Isothermal titration calorimetry of protein-protein interactions
Isothermal titration calorimetry of protein-protein interactions
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DOI：
10.1006/meth.1999.0852
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发表时间：
1999-10-01
期刊：
影响因子：
--
通讯作者：
Nall, BT
中科院分区：
文献类型：
--
作者：
Pierce, MM;Raman, CS;Nall, BT
生物大分子之间的相互作用，无论是蛋白质-DNA、抗体-抗原、抗体-受体等，说明了分子识别的复杂性和多样性。这种相互作用在免疫应答、信号转导级联和基因表达中的重要性怎么强调都不过分。确定稳定这种相互作用的力的性质是非常有意义的。缔合热力学的特征在于相互作用的化学计量（n）、缔合常数（K-a）、自由能（Δ G（B））、焓（Δ H-B）、熵（Δ S-B）和结合热容量（Δ C-p）。结合结构信息，结合的能量学可以提供一个完整的解剖的相互作用，并有助于确定最重要的区域的接口和充满活力的贡献。各种间接方法（ELISA、RIA、表面等离子体共振等）通常用于表征生物学上重要的相互作用。本文介绍了等温滴定量热法（ITC）在蛋白质-蛋白质相互作用研究中的应用，ITC是目前测定蛋白质-蛋白质相互作用热力学性质的最定量手段。ITC通过测定配体与其结合配偶体缔合时产生的热来直接测量结合平衡。在单个实验中，确定结合常数（K-a）、化学计量（n）和结合焓（Δ H-b）的值。结合的自由能和熵由缔合常数确定。通过在不同温度下进行滴定测量的Δ H-b参数的温度依赖性描述了Δ C-p项。作为该方法的实际应用，我们描述了使用ITC研究细胞色素c和两种单克隆抗体之间的相互作用，（C）1999年学术出版社。
The interaction of biological macromolecules, whether protein-DNA, antibody-antigen, hormone-receptor, etc., illustrates the complexity and diversity of molecular recognition. The importance of such interactions in the immune response, signal transduction cascades, and gene expression cannot be overstated, It is of great interest to determine the nature of the forces that stabilize the interaction. The thermodynamics of association are characterized by the stoichiometry of the interaction (n), the association constant (K-a), the free energy (Delta G(b)), enthalpy (Delta H-b), entropy (Delta S-b), and heat capacity of binding (Delta C-p). In combination with structural information, the energetics of binding can provide a complete dissection of the interaction and aid in identifying the most important regions of the interface and the energetic contributions. Various indirect methods (ELISA, RIA, surface plasmon resonance, etc.) are routinely used to characterize biologically important interactions. Here we describe the use of isothermal titration calorimetry (ITC) in the study of protein-protein interactions, ITC is the most quantitative means available for measuring the thermodynamic properties of a protein-protein interaction. ITC measures the binding equilibrium directly by determining the heat evolved on association of a ligand with its binding partner. In a single experiment, the values of the binding constant (K-a), the stoichiometry (n), and the enthalpy of binding (Delta H-b) are determined. The free energy and entropy of binding are determined from the association constant. The temperature dependence of the Delta H-b parameter, measured by performing the titration at varying temperatures, describes the Delta C-p term. As a practical application of the method, we describe the use of ITC to study the interaction between cytochrome c and two monoclonal antibodies, (C) 1999 Academic Press.