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Monosodium glutamate (MSG) is the world's most extensively used food additive and is generally recognized as safe according to the FDA. However, it is well reported that MSG is associated with a number of neurological diseases, and in turn, neurological diseases are associated with protein aggregation. This study rationalized the role of MSG in protein aggregation using different biophysical techniques such as absorption, far-UV CD, DLS, and ITC. Kinetic measurements revealed that MSG causes significant enhancement of aggregation of BSA through a nucleation-dependent polymerization mechanism. Also, CTAB-BSA aggregation is enhanced by MSG significantly. MSG-induced BSA aggregation also exhibits the formation of irreversible aggregates, temperature dependence, non-Arrhenius behavior, and enhancement of hydrodynamic diameter. From the isothermal titration calorimetry measurement, the significant endothermic heat of the interaction of BSA-MSG indicates that protein aggregation may be due to the coupling of MSG with the protein. The determined enthalpy change (Δ H) is largely positive, also suggesting an endothermic nature, whereas entropy change (Δ S) is positive and Gibbs free energy change (Δ G) is largely negative, suggesting the spontaneous nature of the interaction. Furthermore, even a low concentration of MSG is involved in the unfolding of the secondary structure of protein with the disappearance of original peaks and the formation of a unique peak in the far-UV CD, which is an attention-grabbing observation. This is the first investigation which links the dietary MSG with protein aggregation and thus will be very instrumental in understanding the mechanism of various MSG-related human physiological as well as neurological diseases. Kinetic measurements indicating that monosodium glutamate causes significant enhancement of aggregation of protein through nucleation-dependent polymerization mechanism. Even low concentration of MSG is involved in the unfolding of secondary structure of protein with the disappearance of original peaks (208 and 222 nm) and formation of unique peak (226 nm) in the far-UV CD. [ABSTRACT FROM AUTHOR]

To understand protein folding problem under physiological condition, usually taken as dilute aqueous buffer at pH 7.0 and 25 °C, knowledge of properties of folding intermediates is important, such as molten globule (MG). We observed that polyethylene glycol 400 Da (PEG 400) induces molten globule state conformation in cytochrome c at pH 7.0 and 25 °C. This PEG-induced MG state has: (i) native tertiary structure partially perturbed, (ii) unperturbed native secondary structure, (iii) newly exposed hydrophobic patches, and (iv) has 1.58 times more hydrodynamic volume than that of the native protein. Isothermal titration calorimetry and docking studies showed specific binding between PEG 400 and cytochrome c. The study delineates that PEG-protein interactions are more complex than the excluded-volume. The soft interactions need to be seriously studied in crowding milieu that leads to destabilization of protein and overcome stabilizing exclusion volume effect. This study not only can help in unraveling the mystery of steps involved in the proper folding of proteins to solve the massively complicated problems of protein folding but also provides novel insights towards importance of structural change in proteins inside cell where intermediate states of protein import-export easily via membranes rather than native form of proteins. Unlabelled Image • PEG 400 is a low-level toxic ingredient in drug formulations to augment the solubility and bioavailability of weakly water-soluble drugs. • The PEG 400 yields molten globule of holo-cytochrome c , characterized at physiological pH and 25 oC. • The PEG 400 interacts with cytochrome c and its prosthetic group by soft interactions that lead the structural change in the protein. • The exclusion volume effect is surmounted by soft interactions which leads formation of stabilized state of cytochrome c (molten globule). • Importance of formation of molten globule lies in protein import-export easily via membranes rather than native form of proteins. [ABSTRACT FROM AUTHOR]

