NMR and photo-CIDNP studies of human proinsulin and prohormone processing intermediates with application to endopeptidase recognition

The proinsulin-insulin system provides a general model for the proteolytic processing of polypeptide hormones. Two proinsulin-specific endopeptidases have been defined, a type I activity that cleaves the B-chain/C-peptide junction (Arg31-Arg32) and a type II activity that cleaves the C-peptide/A-chain junction (Lys64-Arg65). These endopeptidases are specific for their respective dibasic target sites; not all such dibasic sites are cleaved, however, and studies of mutant proinsulins have demonstrated that additional sequence or structural features are involved in determining substrate specificity. To define structural elements required for endopeptidase recognition, we have undertaken comparative 1H NMR and photochemical dynamic nuclear polarization (photo-CIDNP) studies of human proinsulin, insulin, and split proinsulin analogues as models of prohormone processing intermediates. The overall conformation of proinsulin is observed to be similar to that of insulin, and the connecting peptide is largely unstructured. In the 1H NMR spectrum of proinsulin significant variation is observed in the line widths of insulin-specific amide resonances, reflecting exchange among conformational substates; similar exchange is observed in insulin and is not damped by the connecting peptide. The aromatic 1H NMR resonances of proinsulin are assigned by analogy to the spectrum of insulin, and assignments are verified by chemical modification. Unexpectedly, nonlocal perturbations are observed in the insulin moiety of proinsulin, as monitored by the resonances of internal aromatic groups. Remarkably, these perturbations are reverted by site-specific cleavage of the connecting peptide at the CA junction but not the BC junction. These results suggest that a stable local structure is formed at the CA junction, which influences insulin-specific packing interactions. We propose that this structure (designated the "CA knuckle") provides a recognition element for type II proinsulin endopeptidase.     

Comparative 2D NMR studies of human insulin and des-pentapeptide insulin: sequential resonance assignment and implications for protein dynamics and receptor recognition

The solution structure and dynamics of human insulin are investigated by 2D 1H NMR spectroscopy in reference to a previously analyzed analogue, des-pentapeptide(B26-B30) insulin (DPI; Hua, Q.X., & Weiss, M.A. (1990) Biochemistry 29, 10545-10555). This spectroscopic comparison is of interest since (i) the structure of the C-terminal region of the B-chain has not been determined in the monomeric state and (ii) the role of this region in binding to the insulin receptor has been the subject of long-standing speculation. The present NMR studies are conducted in the presence of an organic cosolvent (20% acetic acid), under which conditions both proteins are monomeric and stably folded. Complete sequential assignment of human insulin is obtained and leads to the following conclusions. (1) The secondary structure of the insulin monomer (three alpha-helices and B-chain beta-turn) is similar to that observed in the 2-Zn crystal state. (2) The folding of DPI is essentially the same as the corresponding portion of intact insulin, in accord with the similarities between their respective crystal structures. However, differences between insulin and DPI are observed in the extent of conformational broadening of amide resonances, indicating that the presence or absence of residues B26-B30 influences the overall dynamics of the protein on the millisecond time scale. (3) Residues B24-B28 adopt an extended configuration in the monomer and pack against the hydrophobic core as in crystallographic dimers; residues B29 and B30 are largely disordered. This configuration differs from that described in a more organic milieu (35% acetonitrile; Kline, A.D., & Justice, R.M., Jr. (1990) Biochemistry 29, 2906-2913), suggesting that the conformation of insulin in the latter study may have been influenced by solvent composition. (4) The insulin fold is shown to provide a model for collective motions in a protein with implications for the mechanism of protein-protein recognition. To our knowledge, this paper describes the first detailed analysis of a protein NMR spectrum under conditions of extensive conformational broadening. Such an analysis is made possible in the present case by comparative study of an analogue (DPI) with more tractable spectroscopic properties.     

Nonlocal structural perturbations in a mutant human insulin: sequential resonance assignment and 13C-isotope-aided 2D-NMR studies of [PheB24-->Gly]insulin with implications for receptor recognition.

