Department of Chemistry, The Pennsylvania State University, University Park, Philadelphia, PA, United States.
Department of Chemistry, The Pennsylvania State University, University Park, Philadelphia, PA, United States.
Methods Enzymol. 2021;651:23-61. doi: 10.1016/bs.mie.2021.01.044. Epub 2021 Mar 3.
The recent discoveries of the first proteins that bind lanthanides as part of their biological function not only are relevant to the emerging field of lanthanide-dependent biology, but also hold promise to revolutionize the technologically critical rare earths industry. Although protocols to assess the thermodynamics of metal-protein interactions are well established for "traditional" metal ions in biology, the characterization of lanthanide-binding proteins presents a challenge to biochemists due to the lanthanides' Lewis acidity, propensity for hydrolysis, and high-affinity complexes with biological ligands. These properties necessitate the preparation of metal stock solutions with very low buffered "free" metal concentrations (e.g., femtomolar to nanomolar) for such determinations. Herein we describe several protocols to overcome these challenges. First, we present standardization methods for the preparation of chelator-buffered solutions of lanthanide ions with easily calculated free metal concentrations. We also describe how these solutions can be used in concert with analytical methods including UV-visible spectrophotometry, circular dichroism spectroscopy, Förster resonance energy transfer (FRET), and sensitized terbium luminescence, in order to accurately determine dissociation constants (Ks) of lanthanide-protein complexes. Finally, we highlight how application of these methods to lanthanide-binding proteins, such as lanmodulin, has yielded insights into selective recognition of lanthanides in biology. We anticipate that these protocols will facilitate discovery and characterization of additional native lanthanide-binding proteins, will motivate the understanding of their biological context, and will prompt their applications in biotechnology.
最近发现的第一批作为其生物功能一部分结合镧系元素的蛋白质不仅与新兴的镧系元素依赖生物学领域相关，而且有望彻底改变技术上至关重要的稀土产业。尽管评估金属-蛋白质相互作用热力学的方案在生物学中“传统”金属离子中已经建立，但由于镧系元素的路易斯酸度、水解倾向以及与生物配体的高亲和力配合物，镧系元素结合蛋白的特性对生物化学家提出了挑战。这些特性需要制备具有非常低缓冲“游离”金属浓度（例如，皮摩尔至纳摩尔）的金属储备溶液，以便进行此类测定。本文介绍了克服这些挑战的几种方案。首先，我们提出了制备螯合剂缓冲镧系离子溶液的标准化方法，这些溶液具有易于计算的游离金属浓度。我们还描述了如何将这些溶液与分析方法（包括紫外-可见分光光度法、圆二色性光谱法、Förster 共振能量转移（FRET）和敏化铽发光）结合使用，以便准确确定镧系元素-蛋白质配合物的离解常数（Ks）。最后，我们强调了这些方法在镧系元素结合蛋白（如 lanmodulin）中的应用如何为生物中镧系元素的选择性识别提供了新的见解。我们预计这些方案将促进对其他天然镧系元素结合蛋白的发现和表征，激发对其生物学背景的理解，并促使其在生物技术中的应用。
