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Proteins have evolved to be foldable, and yet determinants of foldability may be inapparent once the native state is reached. Insight has emerged from studies of diseases of protein misfolding, exemplified by monogenic diabetes mellitus due to mutations in proinsulin leading to endoplasmic reticulum stress and β-cell death. Cellular foldability of human proinsulin requires an invariant Phe within a conserved crevice at the receptor-binding surface (position B24). Any substitution, even related aromatic residue TyrB24, impairs insulin biosynthesis and secretion. As a seeming paradox, a monomeric TyrB24 insulin analog exhibits a native-like structure in solution with only a modest decrement in stability. Packing of TyrB24 is similar to that of PheB24, adjoining core cystine B19-A20 to seal the core; the analog also exhibits native self-assembly. Although affinity for the insulin receptor is decreased ~20-fold, biological activities in cells and rats were within the range of natural variation. Together, our findings suggest that the invariance of PheB24 among vertebrate insulins and insulin-like growth factors reflects an essential role in enabling efficient protein folding, trafficking, and secretion, a function that is inapparent in native structures. In particular, we envision that the para-hydroxyl group of TyrB24 hinders pairing of cystine B19-A20 in an obligatory on-pathway folding intermediate. The absence of genetic variation at B24 and other conserved sites near this disulfide bridge--excluded due to β-cell dysfunction--suggests that insulin has evolved to the edge of foldability. Nonrobustness of a protein's fitness landscape underlies both a rare monogenic syndrome and "diabesity" as a pandemic disease of civilization. [ABSTRACT FROM AUTHOR]

Insulin is susceptible to fibrillation, a misfolding process leading to well ordered cross-β assembly. Protection from fibrillation in β cells is provided by sequestration of the susceptible monomer within zinc hexamers. We demonstrate that proinsulin is refractory to fibrillation under conditions that promote the rapid fibrillation of zinc-free insulin. Proinsulin fibrils, as probed by Raman microscopy, are nonetheless similar in structure to insulin fibrils. The connecting peptide, although not well ordered in native proinsulin, participates in a fibril-specific β-sheet. Native insulin and proinsulin exhibit similar free energies of unfolding as inferred from guanidine denaturation studies: relative amyloidogenicities are thus not correlated with global stability. Strikingly, the susceptibility of proinsulin to fibrillation is increased by scission of the connecting peptide at single sites. We thus propose that the connecting peptide constrains a large scale conformational change in the misfolded protein. A tethering mechanism is proposed based on a model of an insulin protofilament derived from electron-microscopic image reconstruction. The proposed relationship between cross-β assembly and protein topology is supported by studies of single-chain analogs (mini-proinsulin and insulin-like growth factor I) in which foreshortened connecting peptides further retard fibrillation. In addition to its classic function to facilitate disulfide pairing, the connecting peptide may protect β cells from toxic protein misfolding in the endoplasmic reticulum. [ABSTRACT FROM AUTHOR]