The thermodynamic stability of insulin disulfides is not affected by the C-domain of insulin-like growth factor 1

Both Insulin and insulin-like growth factor 1 are members of insulin superfamily. They share homologous primary and tertiary structure as well as weakly overlapping biological activity. However, their folding behavior is different: insulin and its recombinant precursor (PIP) fold into one unique tertiary structure, while IGF-1 folds into two disulfides isomers with similar thermody-namic stability. To elucidate the molecular mechanism of their different folding behavior, we prepared a single-chain hybrid of insulin and IGF-1, [B10Glu]lns/IGF-1(C), and studied its folding behavior compared with that of PIP and IGF-1. We also separated a major non-native disulfides iso-mer of the hybrid and studied its refolding. The data showed that the C-domain of IGF-1 did not affect the folding thermodynamics of insulin, that is, the primary structure of the hybrid encoded only one thermodynamically stable disulfides linkage. However, the folding kinetics of insulin was affected by the C-domain of IGF-1.     

The different folding behavior of insulin and insulin-like growth factor 1 is mainly controlled by their B-chain/domain

Although insulin and insulin-like growth factor 1 (IGF-1) share homologous sequence, similar tertiary structure, weakly overlapped biological activity, and a common ancestor, the two highly homologous sequences encode different folding behavior: insulin folds into one unique stable tertiary structure while IGF-1 folds into two disulfide isomers with similar thermodynamic stability. To further elucidate the molecular mechanism of their different folding behavior, we prepared two single-chain hybrids of insulin and IGF-1, Ins(A)/IGF-1(B) and Ins(B)/IGF-1(A), as well as a mini-IGF-1 by means of protein engineering and studied their structure as well as folding behavior. Both mini-IGF-1 and Ins(A)/IGF-1(B) fold into two thermodynamically stable disulfide isomers in vivo and in vitro just like that of IGF-1, while Ins(B)/IGF-1(A) folds into one unique thermodynamically stable tertiary structure in vivo and in vitro just like that of insulin. So we deduce that the different folding behavior of insulin and IGF-1 is mainly controlled by their B-chain/domain. By V8 endoproteinase digestion and circular dichroism analysis, as well as insulin receptor binding assay, we deduce that Ins(B)/IGF-1(A), isomer 2 of mini-IGF-1, and isomer 2 of Ins(A)/IGF-1(B) adopt native IGF-1/insulin-like three-dimensional structure with native disulfides, while isomer 1 of mini-IGF-1 and isomer 1 of Ins(A)/IGF-1(B) adopt the swap IGF-1-like three-dimensional structure with swap disulfides.     

The sequence determinant causing different folding behaviors of insulin and insulin-like growth factor-1

Although insulin and insulin-like growth factor-1 (IGF-1) belong to the insulin superfamily and share highly homologous sequences, similar tertiary structure, and a common ancestor molecule, amphioxus insulin-like peptide, they have different folding behaviors: IGF-1 folds into two thermodynamically stable tertiary structures (native and swap forms), while insulin folds into one unique stable structure. To further understand which part of the sequence determines their different folding behavior, based on previous reports from the laboratory, two peptide models, [B9A][1-4]porcine insulin precursor (PIP) and [B10E][1-4]PIP, were constructed. The plasmids encoding the peptides were transformed into yeast cells for expression of the peptides; the results showed that the former peptide was expressed as single component, while the latter was expressed as a mixture of two components (isomer 1 and isomer 2). The expression results together with studies of circular dichoism, disulfide rearrangement, and refolding lead us to deduce that isomer 1 corresponds to the swap form and the isomer 2 corresponds to the native form. We further demonstrate that the sequence 1-4 plus B9 of IGF-1 B-domain can make PIP fold into two structures, while sequence 1-5 of insulin B-chain can make IGF-1 fold into one unique structure. In other words, it is the IGF-1 B-domain sequence that 1-4 allows IGF-1 folding into two thermodynamically stable tertiary structures; this sequence plus its residue B9E can change PIP folding behavior from folding into one unique structure to two thermodynamically stable structures, like that of IGF-1.     

