Cleaved serpin refolds into the relaxed state via a stressed conformer.

Serine proteinase inhibitors (serpins) are believed to fold in vivo into a metastable "stressed" state with cleavage of their P1-P1' bond resulting in reactive center loop insertion and a thermostable "relaxed" state. To understand this unique folding mechanism, we investigated the refolding processes of the P1-P1'-cleaved forms of wild type ovalbumin (cl-OVA) and the R339T mutant (cl-R339T). In the native conditions, cl-OVA is trapped as the stressed conformer, whereas cl-R339T attains the relaxed structure.

C-terminus engineering of soybean proglycinin: improvement of emulsifying properties.

Introduction of the extension region of beta-conglycinin alpha' subunit at the C-terminus of proglycinin A1aB1b results in the improvement of its emulsifying properties. To understand the basic for such improvement, we introduced the alpha' and alpha extension regions to the A2B1a C-terminus, and the alpha extension and A5A4B3 hypervariable regions, and an oligopeptide composed of 20 negatively or positively charged residues to the A1aB1b C-terminus, creating A2B1aalpha', A2B1aalpha, and A1aB1balpha, A1aB1bA4IV, A1aB1bNeg and A1aB1bPos, respectively. All the modified versions were produced in Escherichia coli.

Structure-function relationships of soybean proglycinins at subunit levels.

Glycinin consists of five kinds of subunits, group I (A1aB1b, A1bB2, and A2B1a) and group II (A3B4 and A5A4B3). cDNAs for individual subunits were cloned by reverse transcription-polymerase chain reaction method and expressed in Escherichia coli using pET vector. The recombinant proglycinins were purified by ammonium sulfate fractionation and column chromatography in the form of homotrimers.