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 timedependent 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 ␣-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.
This article presents effective algorithms to generate iso-parametric and iso-scallop tool path of five-axis computer numerically controlled machining for cyclide spline surfaces with flat-end cutters. Optimal tool orientations are generated by making the contact order between the cyclide patch and the cutter envelope surface as high as possible. Based on the optimal tool orientation, efficient methods are proposed to generate iso-scallop and iso-parametric tool paths for cyclide spline surfaces. Together with the existing methods of fitting arbitrary surface with cyclide splines, the proposed method can be used to improve five-axis machining efficiency of free-form surfaces.
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