Insulin's mechanism of receptor binding is not well understood despite extensive study by mutagenesis and X-ray crystallography. Of particular interest are "anomalous" analogues whose bioactivities are not readily rationalized by crystal structures. Here the structure and dynamics of one such analogue (GlyB24-insulin) are investigated by circular dichroism (CD) and isotope-aided 2D-NMR spectroscopy. The mutant insulin retains near-native receptor-binding affinity despite a nonconservative substitution (PheB24-->Gly) in the receptor-binding surface. Relative to native insulin, GlyB24-insulin exhibits reduced dimerization; the monomer (the active species) exhibits partial loss of ordered structure, as indicated by CD studies and motional narrowing of selected 1H-NMR resonance. 2D-NMR studies demonstrate that the B-chain beta-turn (residues B20-23) and beta-strand (residues B24-B28) are destabilized; essentially native alpha-helical secondary structure (residues A3-A8, A13-A18, and B9-B19) is otherwise maintained. 13C-Isotope-edited NOESY studies demonstrate that long-range contacts observed between the B-chain beta-strand and the alpha-helical core in native insulin are absent in the mutant. Implications for the mechanism of insulin's interaction with its receptor are discussed.     

Secretion of human insulin by a transformed yeast cell

A yeast expression plasmid encoding a mini-proinsulin molecule was constructed and transformed into Saccharomyces cerevisiae. The plasmid encoded the sequence: B-Arg-Arg-Leu-Gln-Lys-Arg-A in which B represents the B-chain (30 amino acid residues) and A represents the A-chain (21 amino acid residues) of human insulin. The secreted peptides were shown to be a mixture of human insulin and des(B-30)human insulin. Thus, correct disulphide bridges can be established in proinsulin-like molecules devoid of a normal C-peptide region. Furthermore, the specificity of the yeast processing enzymes is so similar to the proinsulin converting enzymes in the human pancreatic beta-cell that it allows the processing of the mini-proinsulin to insulin.     

Proinsulin is refractory to protein fibrillation: topological protection of a precursor protein from cross-beta assembly

Insulin is susceptible to fibrillation, a misfolding process leading to well ordered cross-beta assembly. Protection from fibrillation in beta 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 beta-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-beta 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 beta cells from toxic protein misfolding in the endoplasmic reticulum.     

Structural analysis of proinsulin hexamer assembly by hydroxyl radical footprinting and computational modeling

Mutations in the insulin gene can impair proinsulin folding and cause diabetes mellitus. Although crystal structures of insulin dimers and hexamers are well established, proinsulin is refractory to crystallization. Although an NMR structure of an engineered proinsulin monomer has been reported, structures of the wild-type monomer and hexamer remain undetermined. We have utilized hydroxyl radical footprinting and molecular modeling to characterize these structures. Differences between the footprints of insulin and proinsulin, defining a "shadow" of the connecting (C) domain, were employed to refine the model. Our results demonstrate that in its monomeric form, (i) proinsulin contains a native-like insulin moiety and (ii) the C-domain footprint resides within an adjoining segment (residues B23-B29) that is accessible to modification in insulin but not proinsulin. Corresponding oxidation rates were observed within core insulin moieties of insulin and proinsulin hexamers, suggesting that the proinsulin hexamer retains an A/B structure similar to that of insulin. Further similarities in rates of oxidation between the respective C-domains of proinsulin monomers and hexamers suggest that this loop in each case flexibly projects from an outer surface. Although dimerization or hexamer assembly would not be impaired, an ensemble of predicted C-domain positions would block hexamer-hexamer stacking as visualized in classical crystal lattices. We anticipate that protein footprinting in combination with modeling, as illustrated here, will enable comparative studies of diabetes-associated mutant proinsulins and their aberrant modes of aggregation.     

Proteasomal Degradation of Proinsulin Requires Derlin-2, HRD1 and p97

Patients with type 1 diabetes (T1D) suffer from beta-cell destruction by CD8⁺ T-cells that have preproinsulin as an important target autoantigen. It is of great importance to understand the molecular mechanism underlying the processing of preproinsulin into these CD8⁺ T-cell epitopes. We therefore studied a pathway that may contribute to the production of these antigenic peptides: degradation of proinsulin via ER associated protein degradation (ERAD). Analysis of the MHC class I peptide ligandome confirmed the presentation of the most relevant MHC class I-restricted diabetogenic epitopes in our cells: the signal peptide-derived sequence A15-A25 and the insulin B-chain epitopes H29-A38 and H34-V42. We demonstrate that specific silencing of Derlin-2, p97 and HRD1 by shRNAs increases steady state levels of proinsulin. This indicates that these ERAD constituents are critically involved in proinsulin degradation and may therefore also play a role in subsequent antigen generation. These ERAD proteins therefore represent interesting targets for novel therapies aiming at the reduction and possibly also prevention of beta-cell directed auto-immune reactions in T1D.     

