Replacement of the interchain disulfide bridge-forming amino acids A7 and B7 by glutamate impairs the structure and activity of insulin

Insulin contains three disulfide bonds, one intrachain bond, A6-A11, and two interchain bonds, A7-B7 and A20-B19. Site-directed mutagenesis results (the two cysteine residues of disulfide A7-B7 were replaced by serine) showed that disulfide A7-B7 is crucial to both the structure and activity of insulin. However, chemical modification results showed that the insulin analogs still retained relatively high biological activity when A7Cys and B7Cys were modified by chemical groups with a negative charge. Did the negative charge of the modification groups restore the loss of activity and/or the disturbance of structure of these insulin analogs caused by deletion of disulfide A7-B7? To answer this question, an insulin analog with both A7Cys and B7Cys replaced by Glu, which has a long side-chain and a negative charge, was prepared by protein engineering, and its structure and activity were analyzed. Both the structure and activity of the present analog are very similar to that of the mutant with disulfide A7-B7 replaced by Ser, but significantly different from that of wild-type insulin. The present results suggest that removal of disulfide A7-B7 will result in serious loss of biological activity and the native conformation of insulin, even if the disulfide is replaced by residues with a negative charge.     

Effects of cysteine to serine substitutions in the two inter-chain disulfide bonds of insulin

Using site-directed mutagenesis we deleted the two inter-chain disulfide bonds of insulin, separately or both, by substitution of the cysteine residues with serine. Deletion of A20-B19 or both of the two inter-chain disulfide bonds resulted in the complete loss of secretion of the mutant single-chain porcine insulin precursor (PIP) from Saccharomyces cerevisiae cells. Removal of the A7-B7 disulfide bond resulted in a large reduction of secretion, but we could obtain the mutant for analysis of its biological and some physico-chemical properties. The A7-B7 disulfide bond deleted insulin mutant retained only 0.1% receptor-binding activity compared with porcine insulin, and its in vivo biological potency measured by mouse convulsion assay was also very low. We also studied some physico-chemical properties of the mutant using circular dichroism, native polyacrylamide gel electrophoresis and reversed-phase HPLC, which revealed some structural changes of the mutant peptides compared to native insulin. The present study shows that the two inter-chain disulfide bonds are important for efficient in vivo folding/secretion of PIP from yeast, especially the A20-B19 disulfide bond, and that the A7-B7 disulfide bond is crucial for maintaining the native conformation and biological activity of insulin.     

Synthesis of A7,B7-dicarbainsulin, an analogue with a noncleavable bond between A- and B-chain. II. Synthesis of the A-chain segments

As part of the total synthesis of [A7,B7-L,L-2,7-diaminosuberoyl]-des-(B26-B30)-insulin B25-amide, an insulin analogue containing a non-cleavable bond between A- and B-chain, the chemical synthesis of the A-chain segments is described. The N-terminal sequence A(1-6), Boc-Gly-Ile-Val-Glu(OBut)-Gln-Cys(SBut)-NH-NH2, was synthesized in solution. The middle segment A(8-16), Ddz-Thr(But)-Ser(But)-Ile-Cys(SBut)-Ser(But)-Leu-Tyr- (But)-Gln-Leu-NH-NH2, was obtained by solid phase synthesis according to the Fmoc strategy. The C-terminal segment A(17-21), Bpoc-Glu(OBut)-Asn-Tyr-Cys(Acm)-Asn-OBut, was prepared in solution.     

Role of disulfide bonds in the structure and activity of human insulin

Insulin contains two inter-chain disulfide bonds between the A and B chains (A7-B7 and A20-B19), and one intra-chain linkage in the A chain (A6-A11). To investigate the role of each disulfide bond in the structure, function and stability of the molecule, three des mutants of human insulin, each lacking one of the three disulfide bonds, were prepared by enzymatic conversion of refolded mini-proinsulins. Structural and biological studies of the three des mutants revealed that all three disulfide bonds are essential for the receptor binding activity of insulin, whereas the different disulfide bonds make different contributions to the overall structure of insulin. Deletion of the A20-B19 disulfide bond had the most substantial influence on the structure as indicated by loss of ordered secondary structure, increased susceptibility to proteolysis, and markedly reduced compactness. Deletion of the A6-A11 disulfide bond caused the least perturbation to the structure. In addition, different refolding efficiencies between the three des mutants suggest that the disulfide bonds are formed sequentially in the order A20-B19, A7-B7 and A6-A11 in the folding pathway of proinsulin.     

