Quantifying the Binding Interactions Between Cu(II) and Peptide Residues in the Presence and Absence of Chromophores.
Quantifying the Binding Interactions Between Cu(II) and Peptide Residues in the Presence and Absence of Chromophores.
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DOI：
10.3791/63668
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发表时间：
2022-04-05
期刊：
影响因子：
--
通讯作者：
Stevenson MJ
中科院分区：
文献类型：
--
作者：
Choi S;San Juan JA;Heffern MC;Stevenson MJ
铜（II）是生物系统中的必需金属，赋予与其相互作用的生物分子独特的化学性质。据报道，它直接与多种肽结合，并发挥必要的和病理性的作用，从介导结构到电子转移性质再到赋予催化功能。定量这些Cu（II）-肽复合物在体外的结合亲和力和热力学提供了对结合的热力学驱动力、不同金属离子之间的肽或不同肽之间的Cu（II）的潜在竞争以及Cu（II）-肽复合物在体内的流行的洞察。然而，定量结合热力学可能是具有挑战性的，由于无数的因素，包括占滴定实验中的所有竞争平衡，特别是在缺乏代表肽，d-区金属离子和它们的相互作用的离散光谱手柄的情况下。在这里，一个强大的一组实验提供了准确的定量Cu（II）-肽热力学。本文着重于使用电子吸收光谱的存在和不存在的发色配体提供所需的光谱处理Cu（II）和使用无标记等温滴定量热法。在这两种实验技术中，都描述了一个过程来解释所有的竞争均衡。虽然这篇文章的重点是Cu（II），所描述的一组实验可以适用于超越Cu（II）-肽的相互作用，并提供了一个框架，在生理相关条件下的其他金属肽系统的准确定量。
Copper(II) is an essential metal in biological systems, conferring unique chemical properties to the biomolecules with which it interacts. It has been reported to directly bind to a variety of peptides and play both necessary and pathological roles ranging from mediating structure to electron transfer properties to imparting catalytic function. Quantifying the binding affinity and thermodynamics of these Cu(II)-peptide complexes in vitro provides insight into the thermodynamic driving force of binding, potential competitions between different metal ions for the peptide or between different peptides for Cu(II), and the prevalence of the Cu(II)-peptide complex in vivo. However, quantifying the binding thermodynamics can be challenging due to a myriad of factors, including accounting for all competing equilibria within a titration experiment, especially in cases where there are a lack of discrete spectroscopic handles representing the peptide, the d-block metal ion, and their interactions. Here, a robust set of experiments is provided for the accurate quantification of Cu(II)-peptide thermodynamics. This article focuses on the use of electronic absorption spectroscopy in the presence and absence of chromophoric ligands to provide the needed spectroscopic handle on Cu(II) and the use of label-free isothermal titration calorimetry. In both experimental techniques, a process is described to account for all competing equilibria. While the focus of this article is on Cu(II), the described set of experiments can apply beyond Cu(II)-peptide interactions, and provide a framework for accurate quantification of other metal-peptide systems under physiologically relevant conditions.
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