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首页> 外文期刊>Biochemistry >Quantitative Characterization of Bivalent Probes for a Dual Bromodomain Protein, Transcription Initiation Factor TFIID Subunit 1
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Quantitative Characterization of Bivalent Probes for a Dual Bromodomain Protein, Transcription Initiation Factor TFIID Subunit 1

机译：双溴琼蛋白的二价探针的定量表征，转录起始因子TFIID亚基1

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摘要

Multivalent binding is an efficient means to enhance the affinity and specificity of chemical probes targeting multidomain proteins in order to study their function and role in disease. While the theory of multivalent binding is straightforward, physical and structural characterization of bivalent binding encounters multiple technical difficulties. We present a case study where a combination of experimental techniques and computational simulations was used to comprehensively characterize the binding and structure–affinity relationships for a series of Bromosporine-based bivalent bromodomain ligands with a bivalent protein, Transcription Initiation Factor TFIID subunit 1 (TAF1 ). Experimental techniques—Isothermal Titration Calorimetry, X-ray Crystallography, Circular Dichroism, Size Exclusion Chromatography-Multi-Angle Light Scattering, and Surface Plasmon Resonance—were used to determine structures, binding affinities, and kinetics of monovalent ligands and bivalent ligands with varying linker lengths. The experimental data for monomeric ligands were fed into explicit computational simulations, in which both ligand and protein species were present in a broad range of concentrations, and in up to a 100 s time regime, to match experimental conditions. These simulations provided accurate estimates for apparent affinities (in good agreement with experimental data), individual dissociation microconstants and other microscopic details for each type of protein–ligand complex. We conclude that the expected efficiency of bivalent ligands in a cellular context is difficult to estimate by a single technique in vitro , due to higher order associations favored at the concentrations used, and other complicating processes. Rather, a combination of structural, biophysical, and computational approaches should be utilized to estimate and characterize multivalent interactions.
机译：多价结合是增强靶向多域蛋白的化学探针的亲和力和特异性的有效方法，以研究其在疾病中的功能和作用。虽然多价结合理论是简单的，但二价结合的物理和结构表征遭遇多种技术难题。我们提出了一种案例研究，其中实验技术和计算模拟的组合用于全面地表征具有二价蛋白的一系列溴孢菌的二价溴阳瘤配体的结合和结构 - 亲和力关系，转录起始因子TFIID亚基1（ TAF1）。实验技术 - 等温滴定量热法，X射线晶体学，圆形二色性，尺寸排阻色谱 - 多角度光散射和表面等离子体共振 - 用于确定具有不同接头的单价配体和二价配体的结构，结合亲和力和与二价配体的动力学长度。将单体配体的实验数据送入明确的计算模拟中，其中配体和蛋白质物种均以广泛的浓度存在，以及高达100秒的时间制度，以匹配实验条件。这些模拟为明显亲和力（与实验数据的良好一致）提供了准确的估计，各种蛋白质配体络合物的单个解离微锁和其他微观细节。我们得出结论，由于在所用浓度的浓度和其他复杂过程中青睐的高阶缔合的阶段，难以在体外难以通过单一技术难以估计细胞环境中二价配体的预期效率。相反，应利用结构，生物物理和计算方法的组合来估计和表征多价相互作用。

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  • 来源
    《Biochemistry》 |2018年第14期|共10页
  • 作者

    JunghyunL. Suh; Brian Watts; Jacob I. Stuckey; Jacqueline L. Norris-Drouin; Stephanie H. Cholensky; Bradley M Dickson; Yi An; Sebastian Mathea; Eidarus Salah; Stefan Knapp; Abid Khan; Alexander T. Adams; Brian D. Strahl; Cari A. Sagum; Mark T. Bedford; Lindsey I. James; Dmitri B. Kireev; Stephen V. Frye;

  • 作者单位

    Center for Integrative Chemical Biology and Drug Discovery Division of Chemical Biology and Medicinal Chemistry UNC Eshelman School of Pharmacy University of North Carolina at Chapel Hill Chapel Hill North Carolina 27599 United States;

    Duke Human Vaccine Institute Duke University School of Medicine Duke University Durham North Carolina 27710 United States;

    Center for Integrative Chemical Biology and Drug Discovery Division of Chemical Biology and Medicinal Chemistry UNC Eshelman School of Pharmacy University of North Carolina at Chapel Hill Chapel Hill North Carolina 27599 United States;

    Center for Integrative Chemical Biology and Drug Discovery Division of Chemical Biology and Medicinal Chemistry UNC Eshelman School of Pharmacy University of North Carolina at Chapel Hill Chapel Hill North Carolina 27599 United States;

    Center for Integrative Chemical Biology and Drug Discovery Division of Chemical Biology and Medicinal Chemistry UNC Eshelman School of Pharmacy University of North Carolina at Chapel Hill Chapel Hill North Carolina 27599 United States;

    Center for Integrative Chemical Biology and Drug Discovery Division of Chemical Biology and Medicinal Chemistry UNC Eshelman School of Pharmacy University of North Carolina at Chapel Hill Chapel Hill North Carolina 27599 United States;

    Center for Integrative Chemical Biology and Drug Discovery Division of Chemical Biology and Medicinal Chemistry UNC Eshelman School of Pharmacy University of North Carolina at Chapel Hill Chapel Hill North Carolina 27599 United States;

    Nuffield Department of Medicine Structural Genomics Consortium Old Road Campus Research Building Oxford University Oxford OX3 7DQ United Kingdom;

    Nuffield Department of Medicine Structural Genomics Consortium Old Road Campus Research Building Oxford University Oxford OX3 7DQ United Kingdom;

    Nuffield Department of Medicine Structural Genomics Consortium Old Road Campus Research Building Oxford University Oxford OX3 7DQ United Kingdom;

    Department of Biochemistry and Biophysics and Lineberger Comprehensive Cancer Center University of North Carolina School of Medicine Chapel Hill North Carolina 27599 United States;

    Department of Biochemistry and Biophysics and Lineberger Comprehensive Cancer Center University of North Carolina School of Medicine Chapel Hill North Carolina 27599 United States;

    Department of Biochemistry and Biophysics and Lineberger Comprehensive Cancer Center University of North Carolina School of Medicine Chapel Hill North Carolina 27599 United States;

    Department of Epigenetics and Molecular Carcinogenesis University of Texas MD Anderson Cancer Center Smithville Texas 78957 United States;

    Department of Epigenetics and Molecular Carcinogenesis University of Texas MD Anderson Cancer Center Smithville Texas 78957 United States;

    Center for Integrative Chemical Biology and Drug Discovery Division of Chemical Biology and Medicinal Chemistry UNC Eshelman School of Pharmacy University of North Carolina at Chapel Hill Chapel Hill North Carolina 27599 United States;

    Center for Integrative Chemical Biology and Drug Discovery Division of Chemical Biology and Medicinal Chemistry UNC Eshelman School of Pharmacy University of North Carolina at Chapel Hill Chapel Hill North Carolina 27599 United States;

    Center for Integrative Chemical Biology and Drug Discovery Division of Chemical Biology and Medicinal Chemistry UNC Eshelman School of Pharmacy University of North Carolina at Chapel Hill Chapel Hill North Carolina 27599 United States;

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  • 正文语种 eng
  • 中图分类 生物化学;
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