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To elucidate the role of metalation in superoxide dismutase (SOD1) misfolding and aggregation, we comprehensively characterized the structural features (apo\/holo forms) of WT\u2010SOD1 and P66R mutant in loop IV. Using computational and experimental methodologies, we assessed the physicochemical properties of these variants and their correlation with protein aggregation at the molecular level. Modifications in apo\u2010SOD1 compared to holo\u2010SOD1 were more pronounced in flexibility, stability, hydrophobicity, and intramolecular interactions, as indicated by molecular dynamics simulations. The enzymatic activities of holo\/apo\u2010WT SOD1 were 1.30 and 1.88\u2010fold of the holo\/apo P66R mutant, respectively. Under amyloid\u2010inducing conditions, decreased ANS fluorescence intensity in the apo\u2010form relative to the holo\u2010form suggested pre\u2010fibrillar species and amyloid aggregate growth due to occluded hydrophobic pockets. FTIR spectroscopy revealed that apo\u2010WT\u2010SOD1 and apo\u2010P66R exhibited a mixture of parallel and intermolecular \u03b2\u2010sheet structures, indicative of aggregation propensity. Aggregate species were identified using TEM, Congo red staining, and ThT\/ANS fluorescence spectroscopy. Thermodynamic analyses with GdnHCl demonstrated that metal deficit, mutation, and intramolecular disulfide bond reduction are essential for initiating SOD1 misfolding and aggregation. These disruptions destabilize the dimer\u2010monomer equilibrium, promoting dimer dissociation into monomers and decreasing the thermodynamic stability of SOD1 variants, thus facilitating amyloid\/amorphous aggregate formation. Our findings offer novel insights into protein aggregation mechanisms in disease pathology and highlight potential therapeutic strategies against toxic protein aggregation, including SOD1.<\/jats:p>","DOI":"10.1002\/prot.26765","type":"journal-article","created":{"date-parts":[[2024,12,6]],"date-time":"2024-12-06T23:45:37Z","timestamp":1733528737000},"page":"885-907","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":13,"title":["Insight Into Factors Influencing the Aggregation Process in Wild\u2010Type and\n                    <scp>P66R<\/scp>\n                    Mutant\n                    <scp>SOD1<\/scp>\n                    : Computational and\u00a0Spectroscopic Approaches"],"prefix":"10.1002","volume":"93","author":[{"given":"Roghayeh","family":"Farrokhzad","sequence":"first","affiliation":[{"name":"Department of Molecular and Cell Biology Faculty of Basic Science, University of Mazandaran  Babolsar Iran"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3854-9328","authenticated-orcid":false,"given":"Bagher","family":"Seyedalipour","sequence":"additional","affiliation":[{"name":"Department of Molecular and Cell Biology Faculty of Basic Science, University of Mazandaran  Babolsar Iran"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Payam","family":"Baziyar","sequence":"additional","affiliation":[{"name":"Department of Molecular and Cell Biology Faculty of Basic Science, University of Mazandaran  Babolsar Iran"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Saman","family":"Hosseinkhani","sequence":"additional","affiliation":[{"name":"Department of Biochemistry Faculty of Biological Sciences, Tarbiat Modares University  Tehran Iran"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2024,12,6]]},"reference":[{"key":"e_1_2_10_2_1","doi-asserted-by":"publisher","DOI":"10.1146\/annurev.biochem.75.101304.123901"},{"key":"e_1_2_10_3_1","doi-asserted-by":"publisher","DOI":"10.1002\/anie.201713416"},{"key":"e_1_2_10_4_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.semcdb.2011.04.002"},{"key":"e_1_2_10_5_1","doi-asserted-by":"publisher","DOI":"10.3390\/ijms140612411"},{"key":"e_1_2_10_6_1","doi-asserted-by":"publisher","DOI":"10.1007\/s11910-996-0008-9"},{"key":"e_1_2_10_7_1","doi-asserted-by":"publisher","DOI":"10.3109\/17482960802566824"},{"key":"e_1_2_10_8_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.0809845106"},{"key":"e_1_2_10_9_1","doi-asserted-by":"publisher","DOI":"10.1111\/j.1432-1033.1991.tb15794.x"},{"key":"e_1_2_10_10_1","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M802083200"},{"key":"e_1_2_10_11_1","doi-asserted-by":"publisher","DOI":"10.3390\/molecules22091429"},{"key":"e_1_2_10_12_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.0406650101"},{"key":"e_1_2_10_13_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.0511266103"},{"key":"e_1_2_10_14_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.febslet.2013.06.046"},{"key":"e_1_2_10_15_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.jmb.2011.12.029"},{"key":"e_1_2_10_16_1","doi-asserted-by":"publisher","DOI":"10.1186\/s13065-024-01289-x"},{"key":"e_1_2_10_17_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.biochi.2022.05.001"},{"key":"e_1_2_10_18_1","first-page":"26","article-title":"Development of in Silico Analysis and Molecular Dynamics Simulation on L67P and D76Y Mutants of the Human Superoxide Dismutase 1 (hSOD1) Related to Amyotrophic Lateral Sclerosis","volume":"20","author":"Baziyar P.","year":"2022","journal-title":"Iranian Journal of Biotechnology"},{"key":"e_1_2_10_19_1","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M109.086074"},{"key":"e_1_2_10_20_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.bbagen.2023.130325"},{"key":"e_1_2_10_21_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.ijbiomac.2023.125289"},{"key":"e_1_2_10_22_1","doi-asserted-by":"publisher","DOI":"10.1007\/s13738-022-02660-2"},{"key":"e_1_2_10_23_1","doi-asserted-by":"publisher","DOI":"10.1080\/07391102.2023.2281641"},{"key":"e_1_2_10_24_1","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0005004"},{"key":"e_1_2_10_25_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.compbiolchem.2023.107967"},{"key":"e_1_2_10_26_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.ijbiomac.2015.01.022"},{"issue":"3","key":"e_1_2_10_27_1","first-page":"92","article-title":"Computational Insight Into In Silico Analysis and Molecular Dynamics Simulation of the Dimer Interface Residues of ALS\u2010Linked hSOD1 Forms in Apo\/Holo States: A Combined Experimental and Bioinformatic Perspective","volume":"13","author":"Zaji H. 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