化学学报 ›› 2013, Vol. 71 ›› Issue (01): 44-50.DOI: 10.6023/A12110982 上一篇    下一篇

研究论文

纳米通道单分子检测P53蛋白与DNA的弱相互作用

应佚伦a, 张星a, 刘钰a, 薛梦竹b, 李洪林b,c, 龙亿涛a   

  1. a 华东理工大学上海市功能性材料化学重点实验室 上海 200237;
    b 华东理工大学药学院 上海 200237;
    c 中国科学院上海药物研究所药物发现与设计中心 上海 201203
  • 收稿日期:2012-11-29 出版日期:2013-01-14 发布日期:2012-12-25
  • 通讯作者: 龙亿涛 E-mail:ytlong@ecust.edu.cn
  • 基金资助:
    项目受国家自然科学基金(No. 91027035)和中央高校基本科研业务费专项(Nos. WK1013002, WB1113005)资助. 龙亿涛教授受国家杰出青年基金(No. 21125522)和上海高校特聘教授(东方学者)资助.

Single Molecule Study of the Weak Biological Interactions Between P53 and DNA

Ying Yiluna, Zhang Xinga, Liu Yua, Xue Mengzhub, Li Honglinb,c, Long Yitaoa   

  1. a Shanghai Key Laboratory of Functional Materials Chemistry, East China University of Science and Technology, Shanghai 200237;
    b School of Pharmacy, East China University of Science and Technology, Shanghai 200237;
    c Drug Discovery and Design Center, Shanghai Institute of Materia Medica, Academia Sinica, Shanghai 201203
  • Received:2012-11-29 Online:2013-01-14 Published:2012-12-25
  • Supported by:
    Project supported by the Natural Science Foundation of China (No. 91027035) and the Fundamental Research Funds for the Central Universities (Nos. WK1013002, WB1113005). Long Yitao is supported by the National Science Fund for Distinguished Young Scholars of China (No. 21125522) and the Program for Professor of Special Appointment (Eastern Scholar) at Shanghai Institutions of Higher Learning.

利用基于α-溶血素(α-HL)的纳米通道单分子检测技术在单分子水平上探测位于肿瘤抑制蛋白P53 DNA结合域的多肽分子(P53-P)与含有p21waf1/cip1基因结合域的双链DNA (B40)之间弱相互作用. 在电场力的作用下, 单个P53-P/B40复合物被α-HL纳米通道捕获并限制在其前庭中. 通过空间限域作用, P53-P与B40之间的弱相互作用被放大为具有显著电压依赖性的复合物-α-HL间强烈的相互作用, 从而产生极易分辨的离子流特征阻断信号. 统计分析特征阻断信号显示P53-P与B40之间的弱相互作用使得B40产生微小的构型变化. 分子对接模拟进一步证明, P53-P能够插入B40的小沟并使得小沟间距变窄. 因此, 基于α-HL的纳米通道单分子检测技术可以作为研究单个生物大分子间弱相互作用的超灵敏分析方法.

关键词: 纳米通道单分子检测技术, α-溶血素, P53蛋白, 弱相互作用

Many important cellular events, including protein-DNA interactions, are attributed to weak interactions. Almost all of the known biological functions of P53 depend critically upon its DNA-binding properties via numerous weak interactions. At the single-molecule level, information about the weak interactions between each residue of the P53 DNA binding domain (P53 DBD) and DNA is essential for understanding the biological function of P53 and for anti-cancer drug design. Here, we used the α-hemolysin (α-HL) pore to detect the weak interaction between a peptide of the P53 DBD (P53-P) and a 40-bp double-stranded DNA (B40) that includes the p21waf1/cip1 DNA response element. The weak interactions in the complex of p53-P and B40 (p53-P:B40) produce a unique current trace through an α-HL nanopore with diagnostic ionic current blockages. Each current trace at a particular potential is related to the characterized behavior of captured p53-P:B40. Nanopore analysis indicates that the conformation of B40 might be changed by binding to p53-P, this change is confirmed by the molecule docking simulation. In the presence of the weak interactions between p53-P and B40, the analyte exhibits an increase in the rate constant of association with the nanopore vestibule. This reveals that the analyte-pore interactions could be enhanced by the weak interactions between p53-P and B40. The distorted B40 might be prone to translocate through the narrow constriction in the nanopore at the higher potential (>+120 mV). Moreover, our findings demonstrate that the structure of distorted B40 in p53-P:B40 could be broken by the electric force. Our results support the possibility of identifying the weak interaction between two biomolecules. In addition, the analyte-pore association rate constant could be used to estimate the weak binding energy between different parts of the p53 binding domain and the target sequence. The signatures of the current trace may assist in the prediction of the conformational changes of biomolecules at the single-molecule level. Our observations suggest that a biological α-HL nanopore could be a candidate biosensor for predicting the conformational changes that result from weak interactions.

Key words: nanopore, α-hemolysin, P53, weak interaction