Characterizing ensembles of intrinsically disordered proteins is experimentally challenging because of the ill-conditioned nature of ensemble determination with limited data and the intrinsic fast dynamics of the conformational ensemble. Amide I two-dimensional infrared (2D IR) spectroscopy has picosecond time resolution to freeze structural ensembles as needed for probing disordered-protein ensembles and conformational dynamics. Also, developments in amide I computational spectroscopy now allow a quantitative and direct prediction of amide I spectra based on conformational distributions drawn from molecular dynamics simulations, providing a route to ensemble refinement against experimental spectra. We performed a Bayesian ensemble refinement method on Ala-Ala-Ala against isotope-edited Fourier-transform infrared spectroscopy and 2D IR spectroscopy and tested potential factors affecting the quality of ensemble refinements. We found that isotope-edited 2D IR spectroscopy provides a stringent constraint on Ala-Ala-Ala conformations and returns consistent conformational ensembles with the dominant ppII conformer across varying prior distributions from many molecular dynamics force fields and water models. The dominant factor influencing ensemble refinements is the systematic frequency uncertainty from spectroscopic maps. However, the uncertainty of conformer populations can be significantly reduced by incorporating 2D IR spectra in addition to traditional Fourier-transform infrared spectra. Bayesian ensemble refinement against isotope-edited 2D IR spectroscopy thus provides a route to probe equilibrium-complex protein ensembles and potentially nonequilibrium conformational dynamics.

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.

Insulin replacement therapy for diabetes mellitus seeks to minimise excursions in blood glucose concentration above or below the therapeutic range (hyper-or hypoglycaemia). To mitigate acute and chronic risks of such excursions, glucose-responsive insulindelivery technologies have long been sought for clinical application in type 1 and long-standing type 2 diabetes mellitus. Such 'smart' systems or insulin analogues seek to provide hormonal activity proportional to blood glucose levels without external monitoring. This review highlights three broad strategies to co-optimise mean glycaemic control and time in range: (1) coupling of continuous glucose monitoring (CGM) to delivery devices (algorithm-based 'closed-loop' systems); (2) glucose-responsive polymer encapsulation of insulin; and (3) mechanism-based hormone modifications. Innovations span control algorithms for CGM-based insulin-delivery systems, glucose-responsive polymer matrices, bio-inspired design based on insulin's conformational switch mechanism upon insulin receptor engagement, and glucose-responsive modifications of new insulin analogues. In each case, innovations in insulin chemistry and formulation may enhance clinical outcomes. Prospects are discussed for intrinsic glucose-responsive insulin analogues containing a reversible switch (regulating bioavailability or conformation) that can be activated by glucose at high concentrations.

Isotope-edited two-dimensional Fourier transform infrared spectroscopy (2D FTIR) can potentially provide a unique probe of protein structure and dynamics. However, general methods for the site-specific incorporation of stable 13C=18O labels into the polypeptide backbone of the protein molecule have not yet been established. Here we describe, as a prototype for the incorporation of specific arrays of isotope labels, the total chemical synthesis – via a key ester insulin intermediate – of 97% enriched [(1-13C=18O)PheB24]human insulin, stable-isotope labeled at a single backbone amide carbonyl. The amino acid sequence as well as the positions of the disulfide bonds and the correctly folded structure were unambiguously confirmed by the X-ray crystal structure of the synthetic protein molecule. In vitro assays of the isotope labeled [(1-13C=18O)PheB24]human insulin showed that it had full insulin receptor binding activity. Linear and 2D IR spectra revealed a distinct red-shifted amide I carbonyl band peak at 1595 cm−1 resulting from the (1-13C=18O)PheB24 backbone label. This work illustrates the utility of chemical synthesis to enable the application of advanced physical methods for the elucidation of the molecular basis of protein function.

Abnormal interactions between misfolded mutant and wild-type (WT) proinsulin (PI) in the endoplasmic reticulum (ER) drive the molecular pathogenesis of mutant INS gene-induced diabetes of youth (MIDY). How these abnormal interactions are initiated remains unknown. Normally, PI-WT dimerizes in the ER. Here, we suggest that the normal PI-PI contact surface, involving the B-chain, contributes to dominant-negative effects of misfolded MIDY mutants. Specifically, we find that PI B-chain tyrosine-16 (Tyr-B16), which is a key residue in normal PI dimerization, helps confer dominant-negative behavior of MIDY mutant PI-C(A7)Y. Substitutions of Tyr-B16 with either Ala, Asp, or Pro in PI-C(A7)Y decrease the abnormal interactions between the MIDY mutant and PI-WT, rescuing PI-WT export, limiting ER stress, and increasing insulin production in b-cells and human islets. This study reveals the first evidence indicating that noncovalent PI-PI contact initiates dominant-negative behavior of misfolded PI, pointing to a novel therapeutic target to enhance PI-WT export and increase insulin production.

