Soft interactions versus hard core repulsions: A journey of cytochrome c from acid-induced denaturation to native protein via pre-molten globule and molten globule conformations exploiting dextran and its monomer glucose

Protein attains its biological functional state in a crowded environment, whereas traditional in vitro experiments are carried out in dilute solutions. In this investigation, synthetic crowders (dextran and glu-cose) are exploited to investigate the effect of crowding on the conformation of cytochrome c. Protein folding may occur through many non-native intermediate states. Here, the effect of crowder (dextran) and its monomer (glucose) on the structural conformation and stability of cytochrome c (cyt c) has been studied in an acidic environment using various biophysical techniques such as UV-Vis absorbance, circu-lar dichroism, and fluorescence spectroscopy (Trp and ANS fluorescence), dynamic light scattering and interaction experiments using ITC and molecular docking. These techniques show that the cyt c forms a molten globule in the presence of glucose while the pre-molten globule in the presence of the dextran 70. These results suggest that dextran 70 stabilizes the protein due to macromolecular crowding via soft interactions, while glucose due to co-solute engineering exploiting volume exclusion phenomenon as confirmed by ITC experiments. These investigations inferred that crowders protect protein in harsh con-ditions (acidic pH), which may arise due to various diseased conditions such as acidosis-induced insulin resistance, cardiovascular risks, etc. The current work also has an industrial application to increase the shelf life of therapeutic proteins.(c) 2022 Elsevier B.V. All rights reserved.

Automated summary

In this study, the effect of crowding on the conformation of cytochrome c was investigated using synthetic crowders. The results showed that the presence of glucose led to the formation of a molten globule, while the presence of dextran 70 resulted in a pre-molten globule state. These findings suggest that crowders can protect proteins under harsh conditions and have important biological implications.