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Mitogen-activated protein kinase (MAPK) signaling route modulates several processes, such as cell proliferation, cell programming, metabolic changes and stress responses. Within the group of proteins participating in this pathway, the MAPK kinase (MEK1) is a dimeric, 393-residue-long, dual-specificity protein kinase that phosphorylates both tyrosine and threonine residues. In this study, we explored the conformational changes occurring during the unfolding of MEK1, by using orthogonal biophysical techniques. Intrinsic fluorescence, extrinsic 8-anilinonapthalene-1-sulfonic acid (ANS) fluorescence, dynamic light scattering (DLS), and far-ultraviolet (UV) circular dichroism (CD) showed that the protein acquired a native-like conformation within a narrow pH range (8.0 to 9.0). Urea and guanidinium hydrochloride (GdmCl) denaturations followed by intrinsic and ANS fluorescence and far-UV CD, at pH 8.1, where the protein acquired a native-like conformation, showed that: (i) the apparent conformational stability of isolated MEK1 was low; and (ii) the unfolding occurred through the presence of intermediates. The presence of several unfolding intermediates was also evidenced through: (i) differential scanning calorimetry (DSC) in the absence of the ligand ATP; and (ii) unfolding simulations with the help of computational techniques based on constraint network analysis (CNA). We propose that the apparent low stability of this protein was related to its flexibility and modulates its ability to interact with diverse molecular partners.<\/jats:p>","DOI":"10.3390\/biom16060845","type":"journal-article","created":{"date-parts":[[2026,6,11]],"date-time":"2026-06-11T05:50:58Z","timestamp":1781157058000},"page":"845","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Unfolding Behavior and Conformational Changes Under Different Denaturing Conditions of MAPK 1 (MEK1)"],"prefix":"10.3390","volume":"16","author":[{"ORCID":"https:\/\/orcid.org\/0009-0008-1526-7847","authenticated-orcid":false,"given":"Maria Gabriela","family":"\u00c1lvarez-Rodr\u00edguez","sequence":"first","affiliation":[{"name":"Instituto de Biotecnologia Sanitaria de Elche (IDIBE), Universidad Miguel Hern\u00e1ndez, 03202 Elche, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sonia","family":"Vega","sequence":"additional","affiliation":[{"name":"Instituto de Biocomputaci\u00f3n y F\u00edsica de Sistemas Complejos (BIFI), Universidad de Zaragoza, 50018 Zaragoza, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9360-4086","authenticated-orcid":false,"given":"Felipe","family":"Hornos","sequence":"additional","affiliation":[{"name":"Instituto de Biotecnologia Sanitaria de Elche (IDIBE), Universidad Miguel Hern\u00e1ndez, 03202 Elche, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5702-4538","authenticated-orcid":false,"given":"Adrian","family":"Velazquez-Campoy","sequence":"additional","affiliation":[{"name":"Instituto de Biocomputaci\u00f3n y F\u00edsica de Sistemas Complejos (BIFI), Universidad de Zaragoza, 50018 Zaragoza, Spain"},{"name":"Instituto de Investigaci\u00f3n Sanitaria Arag\u00f3n (IIS Arag\u00f3n), 50009 Zaragoza, Spain"},{"name":"Centro de Investigaci\u00f3n Biom\u00e9dica en Red de Enfermedades Hep\u00e1ticas y Digestivas (CIBEREHD), 28029 Madrid, Spain"},{"name":"Departamento de Bioqu\u00edmica y Biolog\u00eda Molecular y Celular, Universidad de Zaragoza, 50009 Zaragoza, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1110-764X","authenticated-orcid":false,"given":"Bruno","family":"Rizzuti","sequence":"additional","affiliation":[{"name":"Instituto de Biocomputaci\u00f3n y F\u00edsica de Sistemas Complejos (BIFI), Universidad de Zaragoza, 50018 Zaragoza, Spain"},{"name":"CNR-NANOTEC, SS Rende (CS), Department of Physics, University of Calabria, 87036 Rende, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4933-0428","authenticated-orcid":false,"given":"Jos\u00e9 L.","family":"Neira","sequence":"additional","affiliation":[{"name":"Instituto de Biotecnologia Sanitaria de Elche (IDIBE), Universidad Miguel Hern\u00e1ndez, 03202 Elche, Spain"},{"name":"Instituto de Biocomputaci\u00f3n y F\u00edsica de Sistemas Complejos (BIFI), Universidad de Zaragoza, 50018 Zaragoza, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,6,9]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"271","DOI":"10.3892\/ijmm.2017.3036","article-title":"Lo The Crucial Role of Protein Phosphorylation in Cell Signaling and Its Use as Targeted Therapy (Review)","volume":"40","author":"Ardito","year":"2017","journal-title":"Int. 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