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Polyethylene Glycol as a Crowder: Modulating Refolding Kinetics of Crabp I Protein

SSRN, ISSN: 1556-5068
2023
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Article Description

Crowding environment has a significant impact on the folding and stability of protein in biological systems. In this work, we have used four different sizes of a molecular crowder, polyethylene glycol (PEG), to analyze the unfolding and refolding kinetics of an iLBP protein, CRABP I, using urea as chemical denaturant. In general, the stability of the native state of the protein is boosted by the presence of crowding agents in the solution. However, our findings show that not only the type of crowder but also the crowder size played a key role in the effects of excluded volume. In case of lower molecular weight of PEG (M.W. 400), even at 200 g/L concentration only the viscosity effect is observed whereas, for higher molecular weight of PEG (M.W. 1000), along with viscosity effect, excluded volume effect is noticed and, even at higher concentration (200 g/L) of PEG 1000, excluded volume predominates over the viscosity effect. Using the transition state theory, we were also able to determine the free energies of activation for the unfolding and refolding studies from their respective rate constants. Additionally, MD simulation studies provide strong support for our experimental observation. The secondary structure propensity (SSP) indicates the decrease of structural elements (β-sheet, β-bridge, turn, and α-helix) from 81% to 43% over the 1 μs time scale in the unfolding MD simulation at 8 M urea. On the other hand, for a 200 ns refolding simulation, the rate of refolding increased at a concentration of 200 g/L PEG 1000.

Bibliographic Details

Harekrushana Sahoo; Suchismita Subadini; Krishnendu Bera; Devi Prasanna Behera; Jozef Hritz

Elsevier BV

Multidisciplinary; macromolecular crowder; Excluded volume effect; PEG; protein refolding kinetics

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