An "Onion-like" Model of Protein Unfolding: Collective versus Site Specific Approaches.

Approximating protein unfolding by an all-or-none cooperative event is a convenient assumption that can provide precious global information on protein stability. It is however quickly emerging that the scenario is far more complex and that global denaturation curves often hide a rich heterogeneity of states that are largely probe dependent. In this review, we revisit the importance of gaining site-specific information on the unfolding process. We focus on nuclear magnetic resonance, as this is the main technique able to provide site-specific information. We review historical and most modern approaches that have allowed an appreciable advancement of the field of protein folding. We also demonstrate how unfolding is a reporter dependent event, suggesting the outmost importance of selecting the reporter carefully.

Effects of heat treatment on the antigenicity, antigen epitopes, and structural properties of ß-conglycinin.

In this study, the effects of heat treatment on antigenicity, antigen epitopes, and structural changes in ß-conglycinin were investigated. Results showed that the IgG (Immunoglobulin G) binding capacity of heated protein was inhibited with increased temperature, although IgE (Immunoglobulin E) binding capacity increased. Linear antigen epitopes generally remained intact during heat treatment. After heat treatment, ß-conglycinin was more easily hydrolyzed by digestive enzymes, and a large number of linear epitopes was destroyed. In addition, heat denaturation of ß-conglycinin led to the formation of protein aggregates and reduction of disulfide bonds. The contents of random coils and ß-sheet of heated ß-conglycinin decreased, but the contents of ß-turn and α-helix increased. Moreover, the protein structure of heated ß-conglycinin unfolded, more hydrophobic regions were exposed, and the tertiary structure of ß-conglycinin was destroyed. Heat treatment affected the antigenicity and potential sensitization of ß-conglycinin by changing its structure.

Probing the Effects of Heterogeneous Oxidative Modifications on the Stability of Cytochrome c in Solution and in the Gas Phase.

Covalent modifications by reactive oxygen species can modulate the function and stability of proteins. Thermal unfolding experiments in solution are a standard tool for probing oxidation-induced stability changes. Complementary to such solution investigations, the stability of electrosprayed protein ions can be assessed in the gas phase by collision-induced unfolding (CIU) and ion-mobility spectrometry. A question that remains to be explored is whether oxidation-induced stability alterations in solution are mirrored by the CIU behavior of gaseous protein ions. Here, we address this question using chloramine-T-oxidized cytochrome c (CT-cyt c) as a model system. CT-cyt c comprises various proteoforms that have undergone MetO formation (+16 Da) and Lys carbonylation (LysCH2-NH2 → LysCHO, -1 Da). We found that CT-cyt c in solution was destabilized, with a ∼5 °C reduced melting temperature compared to unmodified controls. Surprisingly, CIU experiments revealed the opposite trend, i.e., a stabilization of CT-cyt c in the gas phase. To pinpoint the source of this effect, we performed proteoform-resolved CIU on CT-cyt c fractions that had been separated by cation exchange chromatography. In this way, it was possible to identify MetO formation at residue 80 as the key modification responsible for stabilization in the gas phase. Possibly, this effect is caused by newly formed contacts of the sulfoxide with aromatic residues in the protein core. Overall, our results demonstrate that oxidative modifications can affect protein stability in solution and in the gas phase very differently.

Physicochemical and emulsifying properties of mussel water-soluble proteins as affected by lecithin concentration.

The effects of lecithin addition at different concentrations (0-2.0%) on the physicochemical and emulsifying properties of mussel water-soluble proteins (MWP) were investigated. In solution system, low lecithin concentration (0.5%-1.0%) induced the aggregation and increased turbidity of composite particles. Lecithin addition caused changes in secondary structure and induced partial unfolding of MWP. Hydrophobic interactions between MWP and lecithin may contribute to the exposure of chromophores and hydrophobic groups of MWP. The interfacial tension decreased with lecithin addition. However, at a high lecithin concentration (1.5%-2.0%), the degree of aggregation and state of unfolding alleviated due to competitive adsorption. In emulsion system, with the low concentration of lecithin addition (0.5%-1.0%), droplet size and surface charge of emulsion decreased. The emulsion activity index, emulsion stability index, percentage of adsorbed protein increased. Both creaming stability and viscoelastic properties improved. At an intermediate lecithin concentration (1.0%), the emulsion showed the highest physical stability, while further addition of lecithin caused a slight deterioration in emulsifying properties. Overall, these results indicated the possibility that the lecithin-MWP mixed emulsifiers can be used to obtain emulsions with desirable properties.