A protein caught in a kinetic trap: structures and stabilities of insulin disulfide isomers

Proinsulin contains six cysteines whose specific pairing (A6-A11, A7-B7, and A20-B19) is a defining feature of the insulin fold. Pairing information is contained within A and B domains as demonstrated by studies of insulin chain recombination. Two insulin isomers containing non-native disulfide bridges ([A7-A11,A6-B7,A20-B19] and [A6-A7,A11-B7,A20-B19]), previously prepared by directed chemical synthesis, are metastable and biologically active. Remarkably, the same two isomers are preferentially formed from native insulin or proinsulin following disulfide reassortment in guanidine hydrochloride. The absence of other disulfide isomers suggests that the observed species exhibit greater relative stability and/or kinetic accessibility. The structure of the first isomer ([A7-A11,A6-B7,A20-B19], insulin-swap) has been described [Hua, Q. X., Gozani, S. N., Chance, R. E., Hoffmann, J. A., Frank, B. H., and Weiss, M. A. (1995) Nat. Struct. Biol. 2, 129-138]. Here, we demonstrate that the second isomer (insulin-swap2) is less ordered than the first. Nativelike elements of structure are retained in the B chain, whereas the A chain is largely disordered. Thermodynamic studies of guanidine denaturation demonstrate the instability of the isomers relative to native insulin (DeltaDeltaG(u) > 3 kcal/mol). In contrast, insulin-like growth factor I (IGF-I) and the corresponding isomer IGF-swap, formed as alternative products of a bifurcating folding pathway, exhibit similar cooperative unfolding transitions. The insulin isomers are similar in structure and stability to two-disulfide analogues whose partial folds provide models of oxidative folding intermediates. Each exhibits a nativelike B chain and less-ordered A chain. This general asymmetry is consistent with a hierarchical disulfide pathway in which nascent structure in the B chain provides a template for folding of the A chain. Structures of metastable disulfide isomers provide probes of the topography of an energy landscape.     

Synthetic insulin fragment with insulin-like biological activity.

An insulin fragment, representing the C-terminal functionally important site of its molecule and responsible for receptor binding, was synthesized. The fragment consists of two peptides: a dipeptide (A 20-21) and an octapeptide (B 19-26), linked with a disulfide bond (A20-B19). The biological activity of the newly synthesized fragment relative to insulin was assayed for the influence on glycogenesis and for the ability to stimulate glucose uptake. Comparative tests for the biological activity of the synthesized fragment and of the intact hormone allowed us to conclude that the fragment has insulin-like properties.     

Putative disulfide-forming pathway of porcine insulin precursor during its refolding in vitro

Although the structure of insulin has been well studied, the formation pathway of the three disulfide bridges during the refolding of insulin precursor is ambiguous. Here, we reported the in vitro disulfide-forming pathway of a recombinant porcine insulin precursor (PIP). In redox buffer containing L-arginine, the yield of native PIP from fully reduced/denatured PIP can reach 85%. The refolding process was quenched at different time points, and three distinct intermediates, including one with one disulfide linkage and two with two disulfide bridges, have been captured and characterized. An intra-A disulfide bridge was found in the former but not in the latter. The two intermediates with two disulfide bridges contain the common A20-B19 disulfide linkage and another inter-AB one. Based on the time-dependent formation and distribution of disulfide pairs in the trapped intermediates, two different forming pathways of disulfide bonds in the refolding process of PIP in vitro have been proposed. The first one involves the rapid formation of the intra-A disulfide bond, followed by the slower formation of one of the inter-AB disulfide bonds and then the pairing of the remaining cysteines to complete the refolding of PIP. The second pathway begins first with the formation of the A20-B19 disulfide bridge, followed immediately by another inter-AB one, possibly nonnative. The nonnative two-disulfide intermediates may then slowly rearrange between CysA6, CysA7, CysA11, and CysB7, until the native disulfide bond A6-A11 or A7-B7 is formed to complete the refolding of PIP. The proposed refolding behavior of PIP is compared with that of IGF-I and discussed.     

