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A molecular understanding of the factors affecting protein stability is essential for comprehending the respective structure\u2013function relationships. This study aims to investigate the influence of native cysteine residues, ions, and glycerol on the stability of GlpF, the aqua(glycerol)porin of\n                    <jats:italic>E. coli<\/jats:italic>\n                    , under well-controlled conditions. To this end, the wild-type protein and its variant with four cysteine residues in the transmembrane helix bundle mutated to glycines were overexpressed and purified. The thermal unfolding of GlpF occurs at a transition temperature of 59.2\u2009\u00b1\u20091.4\u00a0\u00b0C, corresponding to an apparent Gibbs free energy of unfolding of approximately 10\u00a0kcal\/mol. In contrast to the wild-type protein, the mutated GlpF exhibits a second unfolding transition, lowered by approximately 10\u00a0\u00b0C, and a decreased Gibbs free energy of unfolding of 5\u00a0kcal\/mol. Our findings highlight that divalent ions and glycerol have a more pronounced stabilizing effect on the mutated GlpF than the wild-type protein. The structural consequences of cysteine to glycine mutations in terms of transmembrane helix rearrangement, the inter- and intra-chain H-bond network, and the reduced inter-chain interaction free energy, as well as intra-chain electrostatic energy and van der Waals energy, are revealed by molecular dynamics simulations. In conclusion, our findings illustrate the significance of cysteine residues distant from each other on protein oligomerization and stability, and the amplified impact of (de)stabilizing agents on less stable protein variants.\n                  <\/jats:p>","DOI":"10.1007\/s00249-025-01803-6","type":"journal-article","created":{"date-parts":[[2025,11,3]],"date-time":"2025-11-03T04:15:16Z","timestamp":1762143316000},"page":"847-860","update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Cysteine-mediated structural stabilization of the tetrameric GlpF"],"prefix":"10.1007","volume":"55","author":[{"given":"Christine","family":"Siligan","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sascha","family":"Gratzl","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kristyna","family":"Pluhackova","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nikolaus","family":"Goessweiner-Mohr","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Peter","family":"Pohl","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5328-1745","authenticated-orcid":false,"given":"Andreas","family":"Horner","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2025,11,3]]},"reference":[{"key":"1803_CR1","doi-asserted-by":"publisher","DOI":"10.1016\/j.softx.2015.06.001","volume":"1\u20132","author":"MJ Abraham","year":"2015","unstructured":"Abraham MJ, Murtola T, Schulz R, P\u00e1ll S, Smith JC, Hess B, Lindahl E (2015) GROMACS: high performance molecular simulations through multi-level parallelism from laptops to supercomputers. 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