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Mechanistic Dissection of PEG-Induced Tuning of β-Barrel Protein Stability via Chemical Denaturation
Suchismita Subadini1, Krishnendu Bera2,3,4, Devi Prasanna Behera1
1Biophysical and Protein Chemistry Lab, Department of Chemistry, NIT Rourkela, Rourkela 769008, India.
Abstract:
Macromolecular crowding plays a pivotal role in shaping protein stability and bridges insights from in vitro studies to the cellular environment. We investigated the stability of CRABP I through urea melt studies in the presence of PEG 2000 and PEG 4000, monitoring fluorescence wavelength shifts as sensitive indicators of structural transitions. In the absence of crowding agents, CRABP I unfolded with Cm at 4.39 M urea, whereas both PEG variants shifted the unfolding transition to higher concentrations, indicating an enhanced stability. This stabilization reflects crowding-induced reshaping of the free energy landscape, where excluded-volume effects entropically favor the native compact state. PEG 4000, with its larger size, imposed stronger steric constraints and augmented preferential hydration, thereby reinforcing intramolecular interactions and restricting the access of urea to the hydrophobic core. Complementary molecular dynamics simulations corroborated these mechanisms, highlighting how macromolecular crowding governs protein folding pathways and stability under physiologically relevant conditions.
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