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Involvement of molten globule state on the folding process of secretary proteins

熔球状态对分泌蛋白折叠过程的影响

基本信息

批准号：
02660094
负责人：
HIROSE Masaaki
金额：
$ 1.47万
依托单位：
Kyoto University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1990
资助国家：
日本
起止时间：
1990 至 1991
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-02660094/
关键词：
molten globule secretary protein formation of conformation 高次構造再生

项目摘要

Human serum albumin(HSA), N-terminal half-molecule of ovotransferrin(N-OVT), and ovalbumin(OVA)were reduced and denatured in the presence of dithiothreitol and 8 M urea. The samples were diluted with a neutral buffer, and protein conformation was evaluated by CD-spectrum. With regards to HSA and N-OVT, the refolded, disulfide-reduced form was found to take a partially folded molten globule-like conformation. When oxidized form of gultathione(GSSG)was added to the state, the intrachain protein disulfide bonds were regenerated. These data indicated that HSA as well as N-OVT take a molten globulelike state during oxidative refolding as an intermediate. In contrast, OVA showed the native-like conformation in its disulfide-reduced state as evaluated by CD-spectrum. The reactivity of cysteine sulfhydryls, however, was significantly different between the native form and the refolded, disulfide-reduced form : no reactive sulfhydryl was detected in the former form, but two sulfhydryls were detected in the latter form. By the addition of GSSG to the reduced state, ovalbumin was transformed to the disulfide bonded form with native cyteine pairing(Cys73-CYsl2O). From these data, we concluded that a protein with many disulfide and domain structure, such as HSA and N-OVT, takes a molten globule-like conformation as a intermediate for oxidative refolding, while a protein with single disulfide and single domain, such as OVA, takes a naive-like conformation in the disulfide-reduced state.

人血清白蛋白（HSA）、卵转铁蛋白（N-OVT）的N-末端半分子和卵清蛋白（OVA）在二硫苏糖醇和8 M尿素存在下还原和变性。用中性缓冲液稀释样品，并通过CD光谱评价蛋白质构象。关于HSA和N-OVT，发现重折叠的二硫化物还原形式采取部分折叠的熔融球状构象。当加入氧化形式的谷氨酰胺（GSSG）时，链内蛋白质二硫键再生。这些数据表明，HSA以及N-OVT在氧化复性过程中作为中间体采取熔融球状状态。与此相反，卵清蛋白显示天然样构象在其二硫键还原状态的CD光谱评价。半胱氨酸巯基的反应性，然而，是显着不同的天然形式和重折叠，二硫键还原的形式之间：没有反应性巯基被检测到在前一种形式，但两个巯基被检测到在后一种形式。通过添加GSSG至还原状态，卵清蛋白转化为具有天然半胱氨酸配对的二硫键形式（Cys 73-CYsl 20）。从这些数据中，我们得出结论，具有许多二硫键和结构域结构的蛋白质，如HSA和N-OVT，以熔融球状构象作为氧化重折叠的中间体，而具有单个二硫键和单个结构域的蛋白质，如OVA，在二硫键还原状态下呈天然样构象。