Abstract Myoglobin is known to show formation of intermediate states under various environmental conditions, in spite of that, this is the first evidence of formation pre-molten globule (PMG) in myoglobin. Polyethylene glycol (PEG) of various molecular sizes shows assorted effects on different proteins. Out of too short and too long PEGs, only PEGs of optimal size interact with proteins leading to change in protein structure that form intermediate state. We are the first one to report the formation of PMG in a protein in the presence of a crowding agent. The PEG-induced intermediate state was characterized by various techniques like absorption, fluorescence, near- and far-UV circular dichroism spectroscopy, ANS binding, and dynamic light scattering measurements to be PMG. Isothermal titration calorimetry and docking studies were further carried out to delineate the mechanism of formation of PMG in myoglobin in physiological conditions. The intermediate formed due to interaction of PEG with myoglobin has physiological implications which are essential to unravel the mystery to solve the massively complicated problems involved in the proper folding of proteins in vivo. Further, outcomes from this study are expected to gain mechanistic insights on the native structure and functions of proteins under in vivo conditions. Graphical abstract Unlabelled Image Highlights • Myoglobin is known to show formation of intermediate states under various environmental conditions but this is the first evidence of existence of stable pre-molten globule of holo-myoglobin that was well characterized. • This is also the first report of formation of pre-molten globule in any protein by any crowding agent, which implies greater role of crowding and intermediate states under in vivo conditions. • PEG 400 shows soft interactions with myoglobin and its prosthetic group, which lead to structural change in the protein. • The low molecular size PEG 400 Da shows more structural change than the higher molecular size PEG 10 kDa previously reported. • Soft interaction overcomes the exclusion volume effect, which leads destabilization of the myoglobin. [ABSTRACT FROM AUTHOR]

The macromolecular crowding progressively has been gaining prominence in recent years as it acts as a sword with double-edge on protein stability and folding, i.e., showing assorted results of having both stabilizing and destabilizing effects. We studied the effects of different concentrations of polyethylene glycol (PEG-10) on structure and stability of myoglobin. The tertiary structure of myoglobin was found to be perturbed in the presence of polyethylene glycol, however there was insignificant change in the secondary structure. It was observed that polyethylene glycol induces molten globule state in myoglobin, where the intermediate state holds hydrophobic patches and larger hydrodynamic volume as compared to the native protein. In addition, isothermal titration calorimetry (ITC) showed strong binding between myoglobin and polyethylene glycol, at the physiological pH. We hypothesize that polyethylene glycol induces molten globule conformation in myoglobin by interacting with heme group of myoglobin. We caution that the binding of protein with crowder and other soft interactions need to be gravely well thought-out when studying macromolecular crowding. In our case, destabilizing protein-crowder interactions could compete and overcome the stabilizing exclusion volume effect. [ABSTRACT FROM AUTHOR]

• Heparin caused complete inhibition of CTAB-induced aggregation of protein. • Heparin prevents the CTAB-induced structural perturbation of the protein. • ITC measurements showed CTAB binds with the protein, however, CTAB-heparin complex suggested no specific binding. • Formation of CTAB-heparin complex was observed to inhibit the aggregation. Heparin, a member of the glycosaminoglycan (GAG) family, is an extremely acidic sulfur-containing polysaccharide which has been suggested to be coupled with protein aggregation-related diseases including Alzheimer's, Parkinson's and Prion diseases. Here, we study the effect of heparin on the cetyltrimethylammonium bromide (CTAB)-induced aggregation of bovine serum albumin (BSA) exploiting biophysical approaches. Kinetic measurements indicated that heparin caused complete inhibition of CTAB-induced aggregation of protein with a significant decrease in the apparent rate constant. Far- and near-UV CD measurements depicted that CTAB behaves as a protein-aggregating agent. However, the perturbation of secondary and tertiary structure-induced by CTAB and the CTAB-induced amyloid formation of protein were prevented by heparin. ITC measurements revealed that enhancement of aggregation induced by CTAB was as a result of sequential binding in which CTAB binds with the protein and accompanied by exothermic heat of interaction. ITC measurements also showed that CTAB and heparin form complex while the complex does not bind with protein. Thus, we hypothesize that CTAB-Heparin complex is engaged in the inhibition of protein aggregation. Therefore, the mechanism of action of CTAB-heparin complex in inhibition of protein aggregation may be used as a basis of the development of therapeutic strategies of various neurodegenerative complications. [Display omitted] [ABSTRACT FROM AUTHOR]

