A TeZla micromixer for interrogating the early and broad folding landscape of G-quadruplex via multistage velocity descending.

Biomacromolecular folding kinetics involves fast folding events and broad timescales. Current techniques face limitations in either the required time resolution or the observation window. In this study, we developed the TeZla micromixer, integrating Tesla and Zigzag microstructures with a multistage velocity descending strategy. TeZla achieves a significant short mixing dead time (40 µs) and a wide time window covering four orders of magnitude (up to 300 ms). Using this unique micromixer, we explored the folding landscape of c-Myc G4 and its noncanonical-G4 derivatives with different loop lengths or G-vacancy sites. Our findings revealed that c-Myc can bypass folding intermediates and directly adopt a G4 structure in the cation-deficient buffer. Moreover, we found that the loop length and specific G-vacancy site could affect the folding pathway and significantly slow down the folding rates. These results were also cross-validated with real-time NMR and circular dichroism. In conclusion, TeZla represents a versatile tool for studying biomolecular folding kinetics, and our findings may ultimately contribute to the design of drugs targeting G4 structures.

Circular permutation at azurin's active site slows down its folding.

Circular permutation (CP) is a technique by which the primary sequence of a protein is rearranged to create new termini. The connectivity of the protein is altered but the overall protein structure generally remains unperturbed. Understanding the effect of CP can help design robust proteins for numerous applications such as in genetic engineering, optoelectronics, and improving catalytic activity. Studies on different protein topologies showed that CP usually affects protein stability as well as unfolding rates. Though a significant number of proteins contain metals or other cofactors, reports of metalloprotein CPs are rare. Thus, we chose a bacterial metalloprotein, azurin, and its CP within the metal-binding site (cpF114). We studied the stabilities, folding, and unfolding rates of apo- and Zn2+-bound CP azurin using fluorescence and circular dichroism. The introduced CP had destabilizing effects on the protein. Also, the folding of the Zn2+-CP protein was much slower than that of the Zn2+-WT or apo-protein. We compared this study to our previously reported azurin-cpN42, where we had observed an equilibrium and kinetic intermediate. cpF114 exhibits an apparent two-state equilibrium unfolding but has an off-pathway kinetic intermediate. Our study hinted at CP as a method to modify the energy landscape of proteins to alter their folding pathways. WT azurin, being a faster folder, may have evolved to optimize the folding rate of metal-bound protein compared to its CPs, albeit all of them have the same structure and function. Our study underscores that protein sequence and protein termini positions are crucial for metalloproteins. TOC Figure. (Top) Zn2+-azurin WT structure (PDB code: 1E67) and 2-D topology diagram of Zn2+-cpF114 azurin. (Bottom) Cartoon diagram representing folding (red arrows) and unfolding (blue arrows) of apo- and Zn2+- WT and cpF114 azurins. The width of the arrows represents the rate of the corresponding processes.

Non-Native Structures of Apomyoglobin and Apoleghemoglobin in Folding Intermediates Related to the Protein Misfolding.

Protein folding is essential for a polypeptide chain to acquire its proper structure and function. Globins are a superfamily of ubiquitous heme-binding α-helical proteins whose function is principally to regulate oxygen homoeostasis. In this review, we explore the hierarchical helical formation in the globin proteins apomyoglobin and leghemoglobin, and we discuss the existence of non-native and misfolded structures occurring during the course of folding to its native state. This review summarizes the research aimed at characterizing and comparing the equilibrium and kinetic intermediates, as well as delineating the complete folding pathway at a molecular level, in order to answer the following questions: "What is the mechanism of misfolding via a folding intermediate? Does the non-native structure stabilize the contemporary intermediate structure? Does the non-native structure induce slower folding?" The role of the non-native structures in the folding intermediate related to misfolding is also discussed.

A Novel Peptide Reagent for Investigating Disulfide-Coupled Folding Intermediates of Mid-Size Proteins.