Two-dimensional NMR studies of Des-(B26-B30)-insulin: sequence-specific resonance assignments and effects of solvent composition.

Des-pentapeptide-insulin (DPI), a monomeric analogue which lacks the C-terminal five residues of the B-chain, provides a tractable model for 2D-NMR studies of insulin under a variety of solvent conditions. In this paper we present the sequential assignment of DPI at pH 1.8 and 25 degrees C in 10% deuterated DMSO/90% H2O; the chemical shifts are in general similar to those recently described in the absence of an organic cosolvent [1], in 20% acetic acid [2] and (for intact insulin) in 35% acetonitrile [3]. Under each of these solvent conditions qualitative analysis of the 2D-NMR data indicates that the major elements of secondary structure observed in the crystal state (three alpha-helices and B-chain beta-turn) are retained in solution. However, there is disagreement in the literature regarding the stability of the insulin fold, as monitored by amide-proton exchange rates and long-range nuclear Overhauser enhancements [1-3]. In contrast to a previous study [1], we observe slowly exchanging amide resonances (in freshly prepared D2O solutions) and nonlocal NOEs under each of the solvent conditions described, implying the existence of a stably folded secondary structure and hydrophobic core. The slowly-exchanging resonances are assigned to the central alpha-helix of the B-chain, the ends of the adjoining beta-turn, and the two A-chain alpha-helices. Qualitative analysis of long-range NOEs indicates that the major features of the crystal state are retained under these solvent conditions.     

X-ray analysis of the single chain B29-A1 peptide-linked insulin molecule. A completely inactive analogue.

A crystal structure of a totally inactive insulin molecule has been determined. For this insulin molecule, the first without detectable activity to be characterized, the A and B-chains are linked by a peptide bond between A1 Gly and B29 Lys. The molecule has retained all its normal self-association properties and it can also accommodate the two different conformations designated T and R, as seen in 4Zn native pig insulin crystals. The hexamers of the crosslinked insulin molecule were crystallized using the 4Zn insulin recipe of Schlichtkrull. The structure has been crystallographically refined with data extending to 2 A using restrained least-square methods. Comparison of the B29-A1 peptide crosslink insulin and the 4Zn native insulin reveals close structural similarities with the native dimer. The analysis of the structure confirms the earlier hypothesis that insulin structures in crystals are not in an active conformation and that a separation of N-terminal A-chain and C-terminal B-chain is required for interaction with the insulin receptor.     

The amino acid sequence of human insulin-like growth factor I and its structural homology with proinsulin

The complete amino acid sequence of human insulin-like growth factor I (IGF-I), a polypeptide isolated from serum, has been determined. IGF-I is a single chain polypeptide of 70 amino acid residues cross-linked by three disulfide bridges. The calculated molecular weight is 7649. IGF-I displays obvious homology to proinsulin: positions 1 to 29 are homologous to insulin B chain and positions 42 to 62 to insulin A chain. A shortened "connecting" peptide with 12 residues (positions 30 to 41) compared to 30 to 35 in proinsulins shows no homology to proinsulin C peptide. An octapeptide sequence at the COOH-terminal end is also a feature not found in proinsulins. The number of differences in amino acid positions between IGF-I and insulins suggests that duplication of the gene of the common ancestor of proinsulin and IGF occurred before the time of appearance of the vertebrates. Of the 19 residues known to be invariant in all insulins so far sequenced, only glutamine A5 and asparagine A21 are replaced in IGF-I by glutamic acid and alanine, respectively. The fact that all half-cystine and glycine residues and most nonpolar core residues of the insulin monomer are conserved is compatible with a three-dimensional structure of IGF-I similar to that of insulin.     

