Abstract
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.
Highlights
It is assumed that the folding methods and biophysical and structural properties of an isolated protein monitored in dilute solutions are similar to the cellular conditions
Many studies have been focused on unfolding holo-Mb under different conditions because of large absorbance changes go with loss of heme and denaturation
The crowded environment was established by providing different concentrations of ficoll 70 or dextran 70
Summary
It is assumed that the folding methods and biophysical and structural properties of an isolated protein monitored in dilute solutions (in vitro) are similar to the cellular conditions (in vivo). There is a complete machinery available in the cell for proper folding of the protein like chaperones, etc. The second and important difference is the presence of the highly crowded intracellular environment, due to the huge quantity of insoluble and soluble biomolecules, which comprises of proteins, nucleic acid, carbohydrates, osmolytes, ribosomes, and the architecture of a cell [1]. Various natural and artificial crowder molecules are used with the purpose to investigate the structural, functional changes and type of interactions induced by crowding, in order to mimic cell like conditions [2,3,4]. The dependence of crowding effect on the concentration, size, shape and nature of crowding agents are very important attributes which must be considered while studying protein folding [8]
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