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infection in children and infants. To date, there is no effective vaccine available against RSV. Heparan sulfate is a type of glycosaminoglycan that aids in the attachment of the RSV to the host cell membrane via the G protein. In the present study, the effect of amino acid substitution on the structure and stability of the ectodomain G protein was studied. Further, it was investigated whether mutation (K117A) in the CX3C motif of G protein alters the binding with heparan sulfate. The point mutation significantly affects the conformational stability of the G protein. The mutant protein showed a low binding affinity with heparan sulfate as compared to the wild-type G protein, as determined by fluorescence quenching, isothermal titration calorimetry (ITC), and molecular docking studies. The low binding affinity and decreased stability suggested that this mutation may play an important role in prevention of attachment of virion to the host cell receptors. Collectively, this investigation suggests that mutation in the CX3C motif of G protein may likely improve the efficacy and safety of the RSV vaccine. [ABSTRACT FROM AUTHOR]

[Display omitted] • Polyethylene glycol (PEG 4 kDa) induces two different intermediate states in myoglobin (Mb). • The intermediate states were characterized using spectroscopic techniques and MD simulations. • This study showed a protein does not follow a singular and specific pathway during folding. • This study foresees the energy landscape theory. • This study shows significance of crowding concentration in cellular organism. Practically proteins perform their functions exposed to various molecules (macro/micro) of different shapes, sizes, and high concentrations. In such conditions, proteins fold through various intermediate states to execute their type of functions. There are various in vitro studies that showed that proteins yield intermediate states (either pre-molten globule, PMG or molten globules, MG) in changing environments (pH, temperature, and co-solute). Here, we investigate that myoglobin (Mb) yields two intermediate states in the presence of polyethylene glycol, PEG (4 kDa molecular weight) at two different concentrations. These intermediates were characterized by various spectroscopic techniques, further; we demonstrated that these changes in the structure of the protein were due to soft interactions which were confirmed by isothermal titration calorimetric and computational studies. Besides, in silico (molecular dynamic simulations) studies were exploited to know the atomic-level details of the protein which are useful to comprehend the functional characteristics of the bio-molecule with structural change and to study atomic motions and inter-atomic interactions in the bio-molecular systems. This is the first time that two intermediates (MG and PMG state) in the protein (Mb) at two different concentrations in the presence of solo size of PEG are yielded. This study shows folding of a protein does not follow a singular and specific pathway but occurs through routes down a folding funnel more like rain flowing down a funnel, hence foresees the energy landscape theory. Moreover, the study provides the significance of crowding concentrations in the cellular organism. [ABSTRACT FROM AUTHOR]

The intracellular environment is overcrowded with a range of molecules (small and large), all of which influence protein conformation. As a result, understanding how proteins fold and stay functional in such crowded conditions is essential. Several in vitro experiments have looked into the effects of macromolecular crowding on different proteins. However, there are hardly any reports regarding small molecular crowders used alone and in mixtures to observe their effects on the structure and stability of the proteins, which mimics of the cellular conditions. Here we investigate the effect of different mixtures of crowders, ethylene glycol (EG) and its polymer polyethylene glycol (PEG 400 Da) on the structural and thermal stability of myoglobin (Mb). Our results show that monomer (EG) has no significant effect on the structure of Mb, while the polymer disrupts its structure and decreases its stability. Conversely, the additive effect of crowders showed structural refolding of the protein to some extent. Moreover, the calorimetric binding studies of the protein showed very weak interactions with the mixture of crowders. Usually, we can assume that soft interactions induce structural perturbations while exclusion volume effects stabilize the protein structure; therefore, we hypothesize that under in vivo crowded conditions, both phenomena occur and maintain the stability and function of proteins. [ABSTRACT FROM AUTHOR]