Role of the COOH-terminal B-chain domain in insulin-receptor interactions. Identification of perturbations involving the insulin mainchain

Previous studies have suggested that the COOH-terminal pentapeptide of the insulin B-chain can play a negative role in ligand-receptor interactions involving insulin analogs having amino acid replacements at position B25 (Nakagawa, S. H., and Tager, H. S. (1986) J. Biol. Chem. 261, 7332-7341). We undertook by the current investigations to identify the molecular site in insulin that induces this negative effect and to explore further the importance of conformational changes that might occur during insulin-receptor interactions. By use of semisynthetic insulin analogs containing amino acid replacements or deletions and of isolated canine hepatocytes, we show here that (a) the markedly decreased affinity of receptor for insulin analogs in which PheB25 is replaced by Ser is apparent for analogs in which up to 3 residues of the insulin B-chain have been deleted, but is progressively reversed in the corresponding des-tetrapeptide and des-pentapeptide analogs, and (b) unlike the case for deletion of TyrB26 and ThrB27, replacement of residue TyrB26 or ThrB27 has no effect to reverse the decreased affinity of full length analogs containing Ser for Phe substitutions at position B25. Additional experiments demonstrated that introduction of a cross-link between Lys epsilon B29 and Gly alpha A1 of insulin decreases the affinity of ligand-receptor interactions whether or not PheB25 is replaced by Ser. We conclude that the negative effect of the COOH-terminal B-chain domain on insulin-receptor interactions arises in greatest part from the insulin mainchain near the site of the TyrB26-ThrB27 peptide bond and that multiple conformational perturbations may be necessary to induce a high-affinity state of receptor-bound insulin.     

A new B-chain mutant of insulin: comparison with the insulin crystal structure and role of sulfonate groups in the B-chain structure.

The solution structure of a new B-chain mutant of bovine insulin, in which the cysteines B7 and B19 are replaced by two serines, has been determined by circular dichroism, 2D-NMR and molecular modeling. This structure is compared with that of the oxidized B-chain of bovine insulin [Hawkins et al. (1995) Int. J. Peptide Protein Res.46, 424-433]. Circular dichroism spectroscopy showed in particular that a higher percentage of helical secondary structure for the B-chain mutant is estimated in trifluoroethanol solution in comparison with the oxidized B-chain. 2D-NMR experiments confirmed, among multiple conformations, that the B-chain mutant presents defined secondary structures such as a alpha-helix between residues B9 and B19, and a beta-turn between amino acids B20 and B23 in aqueous trifluoroethanol. The 3D structures, which are consistent with NMR data and were obtained using a simulated annealing protocol, showed that the tertiary structure of the B-chain mutant is better resolved and is more in agreement with the insulin crystal structure than the oxidized B-chain structure described by Hawkins et al. An explanation could be the presence of two sulfonate groups in the oxidized insulin B-chain. Either by their charges and/or their size, such chemical groups could play a destructuring effect and thus could favor peptide flexibility and conformational averaging. Thus, this study provides new insights on the folding of isolated B-chains.     

Toward the insulin-IGF-I intermediate structures: functional and structural properties of the [TyrB25NMePheB26] insulin mutant

The origins of differentiation of insulin from insulin-like growth factor I (IGF-I) are still unknown. To address the problem of a structural and biological switch from the mostly metabolic hormonal activity of insulin to the predominant growth factor activities of IGF-I, an insulin analogue with IGF-I-like structural features has been synthesized. Insulin residues Phe(B25) and Tyr(B26) have been swapped with the IGF-I-like Tyr(24) and Phe(25) sequence with a simultaneous methylation of the peptide nitrogen of residue Phe(B26). These modifications were expected to introduce a substantial kink in the main chain, as observed at residue Phe(25) in the IGF-I crystal structure. These alterations should provide insight into the structural origins of insulin-IGF-I structural and functional divergence. The [Tyr(B25)NMePhe(B26)] mutant has been characterized, and its crystal structure has been determined. Surprisingly, all of these changes are well accommodated within an insulin R6 hexamer. Only one molecule of each dimer in the hexamer responds to the structural alterations, the other remaining very similar to wild-type insulin. All alterations, modest in their scale, cumulate in the C-terminal part of the B-chain (residues B23-B30), which moves toward the core of the insulin molecule and is associated with a significant shift of the A1 helix toward the C-terminus of the B-chain. These changes do not produce the expected bend of the main chain, but the fold of the mutant does reflect some structural characteristics of IGF-1, and in addition establishes the CO(A19)-NH(B25) hydrogen bond, which is normally characteristic of T-state insulin.     