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.     

Identification of insulin intermediates and sites of cleavage of native insulin by insulin protease from human fibroblasts

We have studied the time sequence degradation of native insulin by insulin protease from human fibroblast using multiple steps involving purification of the products by high performance liquid chromatography, determination of peak composition by amino acid sequence analysis, and confirmation of structure by mass spectrometry and thus elucidated the sites of cleavage of insulin by human insulin protease. We observed that as early as 0.5 min of incubation, three major new peptide peaks, intact insulin, and four smaller peptide peaks can be detected. The major peptides are portions of the insulin molecule, with the amino ends of the A and B chains or the carboxyl ends of the A and B chains still connected by disulfide bonds. Peptide peak I is A1-13-B1-9. Peptide peak II is A1-14-B1-9. Peptide peak III is A14-21-B14-30. The smaller peptide peaks are A14-21-B17-30, A15-21-B14-30, A15-21-B10-30, and A14-21-B10-30. The major peptide bond cleavage sites therefore consist of A13-14, A14-15, B9-10, B13-14, and B10-17. With longer incubation times, peptide peak II appears to lose the A14 tyrosine to form peptide peak I. This peptide I, which is the amino end of the A and B chains, is not further degraded even after 1.5 h of incubation. With longer incubation times, the peptides containing the carboxyl ends of the A and B chains are further degraded to form products from cleavage at the A18-19, B14-15, B25-26, and a small amount of A19-20, B10-11, and B24-25 cleavage and the emergence of 2-5-amino acid peptide chains, tyrosine, alanine, histidine, and leucine-tyrosine. We conclude, based on the three-dimensional structure of insulin, that human insulin protease recognizes the alpha-helical regions around leucine-tyrosine bonds and that final degradation steps to small peptides do not require lysosomal involvement.     

Replacement of disulfides by amide bonds in the relaxin-like factor (RLF/INSL3) reveals a role for the A11-B10 link in transmembrane signaling

The relaxin-like factor (RLF) also named insulin-like 3 (INSL3) consists of two polypeptide chains linked by two interchain and one intrachain disulfide bond. RLF binds to its receptor (LGR8 also named RXFP2) through the B chain and initiates transmembrane communication by activating the adenylate cyclase through the N-terminal region of both chains. Cystine A11-B10 occupies a unique position on the molecular surface just outside the binding region and between the two signaling ports. We have synthesized an RLF analogue in which the disulfide A11-B10 was replaced by a peptide bond and found that cAMP production ceased while receptor binding was not affected. In contrast, replacing the disulfide A24-B22 by a peptide bond reduced potency proportional to the binding affinity and lowered efficacy to 65%, while replacing disulfide A10-A15 by a peptide bond reduced binding affinity to 32% and lowered potency to 7% but maintained 100% efficacy. The exceptional properties of the derivative bearing an A11-B10 isopeptide cross-link suggests that the disulfide has a special role in signal transduction. We propose that disulfide A11-B10 serves as an insulator between the two ports, whereas the amide functionality disturbs the signal transmission complex likely due to changes in polarity. The clear separation between receptor binding and signal activation sites within this small protein permits one to study how the relaxin-like factor initiates the signal on the receptor that induces intracellular cAMP production.     

