Abstract
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.
Highlights
Human insulin is a double-chain peptide that is synthesized in vivo as a single-chain human proinsulin (HPI)
After a series of tests, we have found that several factors in the optimized refolding condition of HPI are obviously different from those of porcine insulin precursor (PIP), the difference of two molecules in sequence is only the length of connecting peptide
Predominance of GSSG in the redox buffer is required for the high yield of HPI, whereas GSH is required for PIP
Summary
Materials—Recombinant HPI with purity greater than 98% was kindly provided by Lilly and confirmed by HPLC on a reverse phase C8 column. The refolding reaction was carried out at 16 °C for 16 h and quenched by adding trifluoroacetic acid to pH 1.0, and the mixture was analyzed on HPLC using a C4 reverse phase column to quantify the refolding yield. For the iodoacetic acid method, the refolding reaction was quenched by adding onefourth volume of freshly prepared 0.5 M IAA in 50 mM Tris-HCl (pH 8.5), and the carboxymethylation reaction was performed at room temperature for 5 min. Before the folding of the intermediates, protein solution and a 2-fold concentration of refolding buffer (200 mM Tris, pH 10.0, 10 mM EDTA, 2 mM GSH, 10 mM GSSG) were incubated at 4 °C for 10 min, respectively. The mixture was analyzed by reverse-phase HPLC on a C4 column (Sephasil peptide, ST 4.6/250 mm; Amersham Biosciences) with a linear gradient of 50 – 80% of solvent B in 30 min. The secondary structure contents of the samples were estimated by using the software J-700 for Windows Secondary Structure Estimation, Version 1.10.00 (JASCO Corp.), equipped with the Jasco-700
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