This is particularly important because information regarding structure, folding and function of the test protein are usually obtained from in vitro studies in dilute solutions but the environment of the test protein inside the cell and that in the dilute buffer (idealized conditions) are totally different from each other. Since the interior of a cell is always occupied by macromolecules, it is interesting to ask how proteins fold and function in such environment. The macromolecules such as proteins, carbohydrates, nucleic acids, and ribosomes are present in a large number, and have evolved to function in a crowded media. 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. 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. Bioinformatics approaches were also used to support all of the in vitro observations. 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. 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. 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. These studies are more significant because their effect can mimicked with in vivo conditions, where the environment is heterogeneous. 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. 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.
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