Determination of disulfide bond arrangement in bombyxin-IV,an insulin superfamily peptide from the silkworm,Bombyx mori,by combination of thermolysin digestion of natural peptide and selective synthesis of disulfide bond isomers

The mode of disulfide linkages in bombyxin-IV, an insulin superfamily peptide consisting of A- and B-chains, was determined as A6–A11, A7–B10, and A20–B22. An intermolecular bond of A20–B22 was identified by sequencing and mass spectrometric analysis of the fragments generated by thermolysin digestion of natural bombyxin-IV. The mode of the remaining two bridges was determined by chemical and selective synthesis of three possible disulfide bond isomers of bombyxin-IV. A- and B-chains were synthesized by solid-phase method, and three disulfide bonds were bridged stepwise and in a fully controlled manner. Retention time on reversed-phase high-performance liquid chromatography (HPLC), thermolysin digests, and biological activity of the synthetic [A6–A11, A7–B10, A20–B22-cystine]-bombyxin-IV revealed that it was identical with the natural bombyxin-IV. Two other isomers with respect to disulfide bond arrangement, [A6–A7, A11–B10, A20–B22-cystine]- and [A6–B10, A7–A11, A20–B22-cystine]-bombyxin-IVs, were distinguishable from the natural one by use of HPLC, thermolysin digestion, and bioassay.     

Effects of deletion and shift of the A20-B19 disulfide bond on the structure, activity, and refolding of proinsulin

To investigate the role of the A20-B19 disulfide bond in the structure, activity and folding of proinsulin, a human proinsulin (HPI) mutant [A20, B19Ala]-HPI was prepared. This mutant, together with another proinsulin mutant previously constructed with an A19Tyr deletion, which can also be taken as shifted mutant of the A20-B19 disulfide bond, were studied for their in vitro refolding, oxidation of free thiol groups, circular dichroism spectra, antibody and receptor binding activities and sensitivity to trypsin digestion in comparison with native proinsulin. The results indicate that deletion of the A20-B19 disulfide bond results in a large decrease in the alpha-helix content of the molecule and higher sensitivity to tryptic digestion. Both the deletion and shift mutations, especially the latter, cause a great decrease in the biological activity of proinsulin analogues. The folding yields of HPI analogues were much lower than that of HPI. And the shift mutant, [Delta A19Tyr]-HPI, was scarcely refolded correctly in vitro and its refolding yield was extremely low. These results suggest that the A20-B19 disulfide bond plays an important role in the structural stabilization and folding of the insulin precursor. By summarizing the refolding studies on proinsulin, a possible folding pathway is proposed.     

Elongation of the interchain disulfide bridges of insulin. A synthetic analog.

The sythesis and isolation in purified form of an analog of insulin with the interchain disulfide bridges elongated by a methylene group is described. This analog differs from the parent molecule in that the cystein residues occupying positions A-7 and A-20 and involved in the formation of the two interchain disulfide bridges of insulin have been replaced by homocysteine residues. For the synthesis of this compound the Hcy-7, 20-A chain of sheep insulin was chemically synthesized and isolated in the S-sulfonated form. Conversion of the latter product to the sulfhydryl derivative and combination with the S-sulfonated form of the B chain of sheep insulin yielded the [Hcy-7, 20-A] insulin. Isolation of the analog from the combination mixture was effected by chromatography on a carboxymethylcellulose column with acetate buffer (pH 3.3) and an exponential sodium chloride gradient. This analog, by the mouse convulsion assay methods and in doses at least 40-fold higher than those normally used for insulin assay, was inactive. By the radioimmunoassay method this synthetic analog was found to possess a potency of 2 i.u./mg. It is concluded that the biological activity of insulin depends critically on a particular geometry conferred on the molecule by the proper placement of the A and B chains.     

Structure-specific effects of protein topology on cross-beta assembly: studies of insulin fibrillation