Met-Lys-双C肽人胰岛素原基因的构建表达及分离纯化

应用 Ｐ Ｃ Ｒ 定点突变方法构建编码 Ｍ ｅｔ Ｌｙｓ 双 Ｃ 肽人胰岛素原基因,并在大肠杆菌中以包含体方式获得表达 表达产物经还原、重组、 Ｓｅｐｈａｄｅｘ Ｇ ７５ 分离纯化,获得 Ｍ ｅｔ Ｌｙｓ 双 Ｃ 肽人胰岛素原,经胰蛋白酶与羧肽酶 Ｂ的酶解, Ｒｅｓｏｕｒｃｅ Ｔ Ｍ Ｑ 阴离子交换柱层析分离制备得人胰岛素,其放免活性、受体结合活性均与猪胰岛素相同      

Conversion of biosynthetic human proinsulin to partially cleaved intermediates by collagenase proteinases adsorbed to isolated rat adipocytes.

Studies of the biological activity of proinsulin have resulted in widely varying conclusions. Relative to insulin, the biological activity of proinsulin has been reported from less than 1% to almost 20%. Many of the assays in vitro for the biological potency of proinsulin have utilized isolated rat adipocytes. To examine further the interaction of proinsulin with rat adipocytes, we prepared specifically-labelled proinsulin isomers that were iodinated on tyrosine residues corresponding to the A14, A19, B16 or B26 residue of insulin. These were incubated with rat adipocytes and their metabolism was examined by trichloroacetic acid precipitation, by Sephadex G-50 chromatography, and by h.p.l.c. chromatography. By trichloroacetic acid-precipitation assay, there was little or no proinsulin degradation. By G-50 chromatography and subsequent h.p.l.c. analysis, however, we found that the labelled proinsulin isomers were converted rapidly and almost completely to materials which eluted differently on h.p.l.c. from intact proinsulin. This conversion was due primarily to proteolytic activity which adsorbed to the fat cells from the crude collagenase used to isolate the cells. Two primary conversion intermediates were found: one with a cleavage at residues 23-24 of proinsulin (the B-chain region of insulin), and one at residues 55-56 in the connecting peptide region. These intermediates had receptor binding properties equivalent to or less than intact proinsulin. These findings show that isolated fat cells can degrade proinsulin to intermediates due to their contamination with proteolytic activity from the collagenase used in their preparation. Thus the previously reported range in biological activities of proinsulin in fat cells may have arisen from such protease contamination. Finally, the present findings demonstrate that a sensitive assay for degradation of hormones is required to examine biological activities in isolated cells.     

Hierarchical protein "un-design": insulin's intrachain disulfide bridge tethers a recognition alpha-helix

A hierarchical pathway of protein folding can enable segmental unfolding by design. A monomeric insulin analogue containing pairwise substitution of internal A6-A11 cystine with serine [[Ser(A6),Ser(A11),Asp(B10),Lys(B28),Pro(B29)]insulin (DKP[A6-A11](Ser))] was previously investigated as a model of an oxidative protein-folding intermediate [Hua, Q. X., et al. (1996) J. Mol. Biol. 264, 390-403]. Its structure exhibits local unfolding of an adjoining amphipathic alpha-helix (residues A1-A8), leading to a 2000-fold reduction in activity. Such severe loss of function, unusual among mutant insulins, is proposed to reflect the cost of induced fit: receptor-directed restoration of the alpha-helix and its engagement in the hormone's hydrophobic core. To test this hypothesis, we have synthesized and characterized the corresponding alanine analogue [[Ala(A6),Ala(A11),Asp(B10),Lys(B28), Pro(B29)]insulin (DKP[A6-A11](Ala))]. Untethering the A6-A11 disulfide bridge by either amino acid causes similar perturbations in structure and dynamics as probed by circular dichroism and (1)H NMR spectroscopy. The analogues also exhibit similar decrements in thermodynamic stability relative to that of the parent monomer as probed by equilibrium denaturation studies (Delta Delta G(u) = 3.0 +/- 0.5 kcal/mol). Despite such similarities, the alanine analogue is 50 times more active than the serine analogue. Enhanced receptor binding (Delta Delta G = 2.2 kcal/mol) is in accord with alanine's greater helical propensity and more favorable hydrophobic-transfer free energy. The success of an induced-fit model highlights the applicability of general folding principles to a complex binding process. Comparison of DKP[A6-A11](Ser) and DKP[A6-A11](Ala) supports the hypothesis that the native A1-A8 alpha-helix functions as a preformed recognition element tethered by insulin's intrachain disulfide bridge. Segmental unfolding by design provides a novel approach to dissecting structure-activity relationships.     

