Yang, YanwuGlidden, Michael D.Dhayalan, BalamuruganZaykov, Alexander N.Chen, Yen-ShanWickramasinghe, Nalinda P.DiMarchi, Richard D.Weiss, Michael A.2023-05-242023-05-242022-03-01Yang Y, Glidden MD, Dhayalan B, et al. Peptide Model of the Mutant Proinsulin Syndrome. II. Nascent Structure and Biological Implications. Front Endocrinol (Lausanne). 2022;13:821091. Published 2022 Mar 1. doi:10.3389/fendo.2022.821091https://hdl.handle.net/1805/33206Toxic misfolding of proinsulin variants in β-cells defines a monogenic diabetes syndrome, designated mutant INS-gene induced diabetes of the young (MIDY). In our first study (previous article in this issue), we described a one-disulfide peptide model of a proinsulin folding intermediate and its use to study such variants. The mutations (LeuB15→Pro, LeuA16→Pro, and PheB24→Ser) probe residues conserved among vertebrate insulins. In this companion study, we describe 1H and 1H-13C NMR studies of the peptides; key NMR resonance assignments were verified by synthetic 13C-labeling. Parent spectra retain nativelike features in the neighborhood of the single disulfide bridge (cystine B19-A20), including secondary NMR chemical shifts and nonlocal nuclear Overhauser effects. This partial fold engages wild-type side chains LeuB15, LeuA16 and PheB24 at the nexus of nativelike α-helices α1 and α3 (as defined in native proinsulin) and flanking β-strand (residues B24-B26). The variant peptides exhibit successive structural perturbations in order: parent (most organized) > SerB24 >> ProA16 > ProB15 (least organized). The same order pertains to (a) overall α-helix content as probed by circular dichroism, (b) synthetic yields of corresponding three-disulfide insulin analogs, and (c) ER stress induced in cell culture by corresponding mutant proinsulins. These findings suggest that this and related peptide models will provide a general platform for classification of MIDY mutations based on molecular mechanisms by which nascent disulfide pairing is impaired. We propose that the syndrome's variable phenotypic spectrum-onsets ranging from the neonatal period to later in childhood or adolescence-reflects structural features of respective folding intermediates.en-USAttribution 4.0 InternationalMonogenic diabetesPeptide chemistryProtein foldingFolding nucleusOxidative folding intermediateNMR spectroscopyArticle