Refolding of amphioxus insulin-like peptide: implications of a bifurcating evolution of the different folding behavior of insulin and insulin-like growth factor 1

Insulin and insulin-like growth factor 1 (IGF-1) share high sequence homology, but their folding behaviors are significantly different: insulin folds into one unique thermodynamically controlled structure, while IGF-1 folds into two thermodynamically controlled disulfide isomers. However, the origin of their different folding behaviors is still elusive. The amphioxus insulin-like peptide (ILP) is thought to be the common ancestor of insulin and IGF-1. A recombinant single-chain ILP has been expressed previously, and now its folding behavior is investigated. The folding behavior of ILP shows the characteristics of both insulin and IGF-1. On one hand, two thermodynamically controlled disulfide isomers of ILP have been identified; on the other hand, the content of isomer 1 (its disulfides are deduced identical to those of swap IGF-1) is much less than that of isomer 2 (its disulfides are deduced identical to those of native IGF-1); that is, more than 96% of ILP folds into the native structure. The present results suggest that the different folding behaviors of insulin and IGF-1 are acquired through a bifurcating evolution: the tendency of forming the thermodynamically controlled non-native disulfide isomer is diminished during evolution from ILP to insulin, while this tendency is amplified during evolution from ILP to IGF-1. Moreover, the N-terminal Gln residue of ILP can spontaneously form a pyroglutamate residue, and its cyclization has a significant effect on the folding behavior of ILP: the percentage of isomer 1 is approximately 2-fold that of isomer 1 of the noncyclized ILP; that is, isomer 1 becomes more favored when the N-terminal residue of ILP is cyclized. So, we deduce that the N-terminal residues have a significant effect on the folding properties of insulin, IGF-1, and ILP.     

Sequences of B-chain/domain 1-10/1-9 of insulin and insulin-like growth factor 1 determine their different folding behavior

Although insulin and insulin-like growth factor-1 (IGF-1) belong to one family, insulin folds into one thermodynamically stable structure, while IGF-1-folds into two thermodynamically stable structures (native and swap forms). We have demonstrated previously that the bifurcating folding behavior of IGF-1 is mainly controlled by its B-domain. To further elucidate which parts of the sequences determine their different folding behavior, by exchanging the N-terminal sequences of mini-IGF-1 and recombinant porcine insulin precursor (PIP), we prepared four peptide models: [1-9]PIP, [1-10]mini-IGF-1, [1-4]PIP, and [1-5]mini-IGF-1 by means of protein engineering, and their disulfide rearrangement, V8 digestion, circular dichroic spectra, disulfide stability, and in vitro refolding were investigated. Among them only [1-9]PIP, like mini-IGF-1/IGF-1, was expressed in yeast as two isomers: isomer 1 (corresponding to swap IGF-1) and isomer 2 (corresponding to native IGF-1), which are supported by the experimental results of disulfide rearrangements, peptide mapping of V8 endoprotenase digests, circular dichroic analysis, in vitro refolding, and disulfide stability analysis. The other peptide models, [1-10]mini-IGF-1, [1-4]PIP, and [1-5]mini-IGF-1, fold into one stable structure as PIP does, which indicates that sequence 1-4 of mini-IGF-1 is important for the folding behavior of mini-IGF-1/IGF-1 but not sufficient to lead to a bifurcating folding. The results demonstrated that the folding information, by which mini-IGF-1/IGF-1-folds into two thermodynamically structures, is encoded/written in its sequence 1-9, while sequences 1-10 of B chain in insulin/PIP play an important role in the guide of its unique disulfide pairing during the folding process.     

The different energetic state of the intra A-chain/domain disulfide of insulin and insulin-like growth factor 1 is mainly controlled by their B-chain/domain

Insulin and insulin-like growth factor 1 (IGF-1) share homologous sequence, similar three-dimensional structure, and weakly overlapping biological activity, but different folding information is stored in their homologous sequences: the sequence of insulin encodes one unique thermodynamically stable three-dimensional structure while that of IGF-1 encodes two disulfide isomers with different three-dimensional structure but similar thermodynamic stability. Their different folding behavior probably resulted from the different energetic state of the intra A-chain/domain disulfide: the intra A-chain disulfide of insulin is a stable bond while that of IGF-1 is a strained bond with high energy. To find out the sequence determinant of the different energetic state of their intra A-chain/domain disulfide, the following experiments were carried out. First, a local chimeric single-chain insulin (PIP) with the A8-A10 residues replaced by the corresponding residues of IGF-1 was prepared. Second, the disulfide stability of two global hybrids of insulin and IGF-1, Ins(A)/IGF-1(B) and Ins(B)/IGF-1(A), was investigated. The local segment swap had no effect on the fidelity of disulfide pairing and the disulfide stability of PIP molecule although the swapped segment is close to the intra A-chain/domain disulfide. In redox buffer which favors the disulfide formation for most proteins, Ins(A)/IGF-1(B) cannot form and maintain its native disulfides just like that of IGF-1, while the disulfides of Ins(B)/IGF-1(A) are stable in the same condition. One major equilibrium intermediate with two disulfides of Ins(A)/IGF-1(B) was purified and characterized. V8 endoproteinase cleavage and circular dichroism analysis suggested that the intra A-chain/domain disulfide was reduced in the intermediate. Our present results suggested that the energetic state of the intra A-chain/domain disulfide of insulin and IGF-1 was not controlled by the A-chain/domain sequence close to this disulfide but was mainly controlled by the sequence of the B-chain/domain.     

