【作者】 何红伟；

【导师】 高音；

【作者基本信息】 首都师范大学 ， 遗传学， 2002， 硕士

【摘要】 蛋白质分子间异肽键交联是生物体内普遍存在的一种蛋白质分子间交联形式。目前对它的研究主要集中在对谷氨酰胺转移酶和泛素-蛋白水解酶复合体途径的研究两个方面。我们以溶菌酶、核糖核酸酶A和蛋白质二硫键异构酶为模型在体外详细地研究了异肽键，提出了解释异肽键形成机理的假说：1)蛋白质发生构象变化包括二级结构的改变；2)蛋白质形成分子间二硫键；3)二硫键的形成帮助蛋白质形成了分子间异肽键。 虽然我们已经有许多证据来支持我们的假说，但还不够。我们构建了PDI删除b’结构域突变体和三个PDI定点突变体。在纯化野生型PDI蛋白中，经热变性、SDS-PAGE，除在55kDa处可见有一条主带外，还出现了二聚体条带。而在相同条件下，纯化删除b’结构域PDI蛋白中完全没有形成二聚体。热变性结果显示，野生型PDI有很强的形成异肽交联二聚体的倾向，而在同样条件下，PDI突变体(删除b’结构域)不形成异肽交联二聚体，说明至少一个参与形成异肽键的氨基酸残基位于被删除区域；为了寻找参与形成异肽键的准确的氨基酸残基，我们构建了三个PDI定点突变体(PDI Glu324Ser,PDI Glu331Gln／Lys333Val／Lys335Arg，PDI Lys291Gln／Lys292Ala)，与纯化野生型PDI蛋白在同样条件下处理，经SDS-PAGE，野生型PDI形成了二聚体，而在纯化PDI突变体(Glu324Ser)蛋白中完全没有形成异肽交联二聚体。说明Glu324是一个参与在体外形成异肽键的氨基酸残基。更多还原

【Abstract】 Interchain isopeptide cross-link is a common kind of molecular cross-links in proteins. Researches about it are focus on transglutaminases and Ubiquitin-proteasome pathway. By using lysozyme, RNase A and protein disulfide isomerase (PDI) as model proteins, we present a hypothesis that protein cross-linking can be accomplished in three concerted steps: (1) a change in protein conformation; (2) formation of interchain disulfide bonds; and (3) formation of interchain isopeptide cross-links.Although we have got much evidence for conforming the mechanism, the details of isopeptide bond cross-linking are not well understood. In this dissertation, we have constructed the b’ domain deletion PDI mutant and three point mutation PDI mutants. The b’ domain deleted mutant completely failed to form dimers on the SDS-polyacrylamide gel, in contrast, dimeric bands were observed in purified preparations of wild-type PDI protein under the same conditions. The thermal unfolding of wild-type PDI protein shows a high propensity toward dimer formation, whereas the b’ domain deleted mutant forms no isopeptide bond dimmer under these conditions as revealed by SDS-PAGE. The results suggest that there is at least one of pivotal amino acids which form the isopeptide bond in the deleted b’ domain. To identify the amino acid residues involved in isopeptide bond in b’ domain, three mutants (PDI Glu324Ser, PDI Glu331Gln/Lys333Val/Lys335Arg, PDI Lys291Gln/Lys292Ala) were made. Unlike wild-type PDI protein, the PDI mutant (Glu324Ser) failed to form isopeptide bond dimmers which revealed by the SDS- polyacrylamide gel after thermal unfolding. The results suggest that Glu324 is one of the pivotal amino acids that form the isopeptide bond.更多还原