When proteins interact with solvent or co-solutes with a high specificity and affinity, protein-ligand complexes may be formed. Such phenomenon may involve the processes like intra- and intermolecular interactions, which result in interaction based protein folding. In this study, cytochrome c (cyt c) was treated with different concentrations of ethylene glycol (EG) in crowded and confined media to check its structural stability using various spectroscopic techniques at pH 7.0 and 25 °C. The various spectroscopic techniques including circular dichroism (Soret, far- and near-UV regions), Fourier transform infrared (FTIR), absorption (UV and visible) and Trp fluorescence shows both secondary and tertiary structure of cyt c increases when treated with EG. The investigations using dynamic light scattering (DLS), time resolved fluorescence and isothermal titration calorimetry (ITC) for binding studies shows weak interaction between EG and cyt c. Small increase in the structure of the protein and insignificant decrease in hydrodynamic radii of the protein was observed from the studies. Molecular docking studies showed that EG has binding site on the protein and interact with few amino acid residues by weak interactions such as van der Waals and hydrogen bonding. This study helps in understanding the protein–ligand interactions, provides facts and the mechanisms that mediates the recognition of binding site for specific ligand to the receptor protein, which make possible of the discovery, design, and development of drugs at molecular level without affecting proteins within an organism. Unlabelled Image • The preferential interaction of ethylene glycol with cytochrome c leads to compaction of the protein. • ITC, CD, fluorescence and molecular docking were exploited to demonstrate protein compaction. • EG acts as kosmotrope (maintains order) on macromolecule, which contributes to stabilization of the protein. [ABSTRACT FROM AUTHOR]

Generally, in vivo function and structural changes are studied by probing proteins in a dilute solution under in vitro conditions, which is believed to be mimicking proteins in intracellular milieu. Earlier, thermal-induced denaturation of myoglobin, in the milieu of crowder molecule showed destabilization of the metal protein. Destabilization of protein by thermal-induced denaturation involves a large extrapolation, so, the reliability is questionable. This led us to measure the effects of macromolecular crowding on its stability by chemical-induced denaturation of the protein using probes like circular dichroism and absorption spectroscopy in the presence of dextran 70 and ficoll 70 at various pHs (acidic: 6.0, almost neutral: 7.0 and basic: 8.0). Observations showed that the degree of destabilization of myoglobin was greater due to ficoll 70 as compared to that of dextran 70 so it can be understood that the nature of the crowder or the shape of the crowder has an important role towards the stability of proteins. Additionally, the degree of destabilization was observed as pH dependent, however the pH dependence is different for different crowders. Furthermore, isothermal titration calorimetry and molecular docking studies confirmed that both the crowders (ficoll and dextran) bind to heme moiety of myoglobin and a single binding site was observed for each. [ABSTRACT FROM AUTHOR]

[Display omitted] • Formation of pre molten globule (PMG) in cyt c in presence of dextran (polymer) due to soft interactions. • Glucose (monomer) induces molten globule (MG) in cyt c due to volume exclusion phenomenon. • Presence of PMG and MG in a same protein under different environmental conditions. • Crowders induce different conformational intermediates in similar environment as evident by interaction studies (ITC & MD). Protein attains its biological functional state in a crowded environment, whereas traditional in vitro experiments are carried out in dilute solutions. In this investigation, synthetic crowders (dextran and glucose) are exploited to investigate the effect of crowding on the conformation of cytochrome c. Protein folding may occur through many non-native intermediate states. Here, the effect of crowder (dextran) and its monomer (glucose) on the structural conformation and stability of cytochrome c (cyt c) has been studied in an acidic environment using various biophysical techniques such as UV–Vis absorbance, circular dichroism, and fluorescence spectroscopy (Trp and ANS fluorescence), dynamic light scattering and interaction experiments using ITC and molecular docking. These techniques show that the cyt c forms a molten globule in the presence of glucose while the pre-molten globule in the presence of the dextran 70. These results suggest that dextran 70 stabilizes the protein due to macromolecular crowding via soft interactions, while glucose due to co-solute engineering exploiting volume exclusion phenomenon as confirmed by ITC experiments. These investigations inferred that crowders protect protein in harsh conditions (acidic pH), which may arise due to various diseased conditions such as acidosis-induced insulin resistance, cardiovascular risks, etc. The current work also has an industrial application to increase the shelf life of therapeutic proteins. [ABSTRACT FROM AUTHOR]