In vitro unfolding of insulin: characterization of intermediates and putative unfolding pathway.

The in vitro insulin unfolding had been studied using the "equilibrium unfolding" method where protein is unfolded by reducing reagents in the presence of trace amounts of oxidants such as oxidized glutathione. Nine intermediates were captured in the unfolding process, named as P1A, P2A, P3A, P4A, P3B, P4B, P5B, P6B, and P7B, which were all either A chain derivatives or B chain derivatives. No intermediate with inter-A-B chain disulfide was captured.

Equilibrium folding of porcine insulin precursor in the presence of redox buffer: implications for the common intermediates shared by its unfolding/ refolding processes.

We use the procedure established for 'disulfide stability analysis in redox system' to investigate the unfolding process of porcine insulin precursor (PIP). Six major unfolding intermediates have been captured, in which four contain two disulfides, two contain one disulfide. Based on the characterization and analysis of the intermediates an unfolding pathway has been proposed, by which the native PIP unfolded through in turn 2SS and 1SS intermediates into fully reduced form.

The sequence determinant causing different folding behaviors of insulin and insulin-like growth factor-1.

Although insulin and insulin-like growth factor-1 (IGF-1) belong to the insulin superfamily and share highly homologous sequences, similar tertiary structure, and a common ancestor molecule, amphioxus insulin-like peptide, they have different folding behaviors: IGF-1 folds into two thermodynamically stable tertiary structures (native and swap forms), while insulin folds into one unique stable structure. To further understand which part of the sequence determines their different folding behavior, based on previous reports from the laboratory, two peptide models, [B9A][1-4]porcine insulin precursor (PIP) and [B10E][1-4]PIP, were constructed. The plasmids encoding the peptides were transformed into yeast cells for expression of the peptides; the results showed that the former peptide was expressed as single component, while the latter was expressed as a mixture of two components (isomer 1 and isomer 2).

Mutational analysis of the absolutely conserved B8Gly: consequence on foldability and activity of insulin.

B8Gly is absolutely conserved in insulins during evolution. Moreover, its corresponding position is always occupied by a Gly residue in other members of insulin superfamily. Previous work showed that Ala replacement of B8Gly significantly decreased both the activity and the foldability of insulin.

1.42A crystal structure of mini-IGF-1(2): an analysis of the disulfide isomerization property and receptor binding property of IGF-1 based on the three-dimensional structure.

Insulin and insulin-like growth factor 1 (IGF-1) share a homologous sequence, a similar three-dimensional structure and weakly overlapping biological activity, but IGF-1 folds into two thermodynamically stable disulfide isomers, while insulin folds into one unique stable tertiary structure. This is a very interesting phenomenon in which one amino acid sequence encodes two three-dimensional structures, and its molecular mechanism has remained unclear for a long time. In this study, the crystal structure of mini-IGF-1(2), a disulfide isomer of an artificial analog of IGF-1, was solved by the SAD/SIRAS method using our in-house X-ray source.

Mutagenesis of the three conserved valine residues: consequence on the foldability of insulin.

Natural polypeptide chain usually can spontaneously fold into tightly compact native structure. This capability is the so-called foldability. However, how the foldability is encoded in the polypeptide chain is still poorly understood.

Contribution of the absolutely conserved B8Gly to the foldability of insulin.

B8Gly is absolutely conserved in insulin from different species, and in other members of the insulin superfamily the corresponding position is always occupied by a Gly residue. However, the reasons for its conservation are still unclear; probably many factors contribute to this phenomenon. In our previous work, B8Gly was replaced by an Ala residue, which suggested that biological activity is one of the factors contributing to its conservation.

Possible Role of B8Gly in Insulin Structural Motif.

In the insulin structural motif n1-Cys-Gly-X10-Cys-n2-Cys-Cys-X3-Cys-X8-Cys-n3, there are seven absolutely conserved amino acid residues, and the only Gly is at position B8. When B8Gly was substituted with Ala by means of site-directed mutagenesis, a mutant insulin, [B8Ala]human insulin was obtained. The receptor binding capacity and in vivo biological activity of the [B8Ala]human insulin were about 2.

[Preparation, characterization and receptor-binding capacity of pig serum transferrin half-molecules containing a single iron-binding site].

N- and C-half molecules containing a single iron-binding site were simultaneously obtained from trypsin digest of iron-saturated pig transferrin. The activities of the pig serum transferrin and of its N- and C-half molecules to bind the human placental membrane transferrin receptor were compared. The results indicate that the receptor-binding site of pig transferrin may be located at the C-half molecule of the transferrin.

Protein Engineering of Insulin: [B9 Glutamic Acid] Human Insulin.

B9Ser of insulin B chain was substituted by Glu using site-directed mutagenesis to obtain a fast-acting insulin-[B9Glu] human insulin. The receptor binding capacity and in vivo biological activity of [B9Glu] human insulin are 21% and 40% as those of porcine insulin respectively.

Two Chain "Insulin/Insulin-like Growth Factor-I" Hybrids.

The two chain "Insulin/Insulin-like Growth Factor-I" hybrids, Ins/IGF-I(8) and Ins/IGF-I(11), were obtained by means of enzymatic semisynthesis, using desoctapeptide insulin (DOI) and the octapeptide and undecapeptide chemically synthesized according to the sequence 22-29 and 22-32 of IGF-I respectively as the starting materials. Comparative studies of the molecules with insulin indicate that the hybrid molecules retain in vivo the full activity of insulin. So the replacement of B27Thr by Asn and B30Ala by Thr, and the exchange of the sequence orders of B25 and B26, B28 and B29, as well as the extension of tripeptide (Gly-Tyr-Gly) at the B30 do not affect insulin activity.

Protein Engineering of Insulin: [B9Glu, B10Asp] Human Insulin.

B9Ser and B10His of the insulin B chain are substituted respectively by Glu and Asp using a gapped duplex DNA approach for site-directed mutagenesis. A mutant insulin-[B9Glu, B10Asp] human insulin was obtained. The receptor binding capacity of the mutant insulin is 34.