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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), both the viscosity effect and excluded volume effect are noticed, and even at a higher concentration (200\u2009g\/L) of PEG 1000, the 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. Analysis of secondary structure propensity (SSP) reveals a marked decline in the presence of structural elements (<jats:italic>\u03b2<\/jats:italic>\u2010sheet, <jats:italic>\u03b2<\/jats:italic>\u2010bridge, turn, and <jats:italic>\u03b1<\/jats:italic>\u2010helix) from 81% to 43% over the 1 \u03bcs time scale unfolding MD simulation under 8 M urea conditions. Conversely, in a 200 ns refolding simulation, the rate of refolding notably increases at a concentration of 200 g\/L PEG 1000.<\/jats:p>","DOI":"10.1002\/bio.4924","type":"journal-article","created":{"date-parts":[[2024,12,2]],"date-time":"2024-12-02T20:04:33Z","timestamp":1733169873000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":6,"title":["Polyethylene Glycol\u2010Based Refolding Kinetic Modulation of CRABP I Protein"],"prefix":"10.1002","volume":"39","author":[{"given":"Suchismita","family":"Subadini","sequence":"first","affiliation":[{"name":"Biophysical and Protein Chemistry lab, Department of Chemistry NIT Rourkela  Rourkela India"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Krishnendu","family":"Bera","sequence":"additional","affiliation":[{"name":"CEITEC MU Masaryk University  Brno Czech Republic"},{"name":"National Centre for Biomolecular Research, Faculty of Science Masaryk University  Brno 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