for describing the physical properties and interactions of material surfaces and biological molecules differ. In particular, FF and water models designed for use in biomolecular simulations are often not directly transferable to surface simulations and vice versa. The adsorption events span a wide range of time- and length-scales that vary from nanoseconds to days, and from nanometers to micrometers, respectively, rendering the use of multi-scale approaches unavoidable. Further, changes in the atomic structure of material surfaces that can lead to surface reconstruction, and in the structure of proteins that can result in complete denaturation of the adsorbed molecules, can create many intermediate structural and energetic states that complicate sampling. In this review, we address the challenges posed to theoretical and computational methods in achieving accurate descriptions of the physical, chemical and mechanical properties of protein-surface systems. In this context, we discuss the applicability of different modeling and simulation techniques ranging from quantum mechanics through all-atom molecular mechanics to coarse-grained approaches. We examine uses of different sampling methods, as well as free energy calculations. Furthermore, we review computational studies of protein-surface interactions and discuss the successes and limitations of current approaches.

Optimization of microchip-based electrophoresis for monoclonal antibody product quality analysis revealed needs for extra surfactants during denaturation

Microchip-based electrophoresis has gained increasing popularity in biopharmaceutical development and testing laboratories because of its automation and rapid analysis capabilities. One application of microchip-based electrophoresis is the assessment of size-based variants for product purity analysis. However, monoclonal antibodies analyzed by this technique sometimes exhibited different electrophoretic behaviors. In this study, when three IgG1 and five IgG4 were analyzed using microchip-based electrophoresis under reducing conditions, one of the IgG1s, denoted as mAb1, exhibited an atypical profile attributed to its specific heterogeneity resulting in separation of its heavy chain into two main species. During investigation of the atypical profile, several parameters that were critical to optimal resolution were evaluated, and the data pointed toward incomplete denaturation of mAb1 due to lack of sufficient surfactant in the vendor provided sample buffer (0.7% surfactant). Denaturation studies demonstrated that, although typical antibody profiles could be achieved at 0.7% surfactant for most antibodies analyzed, five out of eight antibodies were not fully denatured until the surfactant concentration reached 0.9% or higher, and mAb1 required a surfactant concentration of 1.3% for complete denaturation. Molecular modeling analysis revealed features in surface charge, hydrophobicity, and structure from mAb1 that led to its unique surfactant concentration-dependent electrophoretic behaviors observed. The optimized method was further evaluated for specificity, linearity, precision, and limit of quantitation for mAb1, and compared with that of conventional CE-SDS.

Isolating dividing neural and brain tumour cells for gene expression profiling

BackgroundThe characterisation of dividing brain cells is fundamental for studies ranging from developmental and stem cell biology, to brain cancers. Whilst there is extensive anatomical data on these dividing cells, limited gene transcription data is available due to technical constraints. New methodWe focally isolated dividing cells whilst conserving RNA, from culture, primary neural tissue and xenografted glioma tumours, using a thymidine analogue that enables gene transcription analysis. Results5-ethynyl-2-deoxyuridine labels the replicating DNA of dividing cells. Once labelled, cultured cells and tissues were dissociated, fluorescently tagged with a revised click chemistry technique and the dividing cells isolated using fluorescence-assisted cell sorting. RNA was extracted and analysed using real time PCR. Proliferation and maturation related gene expression in neurogenic tissues was demonstrated in acutely and 3 day old labelled cells, respectively. An elevated expression of marker and pathway genes was demonstrated in the dividing cells of xenografted brain tumours, with the non-dividing cells showing relatively low levels of expression. Comparison with existing methodBrdU “immune-labelling”, the most frequently used protocol for detecting cell proliferation, causes complete denaturation of RNA, precluding gene transcription analysis. This EdU labelling technique, maintained cell integrity during dissociation, minimized copper exposure during labelling and used a cell isolation protocol that avoided cell lysis, thus conserving RNA. ConclusionsThe technique conserves RNA, enabling the definition of cell proliferation-related changes in gene transcription of neural and pathological brain cells in cells harvested immediately after division, or following a period of maturation.