Insight into folding mechanisms of proinsulin has been provided by analysis of dominant diabetes-associated mutations in the human insulin gene (INS). Such mutations cause pancreatic β-cell dysfunction due to toxic misfolding of a mutant proinsulin and impairment in trans of wild-type insulin secretion. Anticipated by the “Akita” mouse (a classical model of monogenic diabetes mellitus; DM), this syndrome illustrates the paradigm endoreticulum (ER) stress leading to intracellular proteotoxicity. Diverse clinical mutations directly or indirectly perturb native disulfide pairing leading to protein misfolding and aberrant aggregation. Although most introduce or remove a cysteine (Cys; leading in either case to an unpaired thiol group), non-Cys-related mutations identify key determinants of folding efficiency. Studies of such mutations suggest that the hormone’s evolution has been constrained not only by structure-function relationships, but also by the susceptibility of its single-chain precursor to impaired foldability. An intriguing hypothesis posits that INS overexpression in response to peripheral insulin resistance likewise leads to chronic ER stress and β-cell dysfunction in the natural history of non-syndromic Type 2 DM. Cryptic contributions of conserved residues to folding efficiency, as uncovered by rare genetic variants, define molecular links between biophysical principles and the emerging paradigm of Darwinian medicine: Biosynthesis of proinsulin at the edge of non-foldability provides a key determinant of “diabesity” as a pandemic disease of civilization.

Globular protein sequences encode not only functional structures (the native state) but also protein foldability, i.e. a conformational search that is both efficient and robustly minimizes misfolding. Studies of mutations associated with toxic misfolding have yielded insights into molecular determinants of protein foldability. Of particular interest are residues that are conserved yet dispensable in the native state. Here, we exploited the mutant proinsulin syndrome (a major cause of permanent neonatal-onset diabetes mellitus) to investigate whether toxic misfolding poses an evolutionary constraint. Our experiments focused on an invariant aromatic motif (PheB24–PheB25–TyrB26) with complementary roles in native self-assembly and receptor binding. A novel class of mutations provided evidence that insulin can bind to the insulin receptor (IR) in two different modes, distinguished by a “register shift” in this motif, as visualized by molecular dynamics (MD) simulations. Register-shift variants are active but defective in cellular foldability and exquisitely susceptible to fibrillation in vitro. Indeed, expression of the corresponding proinsulin variant induced endoplasmic reticulum stress, a general feature of the mutant proinsulin syndrome. Although not present among vertebrate insulin and insulin-like sequences, a prototypical variant ([GlyB24]insulin) was as potent as WT insulin in a rat model of diabetes. Although in MD simulations the shifted register of receptor engagement is compatible with the structure and allosteric reorganization of the IR-signaling complex, our results suggest that this binding mode is associated with toxic misfolding and so is disallowed in evolution. The implicit threat of proteotoxicity limits sequence variation among vertebrate insulins and insulin-like growth factors.

The mutant proinsulin syndrome is a monogenic cause of diabetes mellitus due to toxic misfolding of insulin’s biosynthetic precursor. Also designated mutant INS-gene induced diabetes of the young (MIDY), this syndrome defines molecular determinants of foldability in the endoplasmic reticulum (ER) of β-cells. Here, we describe a peptide model of a key proinsulin folding intermediate and variants containing representative clinical mutations; the latter perturb invariant core sites in native proinsulin (LeuB15→Pro, LeuA16→Pro, and PheB24→Ser). The studies exploited a 49-residue single-chain synthetic precursor (designated DesDi), previously shown to optimize in vitro efficiency of disulfide pairing. Parent and variant peptides contain a single disulfide bridge (cystine B19-A20) to provide a model of proinsulin’s first oxidative folding intermediate. The peptides were characterized by circular dichroism and redox stability in relation to effects of the mutations on (a) in vitro foldability of the corresponding insulin analogs and (b) ER stress induced in cell culture on expression of the corresponding variant proinsulins. Striking correlations were observed between peptide biophysical properties, degree of ER stress and age of diabetes onset (neonatal or adolescent). Our findings suggest that age of onset reflects the extent to which nascent structure is destabilized in proinsulin’s putative folding nucleus. We envisage that such peptide models will enable high-resolution structural studies of key folding determinants and in turn permit molecular dissection of phenotype-genotype relationships in this monogenic diabetes syndrome. Our companion study (next article in this issue) employs two-dimensional heteronuclear NMR spectroscopy to define site-specific perturbations in the variant peptides.