A peptide model of insulin folding intermediate with one disulfide

Abstract

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.

Figure 1.

Native pH 8.3 PAGE analysis of the model peptide. 1–4 represents wild-type PIP, the model peptide, [A7Ser, B7Ser]PIP, and [A6Ala, A11Ala]PIP, respectively.

Figure 2.

C4 reversed-phase HPLC analysis of the model peptide. For each analysis, a 5-μg sample was loaded onto the column and eluted by the gradient listed in Materials and Methods.

Figure 3.

CD analysis of the model peptide. The top panel shows the near-UV spectra; the bottom panel shows the far-UV spectra. The open circle represents the spectra of the model peptide; the filled circle represents the spectra of the wild-type PIP.

Figure 4.

In vitro refolding of the model peptide analyzed by C4 reversed-phase HPLC. At the indicated time, 100 μL refolding mixture was removed, acidified to pH 2.0 with TFA, and analyzed by C4 reversed-phase HPLC eluted with the gradient listed in Materials and Methods.

Figure 5.

Disulfide stability of the model peptide in redox buffer. Lanes 1–10 represent that in redox buffer the ratio of GSH to GSSG (mM/mM)) was 0/0, 1/10, 2/5, 5/5, 5/1, 10/1, 20/1, 30/1, 40/1, and 50/1, respectively. When the disulfides were reduced in the redox buffer and then the free thiol groups were carboxymethylated, the molecule carried more negative charges and ran faster on the native PAGE, but the conformation also had an effect on their mobility rate. The gel was stained by Coomassie brilliant blue R250.

Figure 6.

Receptor-binding analysis. The filled circle represents the plot of porcine insulin; the open circle represents the plot of (desB30)[A20–B19]insulin.

Figure 7.

Schematic representation of the early stage of PIP refolding.