Heterogeneous side chain conformation highlights a network of interactions implicated in hysteresis of the knotted protein, minimal tied trefoil

Hysteresis is a signature for a bistability in the native landscape of a protein with significant transition state barriers for the interconversion of stable species. Large global stability, as in GFP, contributes to the observation of this rare hysteretic phenomenon in folding. The signature for such behavior is non-coincidence in the unfolding and refolding transitions, despite waiting significantly longer than the time necessary for complete denaturation. Our work indicates that hysteresis in the knotted protein, the minimal tied trefoil from Thermotoga maritma (MTTTm), is mediated by a network of side chain interactions within a tightly packed core. These initially identified interactions include proline 62 from a tight β-like turn, phenylalanine 65 at the beginning of the knotting loop, and histidine 114 that initiates the threading element. It is this tightly packed region and the knotting element that we propose is disrupted with prolonged incubation in the denatured state, and is involved in the observed hysteresis. Interestingly, the disruption is not linked to backbone interactions, but rather to the packing of side chains in this critical region.

Insights into the unfolding pathway and identification of thermally sensitive regions of phytase from Aspergillus niger by molecular dynamics simulations.

Thermal stability is of great importance in the application of commercial phytases. Phytase A (PhyA) is a monomeric protein comprising twelve α-helices and ten β-sheets. Comparative molecular dynamics (MD) simulations (at 310, 350, 400, and 500K) revealed that the thermal stability of PhyA from Aspergillus niger (A. niger) is associated with its conformational rigidity. The most thermally sensitive regions were identified as loops 8 (residues 83-106), 10 (161-174), 14 (224-230), 17 (306-331), and 24 (442-444), which are present on the surface of the protein. It was observed that solvent-exposed loops denature before or show higher flexibility than buried residues. We observed that PhyA begins to unfold at loops 8 and 14, which further extends to loop 24 at the C-terminus. The intense movement of loop 8 causes the helix H2 and beta-sheet B3 to fluctuate at high temperature. The high flexibility of the H2, H10, and H12 helices at high temperature resulted in complete denaturation. The high mobility of loop 14 easily transfers to the adjacent helices H7, H8, and H9, which fluctuate and partially unfold at high temperature (500K). It was also observed that the salt bridges Asp110-Lys149, Asp205-Lys277, Asp335-Arg136, Asp416-Arg420, and Glu387-Arg400 are important influences on the structural stability but not the thermostability, as the lengths of these salt bridges did not increase with rising temperature. The salt bridges Glu125-Arg163, Asp299-Arg136, Asp266-Arg219, Asp339-Lys278, Asp335-Arg136, and Asp424-Arg428 are all important for thermostability, as the lengths of these bridges increased dramatically with increasing temperature. Here, for the first time, we have computationally identified the thermolabile regions of PhyA, and this information could be used to engineer novel thermostable phytases. Numerous homologous phytases of fungal as well as bacterial origin are known, and these homologs show high sequence similarity. Our findings could prove useful in attempts to increase the thermostability of homologous phytases via protein engineering.

Thermal denaturation behavior of collagen fibrils in wet and dry environment.

We have developed a new minimally invasive technique--integrated low-level energy adhesion technique (ILEAT)--which uses heat, pressure, and low-frequency vibrations for binding living tissues. Because the adhesion mechanism of the living tissues is not fully understood, we investigated the effect of thermal energy on the collagen structure in living tissues using ILEAT. To study the effect of thermal energy and heating time on the structure of the collagen fibril, samples were divided in two categories-wet and dry. Further, atomic force microscopy was used to analyze the collagen fibril structure before and after heating. Results showed that collagen fibrils in water denatured after 1 minute at temperatures higher than 80 °C, while partial denaturation was observed at temperatures of 80 °C and a heating time of 1 min. Furthermore, complete denaturation was achieved after 90 min, suggesting that the denaturation rate is temperature and time dependent. Moreover, the collagen fibrils in dry condition maintained their native structure even after being heated to 120 °C for 90 min in the absence of water, which specifically suppressed denaturation. However, partial denaturation of collagen fibrils could not be prevented, because this determines the adhesion between the collagen molecules, and stabilizes tissue bonding.