In vitro folding/unfolding of insulin/single-chain insulin

Insulin is a double-chain (designated A and B chain respectively) protein hormone containing three disulfides, while insulin is synthesized in vivo as a single-chain precursor and folded well before being released from B-cells. Although the structure and function of insulin have been well characterized, the progress in oxidative folding pathway studies of insulin has been very slow, mainly due to the difficulties brought about by its disulfide-linked double-chain structure. To overcome these difficulties, we recently studied the in vitro oxidative folding process of two single-chain insulins: porcine insulin precursor (PIP) and human proinsulin (HPI). Based on the analysis of the intermediates captured during folding process, the folding pathways have been proposed for PIP and HPI separately. Similarities between the two folding pathways disclose some common principles that govern the insulin folding process. The following unfolding studies of PIP and HPI further indicate that C-peptide might also function during the folding of proinsulin. Here, we gave a brief review on in vitro folding/unfolding process of insulin and single-chain insulin. The implication of these studies on protein folding has also been discussed.     

Peptide models of four possible insulin folding intermediates with two disulfides

The single-chain insulin (PIP) can spontaneously fold into native structure through preferred kinetic intermediates. During refolding, pairing of the first disulfide A20-B19 is highly specific, whereas pairing of the second disulfide is likely random because two two-disulfide intermediates have been trapped. To get more details of pairing property of the second disulfide, four model peptides of possible folding intermediates with two disulfides were prepared by protein engineering, and their properties were analyzed. The four model peptides were named [A20-B19, A7-B7]PIP, [A20-B19, A6-B7]PIP, [A20-B19, A6-A11]PIP, and [A20-B19, A7-A11]PIP according to their remaining disulfides. The four model peptides all adopt partially folded structure with moderate conformational differences. In redox buffer, the disulfides of the model peptides are more easily reduced than those of the wild-type PIP. During in vitro refolding, the reduced model peptides share similar relative folding rates but different folding yields: The refolding efficiency of the reduced [A20-B19, A7-A11]PIP is about threefold lower than that of the other three peptides. The present results indicate that the folding intermediates corresponding to the present model peptides all adopt partially folded conformation, and can be formed during PIP refolding, but the chance of forming the intermediate with disulfide [A20-B19, A7-A11] is much lower than that of forming the other three intermediates.     

A peptide model of insulin folding intermediate with one disulfide

Insulin folds into a unique three-dimensional structure stabilized by three disulfide bonds. Our previous work suggested that during in vitro refolding of a recombinant single-chain insulin (PIP) there exists a critical folding intermediate containing the single disulfide A20-B19. However, the intermediate cannot be trapped during refolding because once this disulfide is formed, the remaining folding process is very quick. To circumvent this difficulty, a model peptide ([A20-B19]PIP) containing the single disulfide A20-B19 was prepared by protein engineering. The model peptide can be secreted from transformed yeast cells, but its secretion yield decreases 2-3 magnitudes compared with that of the wild-type PIP. The physicochemical property analysis suggested that the model peptide adopts a partially folded conformation. In vitro, the fully reduced model peptide can quickly and efficiently form the disulfide A20-B19, which suggested that formation of the disulfide A20-B19 is kinetically preferred. In redox buffer, the model peptide is reduced gradually as the reduction potential is increased, while the disulfides of the wild-type PIP are reduced in a cooperative manner. By analysis of the model peptide, it is possible to deduce the properties of the critical folding intermediate with the single disulfide A20-B19.     