Investigations of protein folding have largely involved the use of disulfide-containing proteins, since the disulfide-coupled folding of proteins allows folding intermediates to be trapped and their conformations determined. However, studies of the folding mechanisms of mid-size proteins face several problems, one of which is that detecting folding intermediates is difficult. Therefore, to solve this issue, a novel peptide reagent, maleimidohexanoyl-Arg5-Tyr-NH2, was designed and applied to the detection of folding intermediates of model proteins. BPTI was chosen as a model small protein to estimate the ability of the novel reagent to detect folding intermediates. In addition, a precursor protein (prococoonase) of Bombyx mori cocoonase was used as a model mid-size protein. Cocoonase is classified as a serine protease and has a high homology with trypsin. We recently found that the propeptide sequence of prococoonase (proCCN) is important for the folding of cocoonase. However, it was difficult to study the folding pathway of proCCN since the folding intermediates could not be separated on a reversed-phase HPLC (RP-HPLC). Therefore, to separate the folding intermediates by RP-HPLC, the novel labeling reagent was used to accomplish this for proCCN. The results indicated that the peptide reagent allowed the intermediates to be captured, separated on SDS-PAGE, and analyzed by RP-HPLC without the occurrence of undesirable disulfide-exchange reactions during the labeling reactions. The peptide reagent reported herein is a practical tool for investigating the mechanisms of disulfide-coupled folding of mid-size proteins.

Folding Mechanism and Aggregation Propensity of the KH0 Domain of FMRP and Its R138Q Pathological Variant.

The K-homology (KH) domains are small, structurally conserved domains found in proteins of different origins characterized by a central conserved ßααß "core" and a GxxG motif in the loop between the two helices of the KH core. In the eukaryotic KHI type, additional αß elements decorate the "core" at the C-terminus. Proteins containing KH domains perform different functions and several diseases have been associated with mutations in these domains, including those in the fragile X mental retardation protein (FMRP). FMRP is an RNA-binding protein crucial for the control of RNA metabolism whose lack or mutations lead to fragile X syndrome (FXS). Among missense mutations, the R138Q substitution is in the KH0 degenerated domain lacking the classical GxxG motif. By combining equilibrium and kinetic experiments, we present a characterization of the folding mechanism of the KH0 domain from the FMRP wild-type and of the R138Q variant showing that in both cases the folding mechanism implies the accumulation of an on-pathway transient intermediate. Moreover, by exploiting a battery of biophysical techniques, we show that the KH0 domain has the propensity to form amyloid-like aggregates in mild conditions in vitro and that the R138Q mutation leads to a general destabilization of the protein and to an increased fibrillogenesis propensity.

The role of the IT-state in D76N ß2-microglobulin amyloid assembly: A crucial intermediate or an innocuous bystander?

The D76N variant of human ß2-microglobulin (ß2m) is the causative agent of a hereditary amyloid disease. Interestingly, D76N-associated amyloidosis has a distinctive pathology compared with aggregation of WT-ß2m, which occurs in dialysis-related amyloidosis. A folding intermediate of WT-ß2m, known as the IT-state, which contains a nonnative trans Pro-32, has been shown to be a key precursor of WT-ß2m aggregation in vitro However, how a single amino acid substitution enhances the rate of aggregation of D76N-ß2m and gives rise to a different amyloid disease remained unclear. Using real-time refolding experiments monitored by CD and NMR, we show that the folding mechanisms of WT- and D76N-ß2m are conserved in that both proteins fold slowly via an IT-state that has similar structural properties. Surprisingly, however, direct measurement of the equilibrium population of IT using NMR showed no evidence for an increased population of the IT-state for D76N-ß2m, ruling out previous models suggesting that this could explain its enhanced aggregation propensity. Producing a kinetically trapped analog of IT by deleting the N-terminal six amino acids increases the aggregation rate of WT-ß2m but slows aggregation of D76N-ß2m, supporting the view that although the folding mechanisms of the two proteins are conserved, their aggregation mechanisms differ. The results exclude the IT-state as the origin of the rapid aggregation of D76N-ß2m, suggesting that other nonnative states must cause its high aggregation rate. The results highlight how a single substitution at a solvent-exposed site can affect the mechanism of aggregation and the resulting disease.