Systemic amyloidoses, an important class of protein misfolding diseases, are often due to fibrillation of disulfide-cross-linked globular proteins otherwise unrelated in sequence or structure. Although cross-beta assembly is regarded as a universal property of polypeptides, it is not understood how such amyloids accommodate diverse disulfide connectivities. Does amyloidogenicity depend on protein topology? A model is provided by insulin, a two-chain protein containing three disulfide bridges. The importance of chain topology is demonstrated by mini-proinsulin (MP), a single-chain analogue in which the C-terminus of the B chain (residue B30) is tethered to the N-terminus of the A chain (A1). The B30-A1 tether impedes the fiber-specific alpha --> beta transition, leading to slow formation of a structurally nonuniform amorphous precipitate. Conversely, fibrillation is robust to interchange of disulfide bridges. Whereas native insulin exhibits pairings [A6-A11, A7-B7, and A20-B19], metastable isomers with alternative pairings [A6-B7, A7-A11, A20-B19] or [A6-A7, A11-B7, A20-B1] readily undergo fibrillation with essentially identical alpha --> beta transitions. Respective pairing schemes are in each case retained. Isomeric fibrils and the amorphous MP precipitate are each able to seed the fibrillation of wild-type insulin, suggesting a structural correspondence between respective nuclei or modes of assembly. Together, our results demonstrate that effects of polypeptide topology on amyloidogenicity depend on structural context. Although the native structures and stabilities of single-chain insulin analogues are similar to those of wild-type insulin, the interchain tether constrains the extent of conformational distortion at elevated temperature, retards initial non-native aggregation, and is apparently incompatible with the mature structure of an insulin protofilament. We speculate that the general danger of fibrillation has imposed a constraint in protein evolution, selecting for topologies unfavorable to amyloid formation.     

Dissecting the role of disulfide bonds on the amyloid formation of insulin

Disulfide bonds play a critical role in the stability and folding of proteins. Here, we used insulin as a model system, to investigate the role of its individual disulfide bond during the amyloid formation of insulin. Tris(2-carboxyethyl)phosphine (TCEP) was applied to reduce two of the three disulfide bonds in porcine insulin and the reduced disulfide bonds were then alkylated by iodoacetamide. Three disulfide bond-modified insulin analogs, INS-2 (lack of A6-A11), INS-3 (lack of A7-B7) and INS-6 (lack of both A6-A11 and A7-B7), were obtained. Far-UV circular dichroism (CD) spectroscopy results indicated that the secondary structure of INS-2 was the closest to insulin under neutral conditions, followed by INS-3 and INS-6, whereas in an acidic solution all analogs were essentially unfolded. To test how these modifications affect the amyloidogenicity of insulin, thioflavin-T (ThT) fluorescence and transmission electronic microscopy (TEM) were performed. Our results showed that all analogs were more prone to aggregation than insulin, with the order of aggregation rates being INS-6>INS-3>INS-2. Cross-linking of unmodified proteins (PICUP) assay results showed that analogs without A6-A11 (INS-2 and INS-6) have a higher potential for oligomerization than insulin and INS-3, which is accompanied with a higher cytotoxicity as the hemolytic assays of human erythrocytes suggested. The results indicated that breakage of A7-B7 induced more unfolding of the insulin structure and a higher amyloidogenicity than breakage of A6-A11, but breakage of A6-A11 caused a significant cytotoxicity increase and a higher potency to form high order toxic oligomers.     

Endosomal proteolysis of internalized insulin at the C-terminal region of the B chain by cathepsin D.

The endosomal compartment of hepatic parenchymal cells contains an acidic endopeptidase, endosomal acidic insulinase, which hydrolyzes internalized insulin and generates the major primary end product A(1--21)-B(1--24) insulin resulting from a major cleavage at residues Phe(B24)-Phe(B25). This study addresses the nature of the relevant endopeptidase activity in rat liver that is responsible for most receptor-mediated insulin degradation in vivo. The endosomal activity was shown to be aspartic acid protease cathepsin D (CD), based on biochemical similarities to purified CD in 1) the rate and site of substrate cleavage, 2) pH optimum, 3) sensitivity to pepstatin A, and 4) binding to pepstatin A-agarose. The identity of the protease was immunologically confirmed by removal of greater than 90% of the insulin-degrading activity associated with an endosomal lysate using polyclonal antibodies to CD. Moreover, the elution profile of the endosomal acidic insulinase activity on a gel-filtration TSK-GEL G3000 SW(XL) high performance liquid chromatography column corresponded exactly with the elution profile of the immunoreactive 45-kDa mature form of endosomal CD. Using nondenaturating immunoprecipitation and immunoblotting procedures, other endosomal aspartic acid proteases such as cathepsin E and beta-site amyloid precursor protein-cleaving enzyme (BACE) were ruled out as candidate enzymes for the endosomal degradation of internalized insulin. Immunofluorescence studies showed a largely vesicular staining pattern for internalized insulin in rat hepatocytes that colocalized partially with CD. In vivo pepstatin A treatment was without any observable effect on the insulin receptor content of endosomes but augmented the phosphotyrosine content of the endosomal insulin receptor after insulin injection. These results suggest that CD is the endosomal acidic insulinase activity which catalyzes the rate-limiting step of the in vivo cleavage at the Phe(B24)-Phe(B25) bond, generating the inactive A(1--21)-B(1--24) insulin intermediate.     

