表面等离子共振
化学
配体结合分析
结合位点
电泳迁移率测定
共识序列
结合选择性
生物物理学
离解常数
DNA
雌激素受体
费斯特共振能量转移
受体
分子生物学
转录因子
生物化学
生物
荧光
基因
纳米技术
纳米颗粒
材料科学
遗传学
物理
癌症
量子力学
乳腺癌
基序列
作者
Huey Fang Teh,Wendy Yen Xian Peh,Xiaodi Su,Jane S. Thomsen
出处
期刊:Biochemistry
[American Chemical Society]
日期:2007-02-01
卷期号:46 (8): 2127-2135
被引量:85
摘要
Specific protein−DNA interactions play a central role in transcription and other biological processes. A comprehensive characterization of protein−DNA interactions should include information about binding affinity, kinetics, sequence specificity, and binding stoichiometry. In this study, we have used surface plasmon resonance spectroscopy (SPR) to study the interactions between human estrogen receptors (ER, α and β subtypes) and estrogen response elements (ERE), with four assay schemes. First, we determined the sequence-dependent receptors' binding capacity by monitoring the binding of ER to various ERE sequences immobilized on a sensor surface (assay format denoted as the direct assay). Second, we screened the relative affinity of ER for various ERE sequences using a competition assay, in which the receptors bind to an ERE-immobilized surface in the presence of competitor ERE sequences. Third, we monitored the assembly of ER−ERE complexes on a SPR surface and thereafter the removal and/or dissociation of the ER (assay scheme denoted as the dissociation assay) to determine the binding stoichiometry. Last, a sandwich assay (ER binding to ERE followed by anti-ER recognition of a specific ER subtype) was performed in an effort to understand how ERα and ERβ may associate and compete when binding to the DNA. With these assay schemes, we reaffirmed that (1) ERα is more sensitive than ERβ to base pair change(s) in the consensus ERE, (2) ERα and ERβ form a heterodimer when they bind to the consensus ERE, and (3) the binding stoichiometry of both ERα− and ERβ−ERE complexes is dependent on salt concentration. With this study, we demonstrate the versatility of the SPR analysis. With the involvement of various assay arrangements, the SPR analysis can be further extended to more than kinetics and affinity study.
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