Initial steps in selenocysteine biosynthesis: The interaction between selenocysteine lyase and selenophosphate synthetase.

The selenocysteine (Sec) incorporation is a co-translational event taking place at an in-frame UGA-codon and dependent on an organized molecular machinery. Selenium delivery requires mainly two enzymes, the selenocysteine lyase (CsdB) is essential for Sec recycling and conversion to selenide, further used by the selenophosphate synthetase (SelD), responsible for the conversion of selenide in selenophosphate. Therefore, understanding the catalytic mechanism involved in selenium compounds delivery, such as the interaction between SelD and CsdB (EcCsdB.EcSelD), is fundamental for the further comprehension of the selenocysteine synthesis pathway and its control. In Escherichia coli, EcCsdB.EcSelD interaction must occur to prevent cell death from the release of the toxic intermediate selenide. Here, we demonstrate and characterize the in vitro EcSelD.EcCsdB interaction by biophysical methods. The EcSelD.EcCsdB interaction occurs with a stoichiometry of 1:1 in presence of selenocysteine and at a low-nanomolar affinity (~1.8 nM). The data is in agreement with the small angle X-ray scattering model fitted using available structures. Moreover, yeast-2-hybrid assays supported the macromolecular interaction in the cellular environment. This is the first report that demonstrates the interaction between EcCsdB and EcSelD supporting the hypothesis that EcSelD.EcCsdB interaction is necessary to sequester the selenide during the selenocysteine incorporation pathway in Bacteria.

Photophysical behavior of heme group: Unfolding of hemoglobin and myoglobin in the presence of Gemini surfactants of different molecular architectures.

Fluorescence studies were performed to determine the photophysical behavior of heme group in the presence of cationic Gemini surfactants of different architectures. Both hemoglobin and myoglobin were used to understand the heme group interactions with Gemini surfactants under the influence of temperature variation and were compared with homologous monomeric surfactants. The results were also supplemented from the size and zeta potential measurements of both proteins. Gemini surfactants showed marked effect on the unfolding behavior of hemoglobin that mainly contributed by the stronger hydrophobic interactions of double hydrocarbon chains as well as methylene spacer in the head group region with the hydrophobic domains of hemoglobin. Myoglobin with single polypeptide chain did not show similar unfolding behavior in the presence of Gemini surfactants rather it was readily solubilized in the surfactant solution and that too in the presence of monomeric surfactants rather than Gemini surfactants. The results highlighted the mechanistic aspects by which water soluble globular proteins interact with amphiphilic molecules of different functionalities and thus, helped to predict the interactions of both hemoglobin and myoglobin with the complex biological molecules possessing similar functionalities.

Application of high-pressure homogenization (HPH) to modify functional, structural and rheological properties of lentil (Lens culinaris) proteins.

The ability of high-pressure homogenization (HPH) to modify the functional, structural and rheological properties of lentil protein isolate (LPI) suspensions were investigated. Protein patterns remained unchanged with HPH treatment. Particle size significantly decreased up to 100 MPa treatment and size distribution was mono-modal after 50 MPa. Microstructural images revealed that increasing pressure from 50 to 150 MPa caused further unfolding of protein particles, which well supported to water solubility, emulsifying, foaming and particle size results. LPI suspensions had shear thinning behavior and results were well fitted to Ostwald de-Waele model (R2 ≥ 0.989). Apparent viscosity and homogenization pressure were modeled with exponential and sigmoidal functions (R2 ≥ 0.983). However, weak gel-like structure was observed from all samples due to G' > G″, and higher homogenization pressures than 50 MPa caused more pronounced gelation after 51.78 °C. These results stated that HPH treatment has a good potential to modify the functional, structural and rheological properties of LPI suspensions.

Non-Arrhenius Reaction-Diffusion Kinetics for Protein Inactivation over a Large Temperature Range.