In vitro refolding of human proinsulin. Kinetic intermediates,putative disulfide-forming pathway folding initiation site,and potential role of C-peptide in folding process

Human insulin is a double-chain peptide that is synthesized in vivo as a single-chain human proinsulin (HPI). We have investigated the disulfide-forming pathway of a single-chain porcine insulin precursor (PIP). Here we further studied the folding pathway of HPI in vitro. While the oxidized refolding process of HPI was quenched, four obvious intermediates (namely P1, P2, P3, and P4, respectively) with three disulfide bridges were isolated and characterized. Contrary to the folding pathway of PIP, no intermediates with one- or two-disulfide bonds could be captured under different refolding conditions. CD analysis showed that P1, P2, and P3 retained partially structural conformations, whereas P4 contained little secondary structure. Based on the time-dependent distribution, disulfide pair analysis, and disulfide-reshuffling process of the intermediates, we have proposed that the folding pathway of HPI is significantly different from that of PIP. These differences reveal that the C-peptide not only facilitates the folding of HPI but also governs its kinetic folding pathway of HPI. Detailed analysis of the molecular folding process reveals that there are some similar folding mechanisms between PIP and HPI. These similarities imply that the initiation site for the folding of PIP/HPI may reside in the central alpha-helix of the B-chain. The formation of disulfide A20-B19 may guide the transfer of the folding information from the B-chain template to the unstructured A-chain. Furthermore, the implications of this in vitro refolding study on the in vivo folding process of HPI have been discussed.     

Precise mapping of an IGF-I-binding site on the IGF-1R

The IGF-1R [type 1 IGF (insulin-like growth factor) receptor] is activated upon binding to IGF-I and IGF-II leading to cell growth, survival and migration of both normal and cancerous cells. We have characterized the binding interaction between the IGF-1R and its ligands using two high-affinity mouse anti-IGF-1R mAbs (monoclonal antibodies), 7C2 and 9E11. These mAbs both block IGF-I binding to the IGF-1R but have no effect on IGF-II binding. Epitope mapping using chimaeras of the IGF-1R and insulin receptor revealed that the mAbs bind to the CR (cysteine-rich) domain of IGF-1R. The epitope was finely mapped using single point mutations in the IGF-1R. Mutation of Phe241, Phe251 or Phe266 completely abolished 7C2 and 9E11 binding. The three-dimensional structure showed that these residues cluster on the surface of the CR-domain. BIAcore analyses revealed that IGF-I and a chimaeric IGF-II with the IGF-I C-domain competed for the binding of both mAbs with the IGF-1R, whereas neither IGF-II nor a chimaeric IGF-I with the IGF-II C-domain affected antibody binding. We therefore conclude the IGF-I C-domain interacts with the CR (cysteine-rich) domain of the receptor at the cluster of residues Phe241, Phe251 and Phe266. These results allow precise orientation of IGF-I within the IGF-I-IGF-1R complex involving the IGF-I C-domain binding to the IGF-1R CR domain. In addition, mAbs 7C2 and 9E11 inhibited both IGF-I- and IGF-II-induced cancer cell proliferation, migration and IGF-1R down-regulation, demonstrating that targeting the IGF-1R is an effective strategy for inhibition of cancer cell growth.     

胰岛素折叠、结合与稳定性的核磁共振研究(英)

Insulin is one of the most important hormonal regulators of metabolism. Since the diabetes patients increase dramatically, the chemical properties, biological and physiological effects of insulin had been extensively studied. In last decade the development of NMR technique allowed us to determine the solution structures of insulin and its variety mutants in various conditions, so that the knowledge of folding, binding and stability of insulin in solution have been largely increased. The solution structure of insulin monomers is essentially identical to those of insulin monomers within the dimer and bexamer as determined by X-ray diffraction. The studies of insulin mutants at the putative residues for receptor binding explored the possible conformational change and fitting between insulin and its receptor. The systematical studies of disulfide paring coupled insulin folding intermediates revealed that in spite of the conformational variety of the intermediates, one structural feature is always remained: a “native-like B chain super-secondary structure“, which consists of B9-B19 helix with adjoining B23-B26 segment folded back against the central segment of B chain, an internal cystine A20-B19 disulfide bridge and a short a-helix at C-terminal of A chain linked. The “super-secondary structure“ might be the “folding nucleus“ in insulin folding mechanism. Cystine A20-B19 is the most important one among three disulfides to stabilize the nascent polypeptide in early stage of the folding. The NMR structure of C. elegans insulin-like peptide resembles that of human insulin and the peptide interacts with human insulin receptor. Other members of insulin superfamily adopt the “insulin fold“ mostly. The structural study of insulin-insulin receptor complex, that of C elegans and other invertebrate insulin-like peptide, insulin fibril study and protein disulfide isomerase (PDI) assistant proinsulin folding study will be new topics in future to get insight into folding, binding, stability, evolution and fibrillation of insulin in detail.     

