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Intra-A chain disulphide bond forms first during insulin precursor folding.

ABSTRACT: In this study, we investigated the folding pathway of insulin precursor and compared it with that of insulin-like growth factor I (IGF-I). The intra-A chain disulphide bond was found to form early in insulin precursor folding, whereas the corresponding disulphide bond in IGF-I formed late. Intra-A chain disulphide-bond deleted [A6, A11-Ser] proteins, including proinsulin, insulin, and A chain, were employed for this investigation. Under the same conditions the recombination yield of insulin from S-sulphonates of native A and B chains was 22%, while the yield of [A6, A11-Ser] insulin from S-sulphonates of [A6, A11-Ser] A chain and native B chains was only approx. 7%. This indicated that the intra-A chain disulphide bond may serve to stabilize the A chain folding intermediate so as to facilitate the correct recognition and pairing with the B chain. Time courses of oxidation of reduced insulin A chains, reduced A and B chains, and reduced proinsulins showed that the intra-A chain disulphide bond formed first during insulin precursor folding. The formation of intra-A chain disulphide bond further accelerated the formation of the other two inter-chain disulphide bonds. The time course of helix structure formation of insulin A chains also indicated that the intra-A chain disulphide bond formed first, and could stabilize partially folded A chain helix structure. The rate of intra-A chain disulphide bond formation was almost the same as that for both helix structure formation and insulin molecule formation, indicating that the formation of the intra-A chain disulphide bond was the rate limiting step for the folding of insulin precursor.

SUBMITTER: Yuan Y

PROVIDER: S-EPMC1220534 | biostudies-other | 1999 Oct

REPOSITORIES: biostudies-other

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Project description:In this work we report the effects of continuous UV-light (276 nm, ~2.20 W.m(-2)) excitation of human insulin on its absorption and fluorescence properties, structure and functionality. Continuous UV-excitation of the peptide hormone in solution leads to the progressive formation of tyrosine photo-product dityrosine, formed upon tyrosine radical cross-linkage. Absorbance, fluorescence emission and excitation data confirm dityrosine formation, leading to covalent insulin dimerization. Furthermore, UV-excitation of insulin induces disulphide bridge breakage. Near- and far-UV-CD spectroscopy shows that UV-excitation of insulin induces secondary and tertiary structure losses. In native insulin, the A and B chains are held together by two disulphide bridges. Disruption of either of these bonds is likely to affect insulin's structure. The UV-light induced structural changes impair its antibody binding capability and in vitro hormonal function. After 1.5 and 3.5 h of 276 nm excitation there is a 33.7% and 62.1% decrease in concentration of insulin recognized by guinea pig anti-insulin antibodies, respectively. Glucose uptake by human skeletal muscle cells decreases 61.7% when the cells are incubated with pre UV-illuminated insulin during 1.5 h. The observations presented in this work highlight the importance of protecting insulin and other drugs from UV-light exposure, which is of outmost relevance to the pharmaceutical industry. Several drug formulations containing insulin in hexameric, dimeric and monomeric forms can be exposed to natural and artificial UV-light during their production, packaging, storage or administration phases. We can estimate that direct long-term exposure of insulin to sunlight and common light sources for indoors lighting and UV-sterilization in industries can be sufficient to induce irreversible changes to human insulin structure. Routine fluorescence and absorption measurements in laboratory experiments may also induce changes in protein structure. Structural damage includes insulin dimerization via dityrosine cross-linking or disulphide bond disruption, which affects the hormone's structure and bioactivity.

Project description:Anfinsen's famous experiment showed that the restoration of catalytic activity of a completely unfolded ribonuclease A is only possible when the correct order of events is followed during the refolding process. Inspired by this work, the effect of structural constraints induced by covalent cross-links on the folding of a synthetic polymer chain via hydrogen-bonding interactions is investigated. Hereto, methacrylate-based monomers comprising either benzene-1,3,5-tricarboxamide (BTA)-based or coumarin-based pendants are copolymerized with n-butyl methacrylate in various ratios via reversible addition-fragmentation chain-transfer (RAFT) polymerization. To assess whether the folding and single-chain polymeric nanoparticle (SCPN) formation depend on the order of events, we compare two folding pathways. In the one case, we first covalently cross-link the coumarin pendants within the polymers in a solvent that prevents hydrogen bonding, after which hydrogen bonding is activated, inducing folding of the polymer. In the other case, we induce hydrogen-bonding interactions between tethered BTAs prior to covalent cross-linking of the coumarin pendants. A combination of circular dichroism (CD) spectroscopy, UV-vis spectroscopy, size-exclusion chromatography (SEC), and dynamic light scattering (DLS) is employed to understand the effect of the structural constraints on the folding behavior of these synthetic polymers. The results show that like in ribonuclease A, the order of events matters greatly and determines the outcome. Importantly, a hydrogen-bond-promoting solvent prevents the formation of SCPNs upon covalent cross-linking and results in multichain aggregates. In contrast, covalently cross-linking the polymer when no hydrogen bonds are present followed by inducing hydrogen bonding favors the formation of SCPNs above the UCST of the methacrylate-based polymer. To our surprise, the two systems show a fundamentally different response to changes in temperature, indicating that also in synthetic polymers differences in the folding pathway induce differences in the properties of the resultant nanostructures.

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Intra-A chain disulphide bond forms first during insulin precursor folding.

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