Understanding protein folding and unfolding has been a long-standing fundamental question and has important applications in manipulating protein activity in biological systems. Experimental investigations of protein unfolding have been predominately conducted by small temperature perturbations (e.g., temperature jump), while molecular simulations are limited to small time scales (microseconds) and high temperatures to observe unfolding. Thus, it remains unclear how fast a protein unfolds irreversibly and loses function (i.e., inactivation) across a large temperature range. In this work, using nanosecond pulsed heating of individual plasmonic nanoparticles to create precise localized heating, we examine the protein inactivation kinetics at extremely high temperatures. Connecting this with protein inactivation measurements at low temperatures, we observe that the kinetics of protein unfolding is less sensitive to temperature change at the higher temperatures, which significantly departs from the Arrhenius behavior extrapolated from low temperatures. To account for this effect, we propose a reaction-diffusion model that modifies the temperature-dependence of protein inactivation by introducing a diffusion limit. Analysis of the reaction-diffusion model provides general guidelines in the behavior of protein inactivation (reaction-limited, transition, diffusion-limited) across a large temperature range from physiological temperature to extremely high temperatures. We further demonstrate that the reaction-diffusion model is particularly useful for designing optimal operating conditions for protein photoinactivation. The experimentally validated reaction-diffusion kinetics of protein unfolding is an important step toward understanding protein-inactivation kinetics over a large temperature range. It has important applications including molecular hyperthermia and calls for future studies to examine this model for other protein molecules.

Simulations of mutant p53 DNA binding domains reveal a novel druggable pocket.

The DNA binding domain (DBD) of the tumor suppressor p53 is the site of several oncogenic mutations. A subset of these mutations lowers the unfolding temperature of the DBD. Unfolding leads to the exposure of a hydrophobic ß-strand and nucleates aggregation which results in pathologies through loss of function and dominant negative/gain of function effects. Inspired by the hypothesis that structural changes that are associated with events initiating unfolding in DBD are likely to present opportunities for inhibition, we investigate the dynamics of the wild type (WT) and some aggregating mutants through extensive all atom explicit solvent MD simulations. Simulations reveal differential conformational sampling between the WT and the mutants of a turn region (S6-S7) that is contiguous to a known aggregation-prone region (APR). The conformational properties of the S6-S7 turn appear to be modulated by a network of interacting residues. We speculate that changes that take place in this network as a result of the mutational stress result in the events that destabilize the DBD and initiate unfolding. These perturbations also result in the emergence of a novel pocket that appears to have druggable characteristics. FDA approved drugs are computationally screened against this pocket.

Impact of pH Modification on Protein Polymerization and Structure⁻Function Relationships in Potato Protein and Wheat Gluten Composites.

Wheat gluten (WG) and potato protein (PP) were modified to a basic pH by NaOH to impact macromolecular and structural properties. Films were processed by compression molding (at 130 and 150 °C) of WG, PP, their chemically modified versions (MWG, MPP) and of their blends in different ratios to study the impact of chemical modification on structure, processing and tensile properties. The modification changed the molecular and secondary structure of both protein powders, through unfolding and re-polymerization, resulting in less cross-linked proteins. The ß-sheet formation due to NaOH modification increased for WG and decreased for PP. Processing resulted in cross-linking of the proteins, shown by a decrease in extractability; to a higher degree for WG than for PP, despite higher ß-sheet content in PP. Compression molding of MPP resulted in an increase in protein cross-linking and improved maximum stress and extensibility as compared to PP at 130 °C. The highest degree of cross-linking with improved maximum stress and extensibility was found for WG/MPP blends compared to WG/PP and MWG/MPP at 130 °C. To conclude, chemical modification of PP changed the protein structures produced under harsh industrial conditions and made the protein more reactive and attractive for use in bio-based materials processing, no such positive gains were seen for WG.

Effect of Imidazolium-Based Ionic Liquids on the Structure and Stability of Stem Bromelain: Concentration and Alkyl Chain Length Effect.

In the present work, changes in the structure and stability of stem bromelain (BM) are observed in the presence of a set of four imidazolium-based ionic liquids (ILs) such as 1-ethyl-3-methylimidazolium chloride ([Emim][Cl]), 1-butyl-3-methylimidazolium chloride ([Bmim][Cl]), 1-hexyl-3-methylimidazolium chloride ([Hmim][Cl]), and 1-decyl-3-methylimidazolium chloride ([Dmim][Cl]), using various biophysical techniques. Fluorescence spectroscopy is used to observe the changes taking place in the microenvironment around the tryptophan (Trp) residues of BM and its thermal stability because of its interactions with the ILs at different concentrations. Near-UV circular dichroism results showed that the native structure of BM remained preserved only at lower concentrations of ILs. In agreement with these results, dynamic light scattering revealed the formation of large aggregates of BM at higher concentrations of ILs, indicating the unfolding of BM. In addition to this, the results also show that higher alkyl chain length imidazolium-based ILs have a more denaturing effect on the BM structure as compared to the lower alkyl chain length ILs because of the increased hydrophobic interaction between the ILs and the BM structure. Interestingly, it is noted that low concentrations (0.01-0.10 M) of short alkyl chain ILs only alter the structural arrangement of the protein without any significant effect on its stability. However, high concentrations of all five ILs are found to disrupt the structural stability of BM.