Effect of Acid Treatment on Interfacial and Foam Properties of Soy Proteins

Abstract: The goal of the present work was to study the effects of acid treatment on the foaming properties of a soybean protein isolate (SPI) and its fractions, glycinin (11S) and β-conglycinin (7S). The structural characteristics, interfacial properties, foaming capacity and stability of the treated proteins were studied. Results from surface hydrophobicity and differential scanning calorimetry (DSC) showed that the acid treatment caused the complete denaturation of 11S and a partial denaturation of 7S. This protein unfolding affected their interfacial properties, which led to an improvement in the foaming properties of both protein fractions and isolate. Treated 7S showed the best behavior in the rearrangement process, probably due to its smaller size and its modified structural characteristics. All treated proteins showed stronger interfacial films. The foams of treated proteins were destabilized mostly due to gravitational drainage rather than Ostwald ripening. Key words: Soy proteins, foams, gravitational drainage, Ostwald ripening.

The Influence of Heat Treatment in Liquid Whey at Various pH on Immunoglobulin G and Lactoferrin from Yak and Cows’ Colostrum/Milk

Yak milk is gaining popularity yearly, as a source of nutritious, immune, less likely to cause allergies than cows’ milk and a means of generating income, though ranked behind bovine milk in China. The main objective of the research was to compare the Immunoglobulin G (IgG) and Lactoferrin (LF) concentrations of liquid whey from Yak and dairy Cows’ colostrum or milk. Thereafter, the thermal stability of liquid whey was evaluated by measuring the concentration change in IgG and LF using an ELISA technique, as influenced by temperature and pH after centrifugation. IgG and LF concentrations in liquid whey from colostrums both in yak and cow were significantly higher (p 0.05) between yak and cow’s milk, as well as between yak and cow’s colostrum. Also, LF content was not significant different between yak and cow milk. However, LF concentration was significantly higher in cows’ colostrum than in yak. With reference to IgG content, yak breed observed to produce high quality colostrum for all samples tested compared to cows. The extent of IgG and LF denaturation confirmed to increase with increased protein concentration, temperature and pH change near to pI. Colostral IgG and LF were more denatured compared to milk in both yak and dairy cows. There was significant (p<0.05) influence of pH changes resulted in either partial or complete denaturation or increased tendency to aggregate, which was removed during centrifugation. Liquid whey less affected at lower pH and at mild heat temperatures.

Proteinaceous α-amylase inhibitors of Setaria italica Linn (Co-6) and its effect on α-amylase from human saliva and Bacillus sp

The specific α-amylase inhibitory activity of Setaria italica Linn (Co-6) was determined by using heat-treated and ammonium sulphate precipitated fractions of α-amylase isolated from Bacillus sp. The α-amylase inhibitor from S. italica Linn (Co-6) was precipitated with 1.0 M fraction of ammonium sulphate and used for further purification and characterization. To overcome the endogenous α-amylase activity, the extract was subjected to heat treatment at 60oC. At temperature above 85o C, complete denaturation of α-amylase inhibitory activity of the millet extract was evidenced. The results of the study suggest the use of S. italica Linn (Co-6) flour, which exhibits significant of α-amylase inhibitory activity, as a dose dependent diet for Non-Insulin dependent diabetes mellitus.