Hierarchical protein folding: asymmetric unfolding of an insulin analogue lacking the A7-B7 interchain disulfide bridge

The landscape paradigm of protein folding can enable preferred pathways on a funnel-like energy surface. Hierarchical preferences may be manifest as a nonrandom pathway of disulfide pairing. Stepwise stabilization of structural subdomains among on-pathway intermediates is proposed to underlie the disulfide pathway of proinsulin and related molecules. Here, effects of pairwise serine substitution of insulin's exposed interchain disulfide bridge (Cys(A7)-Cys(B7)) are characterized as a model of a late intermediate. Untethering cystine A7-B7 in an engineered monomer causes significantly more marked decreases in the thermodynamic stability and extent of folding than occur on pairwise substitution of internal cystine A6-A11 [Weiss, M. A., Hua, Q. X., Jia, W., Chu, Y. C., Wang, R. Y., and Katsoyannis, P. G. (2000) Biochemistry 39, 15429-15440]. Although substantially disordered and without significant biological activity, the untethered analogue contains a molten subdomain comprising cystine A20-B19 and a native-like cluster of hydrophobic side chains. Remarkably, A and B chains make unequal contributions to this folded moiety; the B chain retains native-like supersecondary structure, whereas the A chain is largely disordered. These observations suggest that the B subdomain provides a template to guide folding of the A chain. Stepwise organization of insulin-like molecules supports a hierarchic view of protein folding.     

An insulin analogue with gamma-amino butyric acid substitution for A13Leu-A14Tyr.

An insulin A chain analogue, [A13-14 GABA, A21 Ala]A chain, for which the dipeptide Leu-Try at A13-A14 was substituted by a non-coded amino acid, gamma-amino butyric acid (GABA) and A21 Asn by Ala, was prepared by stepwise Fmoc solid-phase manual synthesis and then combined with the natural B chain of porcine insulin to yield an insulin analogue, [A13-14 GABA, A21Ala] porcine insulin (GABA substituted insulin). This insulin analogue still retains 50% in vivo biological activity and 59% in receptor binding capacity. It can also be crystallized. These results indicate that its overall conformation is similar to the native form and that the side chains of A13Leu and A14Tyr are not essential for insulin activity. In addition, the replacement of a normal C-N peptide bond by an unnatural C-C bond may have general meaning in structure and function studies of other proteins.     

Determination of disulfide bond arrangement in bombyxin-IV, an insulin superfamily peptide from the silkworm,Bombyx mori, by combination of thermolysin digestion of natural peptide and selective synthesis of disulfide bond isomers

The mode of disulfide linkages in bombyxin-IV, an insulin superfamily peptide consisting of A- and B-chains, was determined as A6–A11, A7–B10, and A20–B22. An intermolecular bond of A20–B22 was identified by sequencing and mass spectrometric analysis of the fragments generated by thermolysin digestion of natural bombyxin-IV. The mode of the remaining two bridges was determined by chemical and selective synthesis of three possible disulfide bond isomers of bombyxin-IV. A- and B-chains were synthesized by solid-phase method, and three disulfide bonds were bridged stepwise and in a fully controlled manner. Retention time on reversed-phase high-performance liquid chromatography (HPLC), thermolysin digests, and biological activity of the synthetic [A6–A11, A7–B10, A20–B22-cystine]-bombyxin-IV revealed that it was identical with the natural bombyxin-IV. Two other isomers with respect to disulfide bond arrangement, [A6–A7, A11–B10, A20–B22-cystine]- and [A6–B10, A7–A11, A20–B22-cystine]-bombyxin-IVs, were distinguishable from the natural one by use of HPLC, thermolysin digestion, and bioassay.     

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.