Crowded milieu tuning the stability and activity of stem bromelain.

Proteins in vivo are under an extremely crowded environment because of the presence of bulky and large biological macromolecules (known as crowders). These crowders affect the proper functioning and structure of proteins in a cell. During in vitro studies, we often ignore the effect of macromolecular crowding on protein stability. However, if a large concentration of crowder is used to examine protein stability, its effects on the functioning of protein inside the cell in a confined environment, as stated, can be understood. Keeping this in context, we investigated the effects of macromolecular crowding on stem bromelain (BM) with the help of different crowding agents of varying molecular weights such as dextran (40 kDa and 6 kDa) and ficoll (70 kDa). Activity and stability of BM was examined using UV-vis, fluorescence and circular dichroism (CD) spectroscopy. Furthermore, docking methods are used to complement the crowding effects on the stability of BM. We found that stability and activity of BM are dependent on the surrounding crowder molecules. Thermal flouresence results showed that, thermal stability of BM decreses with incresing concentration of crowder except dextran40. It was observed that the decrese in stability and activity can be related to the presence of soft interactions between crowder and BM. Thus,crowding does not always stabilize the native structure, instead, it depends on degree of disorder of protein structure and on two competing effects: the excluded volume, which favors compact states, and soft interactions, which favor extended conformers.

Omega-3 fatty acids increase the unfolded protein response and improve amyloid-ß phagocytosis by macrophages of patients with mild cognitive impairment.

Macrophages (Mϕs) of patients with Alzheimer's disease and mild cognitive impairment (MCI) are defective in amyloid-ß1-42 (Aß) phagocytosis and have low resistance to apoptosis by Aß. Omega-3 fatty acids (ω-3s) in vitro and in vivo and the ω-3 mediator, resolvin D1, in vitro increase Aß phagocytosis by Mϕs of patients with MCI. We have investigated the unfolded protein response (UPR) to endoplasmic reticulum (ER) stress by Mϕs in a longitudinal study of fish-derived, ω-3-supplemented patients with MCI. Patients in the apolipoprotein E (ApoE)e3/e3 subgroup over time exhibited an increase of protein kinase RNA-like ER kinase (PERK) expression, Aß phagocytosis, intermediate M1-M2 Mϕ type, and a Mini-Mental State Examination (MMSE) rate of change of +1.8 points per year, whereas patients in the ApoEe3/e4 subgroup showed individually divergent results with an MMSE rate of change of -3.2 points per year. In vitro treatment of Mϕs by fish-derived ω-3 emulsion increased Aß phagocytosis, PERK expression, and UPR RNA signature, and decreased ER stress signature. Augmented genes in the UPR signature included chaperones, lectins, foldases, and N-linked glycosylation enzymes. In summary, fish-derived ω-3s increase cytoprotective genes and decrease proapoptotic genes, improve immune clearance of Aß, and are associated with an improved MMSE rate of change in ApoEe3/e3 vs. ApoEe3/e4 patients.-Olivera-Perez, H. M., Lam, L., Dang, J., Jiang, W., Rodriguez, F., Rigali, E., Weitzman, S., Porter, V., Rubbi, L., Morselli, M., Pellegrini, M., Fiala, M. Omega-3 fatty acids increase the unfolded protein response and improve amyloid-ß phagocytosis by macrophages of patients with mild cognitive impairment.

The Lys-Asp-Tyr Triad within the Mite Allergen Der p 1 Propeptide Is a Critical Structural Element for the pH-Dependent Initiation of the Protease Maturation.

