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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 Δε 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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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.

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., Competing Interests: The authors declare no conflict of interest.

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., (Copyright © 2018 Elsevier B.V. All rights reserved.)

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., (Copyright © 2017 Elsevier B.V. All rights reserved.)

[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]