Insulin Receptor Ligand Binding Domain Of L1 And L2 Expression And Identification Of Proinsulin Folding And Unfolding

This thesis consists of two parts. The first part is the expression and characterization of the human insulin receptor's ligand-binding domains. The second part is the refolding and unfolding of insulin precursor and proinsulin. In the first chapter of the second part, we studied the disulfide-forming pathway of a single-chain porcine insulin precursor in vitro. In the second chapter, we further studied the folding pathway of human proinsulin in vitro. Finally in the third chapter, we investigate the unfolding of human proinsulin by using disulfide scrambling.The first part of the thesis describes the expression and characterization of the human insulin receptor's ligand-binding domains L1 and L2. The absence of the detailed three-dimensional structure of human insulin receptor's ectodomain remains a hindrance to understanding the molecular mechanism of insulin binding and transmembrane signaling. The cysteine-rich and highly glycosylated (-subunits, which are responsible for insulin binding, are fragile to the X-ray analysis. So, it's necessary to construct a mini-receptor with the ligand-binding ability in order to investigate the X-ray structure of the ligand/receptor complex. We have noticed that the two homologous domains within the (-subunits, L1 and L2, play an important role in ligand binding, so they have the potential to specifically interact with the insulin. We cloned the genes of sL1 (residues 1-119), sL2 (residues 311-328), L1 (residues 1-157) and L2 (residues 311-470) from the cDNA library of human placenta. The antibodies to sL1 and sL2 were prepared. The fusion-expression system was utilized to express the highly hydrophobic L1 and L2 protein. After a series of screening for the suitable culture condition, we successfully expressed in bacteria the two receptor fragments, L1 and L2. At least 1.0 mg receptor fragment protein with the purity more than 95% could be obtained from 500 ml culture after two steps purification. To test whether the L1 and L2 could form a contact site for insulin, we examined the ligand-binding ability of the purified proteins L1 and L2 by using polyethylene glycerol precipitation assay and surface plasmon resonance (SPR) real-time analysis. Among L1, L2 and their combination, none of them could interact with the insulin. The results clearly indicate that only L1 and L2 domains are not sufficient to create a ligand-binding site. During the SPR assay, we accidentally found that L2 could interact with the avidin immobilized on the SA5 sensor chip. In the first chapter of the second part, we studied the refolding process of porcine insulin precursor (PIP) in vitro and proposed the putative disulfide-forming pathwayof PIP. Although the structure of insulin has been well studied, the forming pathway of the three disulfide-bridges during the refolding of insulin precursor is ambiguous. In redox buffer containing L-Arginine, the yield of native PIP from fully reduced/denatured PIP can reach 85%. The refolding process was quenched at different time point and three distinct intermediates, including one with one disulfide linkage and two with two disulfide bridges, have been captured and characterized. Intra-A disulfide bridge was found in the former but not in the later. The two intermediates with two disulfide bridges contain the common A20-B19 disulfide linkage and another inter-AB one. Based on the time-dependent formation and distribution of disulfide pairs in the trapped intermediates, two different forming pathways of disulfide bonds in the refolding process of PIP in vitro have been proposed. The first one involves the rapid formation of the intra-A disulfide bond, followed by the slower formation of one of the inter-AB disulfide bonds and then the pairing of the remaining cysteines to complete the refolding of PIP. The second pathway begins with the formation of A20-B19 disulfide bridge, followed immediately by another inter-AB one, possibly nonnative. The nonnative two-disulfide intermediates may then slowly rearrange between CysA6, CysA7, CysA11 and CysB7, unt...