The major house dust mite allergen, Der p 1, is a papain-like cysteine protease expressed as an inactive precursor, proDer p 1, carrying an N-terminal propeptide with a unique structure. The maturation of the zymogen into an enzymatically-active form of Der p 1 is a multistep autocatalytic process initiated under acidic conditions through conformational changes of the propeptide, leading to the loss of its inhibitory ability and its subsequent gradual cleavage. The aims of this study were to characterize the residues present in the Der p 1 propeptide involved in the initiation of the zymogen maturation process, but also to assess the impact of acidic pH on the propeptide structure, the activity of Der p 1 and the fate of the propeptide. Using various complementary enzymatic and structural approaches, we demonstrated that a structural triad K17p-D51p-Y19p within the N-terminal domain of the propeptide is essential for its stabilization and the sensing of pH changes. Particularly, the protonation of D51p under acidic conditions unfolds the propeptide through disruption of the K17p-D51p salt bridge, reduces its inhibition capacity and unmasks the buried residues K17p and Y19p constituting the first maturation cleavage site of the zymogen. Our results also evidenced that this triad acts in a cooperative manner with other propeptide pH-responsive elements, including residues E56p and E80p, to promote the propeptide unfolding and/or to facilitate its proteolysis. Furthermore, we showed that acidic conditions modify Der p 1 proteolytic specificity and confirmed that the formation of the first intermediate represents the limiting step of the in vitro Der p 1 maturation process. Altogether, our results provide new insights into the early events of the mechanism of proDer p 1 maturation and identify a unique structural triad acting as a stabilizing and a pH-sensing regulatory element.

Temperature-induced unfolding behavior of proteins studied by tensorial elastic network model.

Motivated by single molecule experiments and recent molecular dynamics (MD) studies, we propose a simple and computationally efficient method based on a tensorial elastic network model to investigate the unfolding pathways of proteins under temperature variation. The tensorial elastic network model, which relies on the native state topology of a protein, combines the anisotropic network model, the bond bending elasticity, and the backbone twist elasticity to successfully predicts both the isotropic and anisotropic fluctuations in a manner similar to the Gaussian network model and anisotropic network model. The unfolding process is modeled by breaking the native contacts between residues one by one, and by assuming a threshold value for strain fluctuation. Using this method, we simulated the unfolding processes of four well-characterized proteins: chymotrypsin inhibitor, barnase, ubiquitein, and adenalyate kinase. We found that this step-wise process leads to two or more cooperative, first-order-like transitions between partial denaturation states. The sequence of unfolding events obtained using this method is consistent with experimental and MD studies. The results also imply that the native topology of proteins "encrypts" information regarding their unfolding process. Proteins 2016; 84:1767-1775. © 2016 Wiley Periodicals, Inc.

A Distinct Proof on Interplay between Trehalose and Guanidinium Chloride for the Stability of Stem Bromelain.

Guanidinium chloride (GdnHCl), a potential denaturant, is well-known to denature a number of proteins in vitro as well as in vivo studies. Its deleterious action on stem bromelain (BM) is quite prominent resulting decrease in protein structure and stability. The counteraction of this adverse effect of GdnHCl by the use of osmolytes is scarcely studied and the mechanism is still illusive and not exclusive. For the first time, to test elegant and simple counteraction hypothesis as a general mechanism we utilized fluorescence, circular dichroism, Fourier transform infrared spectroscopy, and dynamic light scattering to study the counteraction of GdnHCl-induced denaturation of BM by the trehalose. It is revealed from the investigation of the results that trehalose is efficiently counteracting GdnHCl undesirable impacts on BM stability at molar ratio 1:1 of trehalose and GdnHCl. On the contrary, proteolytic activity of BM is increased only for the counteraction study of BM at very high concentrations of GdnHCl although still less than BM in buffer. The mutual exclusion of both trehalose and GdnHCl may stand for the counteraction of denaturation of BM resulting in a compact conformation with less solvent exposed surface area and increased secondary and tertiary structures. In addition, a decrease in BM-solvent interactions may also be contributing to some extent as there is little binding of trehalose replacing some water molecules and reducing binding of GdnHCl.

Molecular dynamics simulations and CD spectroscopy reveal hydration-induced unfolding of the intrinsically disordered LEA proteins COR15A and COR15B from Arabidopsis thaliana.

The LEA (late embryogenesis abundant) proteins COR15A and COR15B from Arabidopsis thaliana are intrinsically disordered under fully hydrated conditions, but obtain α-helical structure during dehydration, which is reversible upon rehydration. To understand this unusual structural transition, both proteins were investigated by circular dichroism (CD) and molecular dynamics (MD) approaches. MD simulations showed unfolding of the proteins in water, in agreement with CD data obtained with both HIS-tagged and untagged recombinant proteins. Mainly intramolecular hydrogen bonds (H-bonds) formed by the protein backbone were replaced by H-bonds with water molecules. As COR15 proteins function in vivo as protectants in leaves partially dehydrated by freezing, unfolding was further assessed under crowded conditions. Glycerol reduced (40%) or prevented (100%) unfolding during MD simulations, in agreement with CD spectroscopy results. H-bonding analysis indicated that preferential exclusion of glycerol from the protein backbone increased stability of the folded state.