Studies on insulin/IGF-1 hybrid and IGF-1 growth-promoting functional region

Single-chain insulin/IGF-1 hybrid-[Ins/IGF-1(C)], single-chain porcine insulin precursor-(PIP), and B10Asp PIP were prepared by protein engineering. Their growth-promoting activities in mouse breast cancer cell line GR2H6 are 10, 0.2, and 2 times that of insulin, respectively, and 29%, 0.6%, and 6% of that of IGF-1, indicating that the C domain and 9Glu of IGF-1 are important for its growth-promoting activity. Given these results and previous reports, we propose that the C domain, 9Glu, and 23Phe-26Asn beta bend are involved in the growth-promoting functional region of IGF-1.     

Equilibrium folding of porcine insulin precursor in the presence of redox buffer: implications for the common intermediates shared by its unfolding/ refolding processes

We use the procedure established for 'disulfide stability analysis in redox system' to investigate the unfolding process of porcine insulin precursor (PIP). Six major unfolding intermediates have been captured, in which four contain two disulfides, two contain one disulfide. Based on the characterization and analysis of the intermediates an unfolding pathway has been proposed, by which the native PIP unfolded through in turn 2SS and 1SS intermediates into fully reduced form. Besides, the comparison of the intermediates captured in PIP unfolding process with those intermediates captured in its refolding process revealed that some intermediates captured during both unfolding/refolding processes of PIP have identical disulfide pairing pattern, from which we suggest that the unfolding/refolding processes of PIP share some common intermediates but flow in the opposite direction.     

1.42A crystal structure of mini-IGF-1(2): an analysis of the disulfide isomerization property and receptor binding property of IGF-1 based on the three-dimensional structure

Insulin and insulin-like growth factor 1 (IGF-1) share a homologous sequence, a similar three-dimensional structure and weakly overlapping biological activity, but IGF-1 folds into two thermodynamically stable disulfide isomers, while insulin folds into one unique stable tertiary structure. This is a very interesting phenomenon in which one amino acid sequence encodes two three-dimensional structures, and its molecular mechanism has remained unclear for a long time. In this study, the crystal structure of mini-IGF-1(2), a disulfide isomer of an artificial analog of IGF-1, was solved by the SAD/SIRAS method using our in-house X-ray source. Evidence was found in the structure showing that the intra-A-chain/domain disulfide bond of some molecules was broken; thus, it was proposed that disulfide isomerization begins with the breakdown of this disulfide bond. Furthermore, based on the structural comparison of IGF-1 and insulin, a new assumption was made that in insulin the several hydrogen bonds formed between the N-terminal region of the B-chain and the intra-A-chain disulfide region of the A-chain are the main reason for the stability of the intra-A-chain disulfide bond and for the prevention of disulfide isomerization, while Phe B1 and His B5 are very important for the formation of these hydrogen bonds. Moreover, the receptor binding property of IGF-1 was analyzed in detail based on the structural comparison of mini-IGF-1(2), native IGF-1, and small mini-IGF-1.     

1.42 Å crystal structure of mini-IGF-1(2): an analysis of the disulfide isomerization property and receptor binding property of IGF-1 based on the three-dimensional structure

Insulin and insulin-like growth factor 1 (IGF-1) share a homologous sequence, a similar three-dimensional structure and weakly overlapping biological activity, but IGF-1 folds into two thermodynamically stable disulfide isomers, while insulin folds into one unique stable tertiary structure. This is a very interesting phenomenon in which one amino acid sequence encodes two three-dimensional structures, and its molecular mechanism has remained unclear for a long time. In this study, the crystal structure of mini-IGF-1(2), a disulfide isomer of an artificial analog of IGF-1, was solved by the SAD/SIRAS method using our in-house X-ray source. Evidence was found in the structure showing that the intra-A-chain/domain disulfide bond of some molecules was broken; thus, it was proposed that disulfide isomerization begins with the breakdown of this disulfide bond. Furthermore, based on the structural comparison of IGF-1 and insulin, a new assumption was made that in insulin the several hydrogen bonds formed between the N-terminal region of the B-chain and the intra-A-chain disulfide region of the A-chain are the main reason for the stability of the intra-A-chain disulfide bond and for the prevention of disulfide isomerization, while Phe B1 and His B5 are very important for the formation of these hydrogen bonds. Moreover, the receptor binding property of IGF-1 was analyzed in detail based on the structural comparison of mini-IGF-1(2), native IGF-1, and small mini-IGF-1.     

