biostudies-literatureUnknown11(6)Ranganathan SProinsulin folding requires dynamic positioning of the C-peptide to guide A- and B-chain alignment and disulfide pairing. Mutant INS-gene-induced diabetes of youth (MIDY) arises when single-residue substitutions disrupt this process. We mapped the conformational free-energy landscapes of wild-type (WT) proinsulin and seven MIDY variants using metadynamics and molecular dynamics simulations. WT exhibits a deep free-energy minimum at compact conformations. In contrast, MIDY mutants display a continuum of destabilization: E-(A4)K retains near-WT stability, <i>Akita</i> (C-(A7)-Y), V-(B18)-A, and R-(Cpep + 2)C show moderate loss of the native basin, while H-(B5)-D, L-(A16)-P, and Y-(B26)C collapse the closed-open barrier and populate misfolded open states >50% of the time. Structural analyses ACS omega9890-9901https://www.ebi.ac.uk/biostudies/studies/S-EPMC12917636biostudies-literatureUnraveling C‑Peptide's Role in MIDY: A Structural Perspective.PMC12917636Kilgore PDick KBishop DZavarzadeh PArunagiri ARanganathan SfalseUnraveling C‑Peptide's Role in MIDY: A Structural Perspective.Proinsulin folding requires dynamic positioning of the C-peptide to guide A- and B-chain alignment and disulfide pairing. Mutant INS-gene-induced diabetes of youth (MIDY) arises when single-residue substitutions disrupt this process. We mapped the conformational free-energy landscapes of wild-type (WT) proinsulin and seven MIDY variants using metadynamics and molecular dynamics simulations. WT exhibits a deep free-energy minimum at compact conformations. In contrast, MIDY mutants display a continuum of destabilization: E-(A4)K retains near-WT stability, <i>Akita</i> (C-(A7)-Y), V-(B18)-A, and R-(Cpep + 2)C show moderate loss of the native basin, while H-(B5)-D, L-(A16)-P, and Y-(B26)C collapse the closed-open barrier and populate misfolded open states >50% of the time. Structural analyses 2026-01-01T00:00:00Z2026 Feb2026-07-16T18:01:05.225Z2026-07-09T11:10:40.775ZS-EPMC129176364172659310.1021/acsomega.5c10573