Study of protein folding under native conditions by rapidly switching the hydrostatic pressure inside an NMR sample cell.

In general, small proteins rapidly fold on the timescale of milliseconds or less. For proteins with a substantial volume difference between the folded and unfolded states, their thermodynamic equilibrium can be altered by varying the hydrostatic pressure. Using a pressure-sensitized mutant of ubiquitin, we demonstrate that rapidly switching the pressure within an NMR sample cell enables study of the unfolded protein under native conditions and, vice versa, study of the native protein under denaturing conditions. This approach makes it possible to record 2D and 3D NMR spectra of the unfolded protein at atmospheric pressure, providing residue-specific information on the folding process. 15N and 13C chemical shifts measured immediately after dropping the pressure from 2.5 kbar (favoring unfolding) to 1 bar (native) are close to the random-coil chemical shifts observed for a large, disordered peptide fragment of the protein. However, 15N relaxation data show evidence for rapid exchange, on a ∼100-µs timescale, between the unfolded state and unstable, structured states that can be considered as failed folding events. The NMR data also provide direct evidence for parallel folding pathways, with approximately one-half of the protein molecules efficiently folding through an on-pathway kinetic intermediate, whereas the other half fold in a single step. At protein concentrations above ∼300 µM, oligomeric off-pathway intermediates compete with folding of the native state.

Druggable Transient Pockets in Protein Kinases.

Drug discovery using small molecule inhibitors is reaching a stalemate due to low selectivity, adverse off-target effects and inevitable failures in clinical trials. Conventional chemical screening methods may miss potent small molecules because of their use of simple but outdated kits composed of recombinant enzyme proteins. Non-canonical inhibitors targeting a hidden pocket in a protein have received considerable research attention. Kii and colleagues identified an inhibitor targeting a transient pocket in the kinase DYRK1A during its folding process and termed it FINDY. FINDY exhibits a unique inhibitory profile; that is, FINDY does not inhibit the fully folded form of DYRK1A, indicating that the FINDY-binding pocket is hidden in the folded form. This intriguing pocket opens during the folding process and then closes upon completion of folding. In this review, we discuss previously established kinase inhibitors and their inhibitory mechanisms in comparison with FINDY. We also compare the inhibitory mechanisms with the growing concept of "cryptic inhibitor-binding sites." These sites are buried on the inhibitor-unbound surface but become apparent when the inhibitor is bound. In addition, an alternative method based on cell-free protein synthesis of protein kinases may allow the discovery of small molecules that occupy these mysterious binding sites. Transitional folding intermediates would become alternative targets in drug discovery, enabling the efficient development of potent kinase inhibitors.

Insight into the Folding and Dimerization Mechanisms of the N-Terminal Domain from Human TDP-43.

TAR DNA-binding protein 43 (TDP-43) is a 414-residue long nuclear protein whose deposition into intraneuronal insoluble inclusions has been associated with the onset of amyotrophic lateral sclerosis (ALS) and other diseases. This protein is physiologically a homodimer, and dimerization occurs through the N-terminal domain (NTD), with a mechanism on which a full consensus has not yet been reached. Furthermore, it has been proposed that this domain is able to affect the formation of higher molecular weight assemblies. Here, we purified this domain and carried out an unprecedented characterization of its folding/dimerization processes in solution. Exploiting a battery of biophysical approaches, ranging from FRET to folding kinetics, we identified a head-to-tail arrangement of the monomers within the dimer. We found that folding of NTD proceeds through the formation of a number of conformational states and two parallel pathways, while a subset of molecules refold slower, due to proline isomerism. The folded state appears to be inherently prone to form high molecular weight assemblies. Taken together, our results indicate that NTD is inherently plastic and prone to populate different conformations and dimeric/multimeric states, a structural feature that may enable this domain to control the assembly state of TDP-43.

Determination of the structural ensemble of the molten globule state of a protein by computer simulations.

