| The thesis consists of two parts. The first part investigates the refolding of amphioxus insulin-like peptide (aILP), and the second part is about the foldability of three conserved Val in PIP.Insulin and IGF-1 share homogenous primary and tertiary structure as well as weakly overlapping biological activity, but their folding behaviors are significantly different: insulin/PIP in vitro folds into one unique thermodynamically controlled structure, while IGF-1 in vitro folds into two thermodynamically controlled disulfide isomers. The different folding behavior of Insulin/PIP and IGF-1 is mainly controlled by the B-chain/domain which determinates the different energetic state of the intra-A-chain/domain disulfide. In 1990, Steiner reported an insulin-like peptide cDNA sequence from amphioxus, which had high sequence homology compared with insulin and IGF-1. So aILP is deduced as an ancestor molecule of insulin and IGF-1. To explore the origin of the different folding behavior of insulin and IGF-1, we carry out the experiments of aILP refolding and disulfide reshuffling, and get two disulfide isomers named native and swap with the same molecular mass. In the two experiments, both from native to swap and from swap to native, the identical balance are got, and the ratio of swap: native at 15 ℃ is 1-2%, the ratio at 37 ℃ 3-4%. It shows that the two isomers are thermodynamically controlled. The CD analysis indicates the α- helix content of native ILP is 30%, and theα-helix content of swap ILP is 12%, so the native ILP has more intact structure and more stable than the swap. The V8 peptide map indicates the native and swap remain the disulfide B19-A20 with the different pair of A6, A7, A11, B7. Given the previous reports about insulin swap and IGF-1 swap, we deduce that the disulfide pairs of the native ILP are B19-A20, A6-A11, B7-A7, and the disulfide pairs of the swap ILP are B19-A20, B7-A6, A7-A11. Meanwhile there is an unique structure in insulin refolding and reshuffling, and there are two isomers with a ratio of swap : native 35:65 in IGF-1 refolding at 10℃. The ratio of ILP is between insulin's and IGF-1's. So first it shows that the ILP has non-Anffinsen folding behavior; second it provides a new proof that aILP is the ancestor molecule of insulin and IGF-1. It is thought that in the molecular evolution from ILP to insulin the folding information is more got, finally folding into a unique structure; in the molecular evolution from ILP to IGF-1 the folding information is lost, and in vitro folding into two similar thermodynamical stable isomers (in vivo the IGF-1 folds into a native structure due to the IGF binding proteins.). When the N end of a protein is Glu or Gln, the cyclization may bring about, and create a pyroglutamate residue, which bring much trouble in the protein conservation. Some neural small peptide with a pyroglutamate residue is easier to assembly and filbril. During the fermentation of ILP, two proteins with different retention time are got, named ILP1 with the expected molecular mass 5701 and ILP2 with an unexpected molecular mass less 17 than the expected molecular mass. ILP1 and ILP2 are with the same mobility rate in native pH 8.3 PAGE. The V8 digestion peptide map of ILP1 and ILP2 is identical except the three N-terminal residues. So we think that ILP2 is created by ILP N-terninal Gln cyclization named [pE]ILP. The CD spectrum of ILP and [pE]ILP is very similar, so the pyroglutamate residue hasn't significant affection on the ILP structure. In the [pE]ILP refolding and disulfide reshuffling, two isomers named swap and native are also got, however the ratio of swap: native increases one time, at 15 ℃ the ratio 3-4% and at 37 ℃ the ratio 6-7%, compared with ILP. On the whole, ILP N-terninal Gln cyclization hasn't significant affection on the ILP structure, or the affection on the ILP structure can't be examined by our experiment. However it has an apparent affection on the ILP folding behavior, and makes swap structure more easy to create. The previous result...
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