Identification and Validation of Genetic Variants that Influence Transcription Factor and Cell Signaling Protein Levels

Many genetic variants associated with human disease have been found to be associated with alterations in mRNA expression. Although it is commonly assumed that mRNA expression changes will lead to consequent changes in protein levels, methodological challenges have limited our ability to test the degree to which this assumption holds true. Here, we further developed the micro-western array approach and globally examined relationships between human genetic variation and cellular protein levels. We collected more than 250,000 protein level measurements comprising 441 transcription factor and signaling protein isoforms across 68 Yoruba (YRI) HapMap lymphoblastoid cell lines (LCLs) and identified 12 cis and 160 trans protein level QTLs (pQTLs) at a false discovery rate (FDR) of 20%. Whereas up to two thirds of cis mRNA expression QTLs (eQTLs) were also pQTLs, many pQTLs were not associated with mRNA expression. Notably, we replicated and functionally validated a trans pQTL relationship between the KARS lysyl-tRNA synthetase locus and levels of the DIDO1 protein. This study demonstrates proof of concept in applying an antibody-based microarray approach to iteratively measure the levels of human proteins and relate these levels to human genome variation and other genomic data sets. Our results suggest that protein-based mechanisms might functionally buffer genetic alterations that influence mRNA expression levels and that pQTLs might contribute phenotypic diversity to a human population independently of influences on mRNA expression.

How a protein can remain stable in a solvent with high content of urea: insights from molecular dynamics simulation of Candida antarctica lipase B in urea : choline chloride deep eutectic solvent.

Deep eutectic solvents (DESs) are utilized as green and inexpensive alternatives to classical ionic liquids. It has been known that some of DESs can be used as solvent in the enzymatic reactions to obtain very green chemical processes. DESs are quite poorly understood at the molecular level. Moreover, we do not know much about the enzyme microstructure in such systems. For example, how some hydrolase can remain active and stable in a deep eutectic solvent including 9 M of urea? In this study, the molecular dynamics of DESs as a liquid was simulated at the molecular level. Urea : choline chloride as a well-known eutectic mixture was chosen as a model DES. The behavior of the lipase as a biocatalyst was studied in this system. For comparison, the enzyme structure was also simulated in 8M urea. The thermal stability of the enzyme was also evaluated in DESs, water, and 8M urea. The enzyme showed very good conformational stability in the urea : choline chloride mixture with about 66% urea (9 M) even at high temperatures. The results are in good agreement with recent experimental observations. In contrast, complete enzyme denaturation occurred in 8M urea with only 12% urea in water. It was found that urea molecules denature the enzyme by interrupting the intra-chain hydrogen bonds in a "direct denaturation mechanism". However, in a urea : choline chloride deep eutectic solvent, as a result of hydrogen bonding with choline and chloride ions, urea molecules have a low diffusion coefficient and cannot reach the protein domains. Interestingly, urea, choline, and chloride ions form hydrogen bonds with the surface residues of the enzyme which, instead of lipase denaturation, leads to greater enzyme stability. To the best of our knowledge, this is the first study in which the microstructural properties of a macromolecule are examined in a deep eutectic solvent.

The stability of insulin in the presence of short alkyl chain imidazolium-based ionic liquids

Fibril or aggregation formation in insulin (In) has been a subject of severe biomedical and biotechnological complications. The search for a novel solvent/co-solvent that can provide long term stabilization for In monomeric form has not been completed yet. In this quest, for the first time we have successfully explored the stability of the monomeric form of In in the presence of ammonium-based ionic liquids (ILs) [A. Kumar and P. Venkatesu, RSC Adv., 2013, 3, 362–367]. Further, in continuation, in this study, we have established the stability of In in the presence of imidazolium-based ILs with different anions. These anions represent the Hofmeister series of anions of ILs. In this regard, we have carried out UV-vis, fluorescence, circular dichroism spectral analysis and dynamic light scattering (DLS) measurements of In in various concentrations of these ILs. Our experimental findings reveal that Br− and Cl− ILs stabilized the native state while the rest of the ILs with anions such as SCN−, HSO4−, CH3COO− and I− were denaturants for the In. Further, the results show that IL–In interactions are difficult to classify on the basis of the Hofmeister series, as bromide containing ILs show more stabilizing properties on the In structure. The disulfide bonds were almost intact in the presence of Br− IL as compared to Cl− and the rest of the Hofmeister anions. On the other hand, a strongly hydrated kosmotropic anion like HSO4− interacts with the structure of In, leading it towards complete denaturation of the In structure. Additionally, all ILs failed to protect the native state of In with increasing temperature.