We have used computer simulations to investigate the structural nature of the molten globule (MG) state of canine milk lysozyme. To sample the conformational space efficiently, we performed replica-exchange umbrella sampling simulations with the radius of gyration as a reaction coordinate. We applied the Weighted Histogram Analysis Method to the trajectory of the simulations to obtain the potential of mean force, from which we identified representative structures corresponding to local minima in the free energy surface. The representative structures obtained in this way are in accord with the characteristics of the MG state reported previously by experimental studies. We conjecture that the MG state comprises a series of partially structured states undergoing relatively fast conformational interchange.

FRET studies of various conformational states adopted by transthyretin.

Transthyretin (TTR) is an extracellular protein able to deposit into well-defined protein aggregates called amyloid, in pathological conditions known as senile systemic amyloidosis, familial amyloid polyneuropathy, familial amyloid cardiomyopathy and leptomeningeal amyloidosis. At least three distinct partially folded states have been described for TTR, including the widely studied amyloidogenic state at mildly acidic pH. Here, we have used fluorescence resonance energy transfer (FRET) experiments in a monomeric variant of TTR (M-TTR) and in its W41F and W79F mutants, taking advantage of the presence of a unique, solvent-exposed, cysteine residue at position 10, that we have labelled with a coumarin derivative (DACM, acceptor), and of the two natural tryptophan residues at positions 41 and 79 (donors). Trp41 is located in an ideal position as it is one of the residues of ß-strand C, whose degree of unfolding is debated. We found that the amyloidogenic state at low pH has the same FRET efficiency as the folded state at neutral pH in both M-TTR and W79F-M-TTR, indicating an unmodified Cys10-Trp41 distance. The partially folded state populated at low denaturant concentrations also has a similar FRET efficiency, but other spectroscopic probes indicate that it is distinct from the amyloidogenic state at acidic pH. By contrast, the off-pathway state accumulating transiently during refolding has a higher FRET efficiency, indicating non-native interactions that reduce the Cys10-Trp41 spatial distance, revealing a third distinct conformational state. Overall, our results clarify a negligible degree of unfolding of ß-strand C in the formation of the amyloidogenic state and establish the concept that TTR is a highly plastic protein able to populate at least three distinct conformational states.

Substitutions mimicking deimination and phosphorylation of 18.5-kDa myelin basic protein exert local structural effects that subtly influence its global folding.

Intrinsically-disordered proteins (IDPs) present a complex interplay of conformational variability and multifunctionality, modulated by environment and post-translational modifications. The 18.5-kDa myelin basic protein (MBP) is essential to the formation of the myelin sheath of the central nervous system and is exemplary in this regard. We have recently demonstrated that the unmodified MBP-C1 component undergoes co-operative global conformational changes in increasing concentrations of trifluoroethanol, emulating the decreasing dielectric environment that the protein encounters upon adsorption to the oligodendrocyte membrane [K.A. Vassall et al., Journal of Molecular Biology, 427, 1977-1992, 2015]. Here, we extended this study to the pseudo-deiminated MBP-C8 charge component, one found in greater proportion in developing myelin and in multiple sclerosis. A similar tri-conformational distribution as for MBP-C1 was observed with slight differences in Gibbs free energy. A more dramatic difference was observed by cathepsin D digestion of the protein in both aqueous and membrane environments, which showed significantly greater accessibility of the F42-F43 cut site of MBP-C8, indicative of a global conformational change. In contrast, this modification caused little change in the protein's density of packing on myelin-mimetic membranes as ascertained by double electron-electron resonance spectroscopy [D.R. Kattnig et al., Biochimica et Biophysica Acta (Biomembranes), 1818, 2636-2647, 2012], or in its affinity for Ca(2+)-CaM. Site-specific threonyl pseudo-phosphorylation at residues T92 and/or T95 did not appreciably affect any of the thermodynamic mechanisms of conformational transitions, susceptibility to cathepsin D, or affinity for Ca(2+)-CaM, despite previously having been shown to affect local structure and disposition on the membrane surface.

