de la Torre Micaela, Pomorski Adam
Department of Chemical Biology, Faculty of Biotechnology, University of Wrocław, Wrocław, Poland.
Front Chem. 2024 Apr 12;12:1378447. doi: 10.3389/fchem.2024.1378447. eCollection 2024.
Metal ions can perform multiple roles ranging from regulatory to structural and are crucial for cell function. While some metal ions like Na are ubiquitously present at high concentrations, other ions, especially Ca and transition metals, such as Zn or Cu are regulated. The concentrations above or below the physiological range cause severe changes in the behavior of biomolecules that bind them and subsequently affect the cell wellbeing. This has led to the development of specialized protocols to study metal ion binding biomolecules in bulk conditions that mimic the cell environment. Recently, there is growing evidence of influence of post-transcriptional and post-translational modifications on the affinity of the metal ion binding sites. However, such targets are difficult to obtain in amounts required for classical biophysical experiments. Single molecule techniques have revolutionized the field of biophysics, molecular and structural biology. Their biggest advantage is the ability to observe each molecule's interaction independently, without the need for synchronization. An additional benefit is its extremely low sample consumption. This feature allows characterization of designer biomolecules or targets obtained coming from natural sources. All types of biomolecules, including proteins, DNA and RNA were characterized using single molecule methods. However, one group is underrepresented in those studies. These are the metal ion binding biomolecules. Single molecule experiments often require separate optimization, due to extremely different concentrations used during the experiments. In this review we focus on single molecule methods, such as single molecule FRET, nanopores and optical tweezers that are used to study metal ion binding biomolecules. We summarize various examples of recently characterized targets and reported experimental conditions. Finally, we discuss the potential promises and pitfalls of single molecule characterization on metal ion binding biomolecules.
金属离子具有多种作用，从调节到结构方面，对细胞功能至关重要。虽然像钠这样的一些金属离子以高浓度普遍存在，但其他离子，特别是钙和过渡金属，如锌或铜，则受到调控。生理范围之上或之下的浓度会导致与之结合的生物分子行为发生严重变化，进而影响细胞健康。这促使人们开发专门的实验方案，以在模拟细胞环境的大量条件下研究金属离子结合生物分子。最近，越来越多的证据表明转录后和翻译后修饰对金属离子结合位点的亲和力有影响。然而，要获得经典生物物理实验所需数量的此类靶标却很困难。单分子技术彻底改变了生物物理学、分子生物学和结构生物学领域。它们最大的优势是能够独立观察每个分子的相互作用，而无需同步。另一个好处是其样品消耗量极低。这一特性使得能够对设计的生物分子或来自天然来源的靶标进行表征。所有类型的生物分子，包括蛋白质、DNA和RNA，都使用单分子方法进行了表征。然而，在这些研究中，有一类生物分子的代表性不足。它们就是金属离子结合生物分子。由于实验中使用的浓度差异极大，单分子实验往往需要单独优化。在这篇综述中，我们重点关注用于研究金属离子结合生物分子的单分子方法，如单分子荧光共振能量转移、纳米孔和光镊。我们总结了最近表征的靶标的各种实例以及报道的实验条件。最后，我们讨论了对金属离子结合生物分子进行单分子表征的潜在前景和陷阱。
