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However, this intermediate state is recalcitrant to detailed structural investigation, as it is only transiently and sparsely populated. Here, this work investigates the intermediate state of an amyloid\u2010forming human lysozyme variant (I59T) using CEST and CPMG RD NMR at low pH.\n                    <jats:sup>15<\/jats:sup>\n                    N CEST profiles probe the thermal unfolding of the native state into the denatured ensemble and reveal a distinct intermediate state. Global fitting of\n                    <jats:sup>15<\/jats:sup>\n                    N CEST and CPMG data provides kinetic and thermodynamic parameters, characterizing the intermediate state populated at 0.6%.\n                    <jats:sup>1<\/jats:sup>\n                    H CEST data further confirm the presence of the intermediate state displaying unusually high or low\n                    <jats:sup>1<\/jats:sup>\n                    H\n                    <jats:sub>N<\/jats:sub>\n                    chemical shifts. To investigate the structural details of this intermediate state, this work uses molecular dynamics (MD) simulations, which recapitulate the experimentally observed folding pathway and free energy landscape. This work observes a high\u2010energy intermediate state with a locally disordered \u03b2\u2010domain and C\u2010helix, stabilized by non\u2010native hydrogen bonding and amide\u2010\u03c0 interactions, accounting for its anomalous\n                    <jats:sup>1<\/jats:sup>\n                    H chemical shifts. Together, these NMR and MD data provide the first direct structural information on the intermediate state, offering insights into targeting lysozyme amyloidosis.\n                  <\/jats:p>","DOI":"10.1002\/advs.202503957","type":"journal-article","created":{"date-parts":[[2025,6,25]],"date-time":"2025-06-25T05:30:38Z","timestamp":1750829438000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Amyloid Forming Human Lysozyme Intermediates are Stabilized by Non\u2010Native Amide\u2010\u03c0 Interactions"],"prefix":"10.1002","volume":"12","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-9131-7334","authenticated-orcid":false,"given":"Minkoo","family":"Ahn","sequence":"first","affiliation":[{"name":"School of Biochemistry University of Bristol  University Walk Bristol BS8 1TD UK"},{"name":"Research Department of Structural and Molecular Biology Biosciences University College London  Gower Street London WC1E 6BT 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