Measurement of histidine pKa values and tautomer populations in invisible protein states.

The histidine imidazole side chain plays a critical role in protein function and stability. Its importance for catalysis is underscored by the fact that histidines are localized to active sites in ∼ 50% of all enzymes. NMR spectroscopy has become an important tool for studies of histidine side chains through the measurement of site-specific pK(a)s and tautomer populations. To date, such studies have been confined to observable protein ground states; however, a complete understanding of the role of histidine electrostatics in protein function and stability requires that similar investigations be extended to rare, transiently formed conformers that populate the energy landscape, yet are often "invisible" in standard NMR spectra. Here we present NMR experiments and a simple strategy for studies of such conformationally excited states based on measurement of histidine (13)Cγ, (13)Cδ2 chemical shifts and (1)Hε-(13)Cε one-bond scalar couplings. The methodology is first validated and then used to obtain pKa values and tautomer distributions for histidine residues of an invisible on-pathway folding intermediate of the colicin E7 immunity protein. Our results imply that the side chains of H40 and H47 are exposed in the intermediate state and undergo significant conformational rearrangements during folding to the native structure. Further, the pKa values explain the pH-dependent stability differences between native and intermediate states over the pH range 5.5-6.5 and they suggest that imidazole deprotonation is not a barrier to the folding of this protein.

Proline Residues as Switches in Conformational Changes Leading to Amyloid Fibril Formation.

Here we discuss studies of the structure, folding, oligomerization and amyloid fibril formation of several proline mutants of human stefin B, which is a protein inhibitor of lysosomal cysteine cathepsins and a member of the cystatin family. The structurally important prolines in stefin B are responsible for the slow folding phases and facilitate domain swapping (Pro 74) and loop swapping (Pro 79). Moreover, our findings are compared to ß2-microglobulin, a protein involved in dialysis-related amyloidosis. The assessment of the contribution of proline residues to the process of amyloid fibril formation may shed new light on the critical molecular events involved in conformational disorders.

Effects of Metal Ions, Temperature, and a Denaturant on the Oxidative Folding Pathways of Bovine α-Lactalbumin.

Bovine α-lactalbumin (αLA) has four disulfide (SS) bonds in the native form (N). On the oxidative folding pathways of this protein, two specific SS folding intermediates, i.e., (61-77, 73-91) and des[6-120], which have two and three native SS bonds, respectively, accumulate predominantly in the presence of Ca2+. In this study, we reinvestigated the pathways using a water-soluble cyclic selenoxide reagent, trans-3,4-dihydroxyselenolane oxide (DHSox), as a strong and quantitative oxidant to oxidize the fully reduced form (R). In the presence of ethylenediaminetetraacetic acid (EDTA) (under a metal-free condition), SS formation randomly proceeded, and N did not regenerate. On the other hand, two specific SS intermediates transiently generated in the presence of Ca2+. These intermediates could be assigned to (61-77, 73-91) and des[6-120] having two common SS bonds, i.e., Cys61-Cys77 and Cys73-Cys91, near the calcium binding pocket of the ß-sheet domain. Much faster folding to N was observed in the presence of Mn2+, whereas Na⁺, K⁺, Mg2+, and Zn2+ did not affect the pathways. The two key intermediates were susceptible to temperature and a denaturant. The oxidative folding pathways revealed were significantly different from those of hen egg white lysozyme, which has the same SS-bonding pattern as αLA, suggesting that the folding pathways of SS-containing proteins can alter depending on the amino acid sequence and other factors, even when the SS-bond topologies are similar to each other.

Differential effects of disulfide bond formation in TEM-1 versus CTX-M-9 ß-lactamase.