Mechanism of p27 Unfolding for CDK2 Reactivation.

Cell-cycle regulatory protein, CDK2 is active when bound to its complementary partner protein, CyclinA or E. Recent discovery of the Kip/Cip family of proteins has indicated that the activity of CDK2 is also regulated by a member protein, p27. Although, the mechanism of CDK2 inhibition by p27 binding is known from crystal structure, little is known about the mechanism of CDK2 reactivation. Here, we execute classical and accelerated molecular dynamics simulations of unphosphorylated- and phosphorylated-p27 bound CDK2/CyclinA to unravel the CDK2 reactivation mechanism at molecular-to-atomic detail. Results suggest that the phosphorylation of p27 Y88 residue (pY88-p27) first disrupts the p27/CDK2 hybrid ß-sheet and subsequently ejects the p27 310 helix from CDK2 catalytic cleft. The unbinding of p27 from CDK2/CyclinA complex, thus, follows a two-step unfolding mechanism, where the 310 helix ejection constitutes the rate-limiting step. Interestingly, the unfolding of p27 leaves CDK2/CyclinA in an active state, where the prerequisite CDK2-CyclinA interfacial contacts were regained and ATP achieved its native position for smooth transfer of phosphate. Our findings match very well with NMR chemical shift data that indicated the flip-out of p27 310 helix from CDK2 pocket and kinetic experiments that exhibited significant kinase activity of CDK2 when saturated with pY88-p27.

The aqueous extract of Glycyrrhiza inflata can upregulate unfolded protein response-mediated chaperones to reduce tau misfolding in cell models of Alzheimer's disease.

BACKGROUND: Alzheimer's disease (AD) and several neurodegenerative disorders known as tauopathies are characterized by misfolding and aggregation of tau protein. Although several studies have suggested the potential of traditional Chinese medicine (TCM) as treatment for neurodegenerative diseases, the role of TCM in treating AD and tauopathies have not been well explored. MATERIALS AND METHODS: Tau protein was coupled to the DsRed fluorophore by fusing a pro-aggregation mutant of repeat domain of tau (ΔK280 tauRD) with DsRed. The ΔK280 tauRD-DsRed fusion gene was then used to generate Tet-On 293 and SH-SY5Y cell clones as platforms to test the efficacy of 39 aqueous extracts of TCM in reducing tau misfolding and in neuroprotection. RESULTS: Seven TCM extracts demonstrated a significant reduction in tau misfolding and reactive oxidative species with low cytotoxicity in the ΔK280 tauRD-DsRed 293 cell model. Glycyrrhiza inflata and Panax ginseng also demonstrated the potential to improve neurite outgrowth in the ΔK280 tauRD-DsRed SH-SY5Y neuronal cell model. G. inflata further rescued the upregulation of ERN2 (pro-apoptotic) and downregulation of unfolded-protein-response-mediated chaperones ERP44, DNAJC3, and SERP1 in ΔK280 tauRD-DsRed 293 cells. CONCLUSION: This in vitro study provides evidence that G. inflata may be a novel therapeutic for AD and tauopathies. Future applications of G. inflata on animal models of AD and tauopathies are warranted to corroborate its effect of reducing misfolding and potential disease modification.

Trimethylamine-N-oxide switches from stabilizing nature: A mechanistic outlook through experimental techniques and molecular dynamics simulation.

In adaptation biology of the discovery of the intracellular osmolytes, the osmolytes are found to play a central role in cellular homeostasis and stress response. A number of models using these molecules are now poised to address a wide range of problems in biology. Here, a combination of biophysical measurements and molecular dynamics (MD) simulation method is used to examine the effect of trimethylamine-N-oxide (TMAO) on stem bromelain (BM) structure, stability and function. From the analysis of our results, we found that TMAO destabilizes BM hydrophobic pockets and active site as a result of concerted polar and non-polar interactions which is strongly evidenced by MD simulation carried out for 250 ns. This destabilization is enthalpically favourable at higher concentrations of TMAO while entropically unfavourable. However, to the best of our knowledge, the results constitute first detailed unambiguous proof of destabilizing effect of most commonly addressed TMAO on the interactions governing stability of BM and present plausible mechanism of protein unfolding by TMAO.