{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,4,11]],"date-time":"2026-04-11T05:50:51Z","timestamp":1775886651938,"version":"3.50.1"},"reference-count":78,"publisher":"Wiley","issue":"12","license":[{"start":{"date-parts":[[2025,11,22]],"date-time":"2025-11-22T00:00:00Z","timestamp":1763769600000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"},{"start":{"date-parts":[[2025,11,22]],"date-time":"2025-11-22T00:00:00Z","timestamp":1763769600000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/doi.wiley.com\/10.1002\/tdm_license_1.1"}],"funder":[{"DOI":"10.13039\/501100001843","name":"Science and Engineering Research Board","doi-asserted-by":"publisher","award":["CRG\/2022\/002324"],"award-info":[{"award-number":["CRG\/2022\/002324"]}],"id":[{"id":"10.13039\/501100001843","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["Protein Science"],"published-print":{"date-parts":[[2025,12]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>\n                    The cytoplasm of a cell is inherently crowded with diverse biological macromolecules of varying sizes, which significantly influence thermodynamics and kinetics of essential processes such as protein folding, molecular association, and enzymatic reactions. Similarly, human eye lens is densely packed with crystallin proteins of different sizes, including multimers, oligomers and monomers, creating a highly crowded and concentrated environment. In this study, we explored the effects of poly ethylene glycol (PEG) of varying molecular weights and sizes (PEG\u2010400, PEG\u20102k, PEG\u20106k, PEG\u201035k, and PEG\u2010100k) on stability of H\u03b3DC. A uniform crowder concentration of 100 gL\n                    <jats:sup>\u22121<\/jats:sup>\n                    was maintained in all cases to mimic lens\u2010like crowding conditions. Unfolding experiments were performed at 5\u2009\u03bcM H\u03b3DC in 50\u2009mM phosphate buffer (pH\u20097.4) using CD spectroscopy in the absence and presence of the crowders, monitoring ellipticity at 218\u2009nm at a scan rate of 1\u2009K\/min. Our results revealed that crowders facilitate the thermal unfolding of H\u03b3DC, with the destabilizing effect increasing as the crowder size increases. Thermodynamic analysis showed that small crowders destabilize the protein primarily through enthalpic interactions, counterbalanced to some extent by entropic stabilization (). In contrast, larger crowders (PEG\u201035k, PEG\u2010100k) destabilize the protein almost entirely through entropic contributions, contradicting classical excluded volume theory. To rationalize these findings, we employed the associated water stabilization mechanism hypothesis, highlighting the role of associated water. Overall, our study emphasizes pivotal role of biological water in modulating protein stability, providing mechanistic insight into size\u2010dependent crowding effects in human eye lens.\n                  <\/jats:p>","DOI":"10.1002\/pro.70393","type":"journal-article","created":{"date-parts":[[2025,11,22]],"date-time":"2025-11-22T12:40:42Z","timestamp":1763815242000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Role of associated water in stabilizing human \u03b3\u2010D crystallin under crowded eye lens conditions"],"prefix":"10.1002","volume":"34","author":[{"ORCID":"https:\/\/orcid.org\/0009-0008-1384-441X","authenticated-orcid":false,"given":"Bhupendra","family":"Singh","sequence":"first","affiliation":[{"name":"Department of Chemistry Indian Institute of Technology Kanpur  UP 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