Contribution of the absolutely conserved B8Gly to the foldability of insulin

B8Gly is absolutely conserved in insulin from different species, and in other members of the insulin superfamily the corresponding position is always occupied by a Gly residue. However, the reasons for its conservation are still unclear; probably many factors contribute to this phenomenon. In our previous work, B8Gly was replaced by an Ala residue, which suggested that biological activity is one of the factors contributing to its conservation. In order to identify more factors contributing to this positional conservation, the secretion efficiency, structural stability, disulfide stability, and in vitro refolding of single-chain insulin (PIP) and a mutant with B8Gly replaced by Ala, were investigated. Compared with wild-type PIP, the B8Ala replacement decreased the secretion efficiency, structural stability, disulfide stability, and in vitro refolding efficiency of the PIP sequence. These results suggest that B8Gly is important to the secretion, folding, and stability of the insulin sequence.     

Volume and free energy of folding for troponin C C-domain: linkage to ion binding and N-domain interaction

Troponin C (TnC) is an 18-kDa acidic protein of the EF-hand family that serves as the trigger for muscle contraction. In this study, we investigated the thermodynamic stability of the C-domain of TnC in all its occupancy states (apo, Mg (2+)-, and Ca (2+)-bound states) using a fluorescent mutant with Phe 105 replaced by Trp (F105W/C-domain, residues 88-162) and (1)H NMR spectroscopy. High hydrostatic pressure was employed as a perturbing agent, in combination with urea or without it. On the basis of changes in Trp emission, the C-domain apo state was denatured by pressure (in the range of 1-1000 bar) in the absence of urea. The fluorescence data were corroborated by following the changes in the (1)H NMR signal of Histidine 128. Addition of Ca (2+) or Mg (2+) increased the C-domain stability so that complete denaturation was attained only by the combined use of high hydrostatic pressure and either 7-8 M or 1.5-2 M urea, respectively. The (1)H NMR spectra in the presence of Ca (2+) was typical of a highly structured protein and allowed us to follow the changes in the local environment of several amino-acid residues as a function of pressure at 4 M Urea. Different residues presented different volume changes, but those that are in the hydrophobic core portrayed values very similar to that obtained for tryptophan 105 as measured by fluorescence, indicating that it is indeed a good probe for the overall tertiary structure. From these experiments, we calculated the thermodynamic parameters (Delta G degrees atm and Delta V) that govern the folding of the C-domain in all its possible physiological states and constructed a thermodynamic cycle. Furthermore, a comparison of the volume and free-energy changes of folding of isolated C-domain with those of intact TnC (F105W) revealed that the N-domain has little effect on the structure of the C-domain, even in the presence of Ca (2+). The volume and free-energy diagrams reveal a landscape of different conformations from the less structured, denatured apo form to the highly structured, Ca (2+)-bound form. The large change in folding free energy of the C-domain that takes place when Ca (2+) binds may explain the much higher Ca (2+) affinity of sites III and IV, 2 orders of magnitude higher than the affinity of sites I and II.     

Solution Structure of Proinsulin: CONNECTING DOMAIN FLEXIBILITY AND PROHORMONE PROCESSING*

The folding of proinsulin, the single-chain precursor of insulin, ensures native disulfide pairing in pancreatic β-cells. Mutations that impair folding cause neonatal diabetes mellitus. Although the classical structure of insulin is well established, proinsulin is refractory to crystallization. Here, we employ heteronuclear NMR spectroscopy to characterize a monomeric analogue. Proinsulin contains a native-like insulin moiety (A- and B-domains); the tethered connecting (C) domain (as probed by {¹H}-¹⁵N nuclear Overhauser enhancements) is progressively less ordered. Although the BC junction is flexible, residues near the CA junction exhibit α-helical-like features. Relative to canonical α-helices, however, segmental ¹³C_α/β chemical shifts are attenuated, suggesting that this junction and contiguous A-chain residues are molten. We propose that flexibility at each C-domain junction facilitates prohormone processing. Studies of protease SPC3 (PC1/3) suggest that C-domain sequences contribute to cleavage site selection. The structure of proinsulin provides a foundation for studies of insulin biosynthesis and its impairment in monogenic forms of diabetes mellitus.