The folding nucleus of the insulin superfamily: a flexible peptide model foreshadows the native state

Oxidative folding of insulin-like growth factor I (IGF-I) and single-chain insulin analogs proceeds via one- and two-disulfide intermediates. A predominant one-disulfide intermediate in each case contains the canonical A20-B19 disulfide bridge (cystines 18-61 in IGF-I and 19-85 in human proinsulin). Here, we describe a disulfide-linked peptide model of this on-pathway intermediate. One peptide fragment (19 amino acids) spans IGF-I residues 7-25 (canonical positions B8-B26 in the insulin superfamily); the other (18 amino acids) spans IGF-I residues 53-70 (positions A12-A21 and D1-D8). Containing only half of the IGF-I sequence, the disulfide-linked polypeptide (designated IGF-p) is not well ordered. Nascent helical elements corresponding to native alpha-helices are nonetheless observed at 4 degrees C. Furthermore, (13)C-edited nuclear Overhauser effects establish transient formation of a native-like partial core; no non-native nuclear Overhauser effects are observed. Together, these observations suggest that early events in the folding of insulin-related polypeptides are nucleated by a native-like molten subdomain containing Cys(A20) and Cys(B19). We propose that nascent interactions within this subdomain orient the A20 and B19 thiolates for disulfide bond formation and stabilize the one-disulfide intermediate once formed. Substitutions in the corresponding region of insulin are associated with inefficient chain combination and impaired biosynthetic expression. The intrinsic conformational propensities of a flexible disulfide-linked peptide thus define a folding nucleus, foreshadowing the structure of the native state.     

Toward the solution structure of human insulin: sequential 2D 1H NMR assignment of a des-pentapeptide analogue and comparison with crystal structure

2D 1H NMR studies are presented of des-pentapeptide-insulin, an analogue of human insulin lacking the C-terminal five residues of the B chain. Removal of these residues, which are not required for function, is shown to reduce conformational broadening previously described in the spectrum of intact insulin [Weiss et al. (1989) Biochemistry 28, 9855-9873]. This difference presumably reflects more rapid internal motions in the fragment, which lead to more complete averaging of chemical shifts on the NMR time scale. Sequential 1H NMR assignment and preliminary structural analysis demonstrate retention in solution of the three alpha-helices observed in the crystal state and the relative orientation of the receptor-binding surfaces. These studies provide a foundation for determining the solution structure of insulin.     

Inactive conformation of an insulin despite its wild-type sequence.

The peptide group between residues B24 and B25 of insulin was replaced by an ester bond. This modification only in the backbone was meant to eliminate a structurally important H-bond between the amide proton of B25 and the carbonyl oxygen of A19, and consequently to enhance detachment of the C-terminal B-chain from the body of the molecule, exposing the underlying A-chain. According to a model derived from the effects of side-chain substitutions, main-chain shortening, and crosslinking, this conformational change is prerequisite for receptor binding. Contrary to the expectation that increased flexibility would increase receptor binding and activity, depsi-insulin ([B24-B25 CO-O]insulin) has turned out be only 3-4% potent. In search of an explanation for this observation, the solution structure of depsi-insulin was determined by two-dimensional 1H-NMR spectroscopy. It was found that the loss of the B25-A19 H-bond does not entail detachment of the C-terminal B-chain. On the contrary, it is overcompensated by a gain in hydrophobic interaction achieved by insertion of the Phe B25 side chain into the molecule's core. This is possible because of increased rotational freedom in the backbone owing to the ester bond. Distortion of the B20-B23 turn and an altered direction of the distal B-chain are consequences that also affect self-association. The exceptional position of the B25 side chain is thus the key feature of the depsi-insulin structure. Being buried in the interior, it is not available for guiding the interaction with the receptor, a crucial role attributed to it by the model. This seems to be the main reason why the structure of depsi-insulin represents an inactive conformation.