Peptide models of four possible insulin folding intermediates with two disulfides

Abstract

The single-chain insulin (PIP) can spontaneously fold into native structure through preferred kinetic intermediates. During refolding, pairing of the first disulfide A20-B19 is highly specific, whereas pairing of the second disulfide is likely random because two two-disulfide intermediates have been trapped. To get more details of pairing property of the second disulfide, four model peptides of possible folding intermediates with two disulfides were prepared by protein engineering, and their properties were analyzed. The four model peptides were named [A20-B19, A7-B7]PIP, [A20-B19, A6-B7]PIP, [A20-B19, A6-A11]PIP, and [A20-B19, A7-A11]PIP according to their remaining disulfides. The four model peptides all adopt partially folded structure with moderate conformational differences. In redox buffer, the disulfides of the model peptides are more easily reduced than those of the wild-type PIP. During in vitro refolding, the reduced model peptides share similar relative folding rates but different folding yields: The refolding efficiency of the reduced [A20-B19, A7-A11]PIP is about threefold lower than that of the other three peptides. The present results indicate that the folding intermediates corresponding to the present model peptides all adopt partially folded conformation, and can be formed during PIP refolding, but the chance of forming the intermediate with disulfide [A20-B19, A7-A11] is much lower than that of forming the other three intermediates.

Figure 1.

Native PAGE (at pH 8.3) analysis of the model peptides with two disulfides. Lanes 1–5 represent wild-type PIP, [A20–B19, A6–A11]PIP, [A20–B19, A7–B7]PIP, [A20–B19, A7–A11]PIP, and [A20–B19, A6–B7]PIP, respectively. In each lane, 2 μg of purified sample was loaded. The gel was stained by Coomassie brilliant blue R250.

Figure 2.

C4 reverse-phase HPLC analysis of the model peptides with two disulfides. In each analysis, 100 μL of sample (5 μg) was loaded onto a C4 column and eluted by using the elution gradient listed in Materials and Methods.

Figure 3.

CD analysis in far-UV region (A) and near-UV region (B). The filled circles indicate wild-type PIP; open circles, [A20–B19, A7–B7]PIP; filled triangles, [A20–B19, A6–A11]PIP; open triangles, [A20–B19, A6–B7]PIP; and filled squares, [A20–B19, A7–A11]PIP.

Figure 4.

Disulfide stability analysis of the model peptides with two disulfides. Lanes 1–9 represent that in redox buffer, the ratio of GSH/GSSG (mM/mM)) was 0 : 0, 1 : 10, 1 : 5, 5 : 5, 5 : 1, 7 : 1, 10 : 1, 20 : 1, and 30 : 1, respectively. The gel was stained by Coomassie brilliant blue R250.

Figure 5.

HPLC profiles of the refolding of the four model peptides with two disulfides. At the indicated reaction time, 100 μL of reaction mixture was removed, acidified with TFA, and then loaded onto the C4 reverse-phase column and eluted by using the elution gradient listed in Materials and Methods.

Figure 6.

The actual (A) and normalized (B) refolding curves of the four model peptides with two disulfides. Filled circles indicate [A20–B19, A6–A11]PIP; open circles, [A20–B19, A7–A11]PIP; filled triangles, [A20–B19, A7–B7]PIP; open triangles, [A20–B19, A6–B7]PIP; and filled squares, wild-type PIP. The actual refolding yield was calculated from the peak area of HPLC profiles; the normalized refolding yield is the ratio of actual refolding yield to the highest refolding yield of each model peptide.