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It is known from in vitro studies that macromolecular crowding in the cell effects protein structure, stability and function; but predictive studies are relatively unexplored. There are few reports where the effect of various crowder mixtures has been exploited to discern their combined effect on the structural stability of proteins. These studies are more significant because their effect can mimicked with in vivo conditions, where the environment is heterogeneous. Effects of two crowders, polyethylene glycol (PEG 400 Da), and its monomer ethylene glycol (EG) alone and in mixture on the structural stability of cytochrome c (cyt c) were determined using various spectroscopic and bioinformatics tools. The main conclusions of our study are (i) the monomer EG has a kosmotropic effect on the protein (stabilizes the protein), and has no significant effect on the tertiary structure; (ii) PEG 400 destabilizes the structure as well as the stability of the protein; and (iii) EG counteracts the destabilizing effect of PEG 400. From this investigation, it seems evident that proteins may fold or unfold in the crowded environment of the cell where various interactions assist them to maintain their structure for their functions. Bioinformatics approaches were also used to support all of the in vitro observations. Cyt c is functional protein; if the structure of the protein is modulated due to change in the environment its nature of function will also change. Our research addresses the question by modulating the environment around the protein, and the macromolecule (protein) conformation dynamics and interaction study via in vitro and in silico approaches which indirectly compares with that of the environment in-cellular milieu, which is highly crowded. [ABSTRACT FROM AUTHOR]

Proteins are indispensable to cellular communication and metabolism. The structure on which cells and tissues are developed is deciphered from proteins. To perform functions, proteins fold into a three-dimensional structural design, which is specific and fundamentally determined by their characteristic sequence of amino acids. Few of them have structural versatility, allowing them to adapt their shape to the task at hand. The intermediate states appear momentarily, while protein folds from denatured (D) ⇔ native (N), which plays significant roles in cellular functions. Prolific effort needs to be taken in characterizing these intermediate species if detected during the folding process. Protein folds into its native structure through definite pathways, which involve a limited number of transitory intermediates. Intermediates may be essential in protein folding pathways and assembly in some cases, as well as misfolding and aggregation folding pathways. These intermediate states help to understand the machinery of proper folding in proteins. In this review article, we highlight the various intermediate states observed and characterized so far under in vitro conditions. Moreover, the role and significance of intermediates in regulating the biological function of cells are discussed clearly. [ABSTRACT FROM AUTHOR]

Macromolecular crowding can have significant consequences on the structure and dynamics of a protein. The size and shape of a co-solute molecule and the nature of protein contribute significantly in macromolecular crowding, which results in different outcomes in similar conditions. The structure of apo-myoglobin (apo-Mb) both in the absence and presence of denaturants (GdmCl and urea) was investigated in crowded conditions at pH 7.0, with a comparable size of crowders (~70 kDa) but of different shapes (ficoll and dextran) at various concentrations using spectroscopic techniques like absorption and circular dichroism to monitor changes in secondary and tertiary structure, respectively. The crowders in the absence of denaturants showed structural stabilization of the tertiary structure while no significant change in the secondary structure was observed. The effect of crowders on the stability of the protein was also investigated using probes such as Δ ε 291 and θ 222 using chemical denaturants. The analysis of chemical-induced denaturation curves showed that both the crowders stabilize apo-Mb by increasing the values of the midpoint of transition (C m) and change in free energy in the absence of denaturant (∆ G D °), and it was observed that dextran 70 shows more stabilization than ficoll 70 under similar conditions. In this study apo-Mb showed stabilization under crowded conditions, which is a deviation from earlier work from our group where holo form of the same protein was destabilized. This study emphasizes that volume exclusion is a dominant force in a simple protein while soft interactions may play important role in the proteins that are possessing prosthetic group. Hence, the effect of crowders is protein-dependent, and excluded volume plays a great role in the stabilization of apo-Mb, which does not interact with the crowders. [Display omitted] • Apo myoglobin (apo-Mb) is stabilized in the presence of ficoll 70 and dextran 70 at 25 oC. • Not only sizes but shapes of crowder molecule are significant in macromolecular crowding. • The effect of the crowders in similar conditions depends on nature of the protein (with and without prosthethic group). • In holo-Mb crowders are destabilized due to soft interactions, however apo-Mb is stabilized by excluded volume effect. [ABSTRACT FROM AUTHOR]

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]

[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]