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Protein-protein interactions and methods for identifying interacting proteins and the amino acid sequence at the site of interaction
Protein-protein interactions and methods for identifying interacting proteins and the amino acid sequence at the site of interaction
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机译：蛋白质间相互作用以及鉴定相互作用蛋白和相互作用位点氨基酸序列的方法
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摘要
The invention relates to protein-protein interactions and methods for identifying interacting proteins and the amino acid sequence at the site of interaction. Using overlapping hexapeptides that encode for the entire amino acid sequences of the linker domains of human P-glycoprotein gene 1 and 3 (HP-gp1 and HP-gp3), a direct and specific binding between HP-gp1 and 3 linker domains and intracellular proteins was demonstrated. Three different stretches (617EKGIYFKLVTM627, (SEQ ID NO: 1) 658SRSSLIRKRSTRRSVRGSQA677 (SEQ ID NO: 2) and 694PVSFWRIMKLNLT706 (SEQ ID NO: 3) for HP-gp1 and 618LMKKEGVYFKLVNM631 (SEQ ID NO: 4), 648KAATRMAPNGWKSRLFRHSTQKNLKNS674 (SEQ ID NO: 5), and 695PVSFLKVLKLNKT707 (SEQ ID NO: 6) for HP-gp3) in linker domains bound to proteins with apparent molecular masses of ˜80 kDa, 57 kDa and 30 kDa. The binding of the 57 kDa protein was further characterized. Purification and partial N-terminal amino acid sequencing of the 57 kDa protein showed that it encodes the N-terminal amino acids of alpha and beta-tubulins. The method of the present invention was further validated with Annexin. The present invention thus demonstrates a novel concept whereby the interactions between two proteins are mediated by strings of few amino acids with high and repulsive binding energies, enabling the identification of high affinity binding sites between any interacting proteins.
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机译：本发明涉及蛋白质-蛋白质相互作用以及用于鉴定相互作用蛋白和相互作用位点的氨基酸序列的方法。使用编码人P糖蛋白基因1和3（HP-gp1和HP-gp3）接头结构域的整个氨基酸序列的重叠六肽，HP-gp1和3接头结构域与细胞内蛋白之间直接且特异性结合被证明。三种不同的延伸（ 617 EKGIYFKLVTM 627 ，（SEQ ID NO：1） 658 SRSSLIRKRSTRRSVRGSQA 677 （SEQ ID NO ：2）和 694 PVSFWRIMKLNLT 706 （SEQ ID NO：3）用于HP-gp1和 618 LMKKEGVYFKLVNM 631 （SEQ ID NO：4）， 648 KAATRMAPNGWKSRLFRHSTQKNLKNS 674 （SEQ ID NO：5）和 695 PVSFLKVLKLNKT 707 （SEQ ID NO：6）中，在连接域中与表观分子量为〜80kDa，57kDa和30kDa的蛋白质结合。进一步表征了57kDa蛋白的结合。 57 kDa蛋白的纯化和部分N末端氨基酸测序表明，它编码α和β-微管蛋白的N末端氨基酸。用膜联蛋白进一步验证了本发明的方法。因此，本发明证明了一种新颖的概念，其中两个蛋白质之间的相互作用由具有高和排斥结合能的少数氨基酸串介导，从而能够鉴定任何相互作用蛋白质之间的高亲和力结合位点。
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