To investigate how disulfide bonds can impact protein energy landscapes, we surveyed the effects of adding or removing a disulfide in two ß-lactamase enzymes, TEM-1 and CTX-M-9. The homologs share a structure and 38% sequence identity, but only TEM-1 contains a native disulfide bond. They also differ in thermodynamic stability and in the number of states populated at equilibrium: CTX-M-9 is two-state whereas TEM-1 has an additional intermediate state. We hypothesized that the disulfide bond is the major underlying determinant for these observed differences in their energy landscapes. To test this, we removed the disulfide bridge from TEM-1 and introduced a disulfide bridge at the same location in CTX-M-9. This modest change to sequence modulates the stabilities-and therefore populations-of TEM-1's equilibrium states and, more surprisingly, creates a novel third state in CTX-M-9. Unlike TEM-1's partially folded intermediate, this third state is a higher-order oligomer with reduced cysteines that retains the native fold and is fully active. Sub-denaturing concentrations of urea shifts the equilibrium to the monomeric form, allowing the disulfide bond to form. Interestingly, comparing the stability of the oxidized monomer with a variant lacking cysteines reveals the disulfide is neither stabilizing nor destabilizing in CTX-M-9, in contrast with the observed stabilization in TEM-1. Thus, we can conclude that engineering disulfide bonds is not always an effective stabilization strategy even when analogous disulfides exist in more stable structural homologs. This study also illustrates how homo-oligomerization can result from a small number of mutations, suggesting complex formation might be easily accessed during a protein family's evolution.

Nanopore single-molecule biosensor in protein denaturation analysis.

Unclear issues in protein studies include but not limited to the stability and denaturation mechanism in the presence of denaturants. Herein, we report a dynamic monitoring approach based on nanopore single-molecule biosensor, which can detect the protein's folding and unfolding transitions by recording a nanopore ionic current. When gradually increasing the concentration of denaturant guanidine hydrochloride (GdmCl), sensitive responses were observed with lysozyme unfolding. The emergence of the featured biphasic-pulse demonstrated the existence of a stable intermediate. It was the first time to experimentally confirm the dynamic equilibrium between the intermediate and the native states at single molecule level, therefore consolidating the standpoint of lysozyme denaturation process following the three-state model. Additionally, we got more insights into the conformation about the intermediate as globular-like structure, larger gyration radius, and enhanced positive charge density. We considered that the manner of denaturant toward lysozyme adopts the "direct" model based on stronger electrostatic and van der Waals forces. Nanopore biosensor exhibited excellent sensitivity with a low detection concentration of 280 pM and reproducibility in analysing the folding intermediate of lysozyme.

Structure-activity relationship for the folding intermediate-selective inhibition of DYRK1A.

DYRK1A phosphorylates proteins involved in neurological disorders in an intermolecular manner. Meanwhile, during the protein folding process of DYRK1A, a transitional folding intermediate catalyzes the intramolecular autophosphorylation required for the "one-off" inceptive activation and stabilization. In our previous study, a small molecule termed FINDY (1) was identified, which inhibits the folding intermediate-catalyzed intramolecular autophosphorylation of DYRK1A but not the folded state-catalyzed intermolecular phosphorylation. However, the structural features of FINDY (1) responsible for this intermediate-selective inhibition remain elusive. In this study, structural derivatives of FINDY (1) were designed and synthesized according to its predicted binding mode in the ATP pocket of DYRK1A. Quantitative structure-activity relationship (QSAR) of the derivatives revealed that the selectivity against the folding intermediate is determined by steric hindrance between the bulky hydrophobic moiety of the derivatives and the entrance to the pocket. In addition, a potent derivative 3 was identified, which inhibited the folding intermediate more strongly than FINDY (1); it was designated as dp-FINDY. Although dp-FINDY (3) did not inhibit the folded state, as well as FINDY (1), it inhibited the intramolecular autophosphorylation of DYRK1A in an in vitro cell-free protein synthesis assay. Furthermore, dp-FINDY (3) destabilized endogenous DYRK1A in HEK293 cells. This study provides structural insights into the folding intermediate-selective inhibition of DYRK1A and expands the chemical options for the design of a kinase inhibitor.

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

Toxic misfolding of proinsulin variants in ß-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 ß-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.

Peptide Model of the Mutant Proinsulin Syndrome. I. Design and Clinical Correlation.

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 ß-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.