Peptide Model of the Mutant Proinsulin Syndrome. II. Nascent Structure and Biological Implications

Toxic misfolding of proinsulin variants in β-cells defines a monogenic diabetes syndrome, designated (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 (Leu→Pro, Leu→Pro, and Phe→Ser) probe residues conserved among vertebrate insulins. In this companion study, we describe H and H-C NMR studies of the peptides; key NMR resonance assignments were verified by synthetic C-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 Leu, Leu and Phe at the nexus of nativelike α-helices α and α (as defined in native proinsulin) and flanking β-strand (residues B24-B26). The variant peptides exhibit successive structural perturbations in order: parent (most organized) > Ser >> Pro > Pro (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.