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Investigation of changes in thermal stabilities and structures of proteins in the presence of different co-solutes (ligands) is an integral part in the basic research, discovery, and development of drugs. Ethylene glycol (EG) is known to be toxic and causes teratogenic, inducing primarily skeletal and external malformations and other diseases. The effect of EG on the structure and thermal stability of myoglobin (Mb) was studied using various spectroscopic techniques at pH 7.0 and two different temperatures. As revealed by circular dichroism, Trp fluorescence, nano-DSF, and absorption (UV and visible) measurements, EG (i) has no significant effect on secondary and tertiary structures of Mb at 25 °C, and (ii) it decreases the thermal stability of the protein, which increases with increasing concentration of EG. As revealed by ANS (8-anilino-1-naphthalene sulfonic acid) fluorescence measurements, heat-induced denatured protein has newly exposed hydrophobic patches that bind to ANS. Isothermal titration calorimetry revealed that the interaction between EG and Mb is temperature dependent; the preferential interaction of EG is entropy driven at low temperature, 298 K (25 °C), and it is enthalpy driven at higher temperature, 343 K (70 °C). Molecular docking study showed that EG interacts with side chains of amino acid residues of Mb through van der Waals interactions and hydrogen bonding.

One of the significant proteins that have attracted research groups due to virtue of being a potent selective anticancer drug target and property of triggering apoptosis upon release in cytoplasm is cytochrome c (cyt c). The mechanical transformations due to the macromolecular crowding in membrane in the mammalian cell are proposed to be useful inductors of changes in volume. It is very interesting to know that mitochondrial function were observed to be improved by polyethylene glycol (PEG) interaction, which in turn inhibits the cyt c (a pro-apoptotic cell death factor). In this work, the effect of polyethylene glycol of molecular weight 4 kilo Dalton (PEG 4 kDa) was investigated to highlight the structural transformations (tertiary and secondary structure) in cyt c using a choice of spectroscopic techniques (including UV–Vis absorption, near-UV, far-UV and Soret circular dichroism and fluorescence spectroscopy), which shows noteworthy shifts in the secondary and tertiary structures at higher concentrations of PEG 4 kDa with small changes in the heme-globular interactions. The size distribution changes of native protein treated with various concentrations of the crowder were observed and analyzed by dynamic light scattering (DLS). The interaction studies of the crowder with the protein was observed and analyzed by FTIR, isothermal titration calorimetry, time resolved fluorescence and molecular docking. The investigations suggested that the structural changes in the protein occurred due to soft interactions of PEG 4 kDa, which usually destabilizes proteins. The experimental evidence in this study proposed that crowding could be another approach to mechanical super-competition and free of certain markers that could aid in the identification and control of various diseases. This study suggests that crowders at specific concentrations, which softly interact with proteins, can be exploited as remedy for various diseases.

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