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Folding Units of Reduced and S-Protected Lysozyme

还原和 S-保护的溶菌酶的折叠单位

基本信息

批准号：
63420054
负责人：
SEGAWA Shin-ichi
金额：
$ 0.64万
依托单位：
Kwansei Gakuin University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (A)
财政年份：
1988
资助国家：
日本
起止时间：
1988 至 1989
项目状态：
已结题

项目摘要

At the present, it in accepted that protein folding proceeds through early folding of short segments of a polypeptide chain and the subsequent assembly -of them into the final tertiary structure. Lysozyme is a protein containing four disulfide bridges, and its tertiary structure in very stable in the aqueous solution. When it loses the disulfide bridges, however, its tertiary structure can not be kept. In such an unfolded state of lysozyme, local structures are expected to be prefferentailly formed through short range interactions within shoot segments of polypeptide chain. It is important for the study of protein folding to find such preferential folding units and to identify their positions along a polypeptide chain.Reduced and S-3-(trimethylated amino) propylated lysozyme (abbreviated to TMAP-lysozyme) was used for our experiments. Circular dichroism spectra off TMAP-lysozyme were measured in various solutions containing GuHC1 or sorbitor. The addition of sorbitor was found to increase the negative ellipticity in the 190-260 nm region. The limited proteolysis of this TMAP-lysozyme by trypsin was performed with the aim of identifying the segments of polypeptide chain havens the preferential local structures. All the tryptic peptides of TNAP-lyscyzyme were identified on the reverse phase chromaturams. The time course of production of their tryptic peptides was followed by plotting their peak areas against the reaction time with trypsin. Also, intermediate products consisting of several tryptic peptides were found on the same chromatgrams. Kinetics of their production and digestion was also observed. Hydrolysis rate constants by trypsin were estimated at all cleavage sites according to the Michaelis Menten mechanism. As a result, it was found that the sites of resistance to trypsin concentrate in the N-terminal half of the polypeptide chain of TMAP-lysozyme.

目前，人们普遍认为蛋白质折叠是通过多肽链的短片段的早期折叠和随后的组装成最终的三级结构进行的。溶菌酶是一种含有四个二硫桥的蛋白质，其三级结构在水溶液中非常稳定。然而，当失去二硫桥时，其三级结构就不能保持了。在这种未折叠的溶菌酶状态下，局部结构预计更倾向于通过多肽链的短段相互作用形成。寻找这些优先折叠单元并确定它们在多肽链上的位置对蛋白质折叠的研究具有重要意义。我们的实验使用还原和S-3-（三甲基化氨基）丙基溶菌酶（简称tmap -溶菌酶）。在含GuHC1或吸收剂的不同溶液中测定了tmap -溶菌酶的圆二色性光谱。吸收剂的加入增加了190 ~ 260 nm区域的负椭圆率。胰蛋白酶对这种tmap -溶菌酶进行有限的蛋白水解，目的是识别具有优先局部结构的多肽链片段。在反相色谱上鉴定了所有的tnap -溶菌酶的色氨酸。通过绘制其峰面积与胰蛋白酶反应时间的关系，跟踪了它们的胰蛋白酶肽生产的时间过程。在同一层析图上还发现了由几种色氨酸组成的中间产物。还观察了它们的产生和消化动力学。根据Michaelis Menten机制估计了胰蛋白酶在所有裂解位点的水解速率常数。结果发现，对胰蛋白酶的耐药位点集中在tmap -溶菌酶多肽链的n端。