Interactions between PHD3-Bromo of MLL1 and H3K4me3 Revealed by Single-Molecule Magnetic Tweezers in a Parallel DNA Circuit.,Bioconjugate Chemistry

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Interactions between PHD3-Bromo of MLL1 and H3K4me3 Revealed by Single-Molecule Magnetic Tweezers in a Parallel DNA Circuit.
Bioconjugate Chemistry ( IF 4.0 ) Pub Date : 2019-11-15 , DOI: 10.1021/acs.bioconjchem.9b00665
Xiaofeng Ma ₁ , Manning Zhu ₁ , Jianyu Liu ₂ , Xu Li ₁ , Lihua Qu ₁ , Lin Liang ₁ , Wei Huang ₁ , Junli Wang ₁ , Ning Li ₁ , Jun-Hu Chen ₃ , Wenke Zhang ₂ , Zhongbo Yu ₁

Affiliation

Single-molecule force spectroscopy is a powerful tool to directly measure protein-protein interactions (PPI). The high specificity and precision of PPI measurements made it possible to reveal detailed mechanisms of intermolecular interactions. However, protein aggregation due to specific or nonspecific interactions is among the most challenging problems in PPI examination. Here, we propose a strategy of a parallel DNA circuit to probe PPI using single-molecule magnetic tweezers. In contrast to PPI examination using atomic force microscopy, microspheres as probes used in magnetic tweezers avoided the single-probe issue of a cantilever. Negatively charged DNA as a linker circumvented the severe aggregation in the PPI construct with a protein linker. The unnatural amino acid encoded in proteins of interest expanded the choices of biorthogonal conjugation. We demonstrated how to apply our strategy to probe the PPI between the PHD3-Bromo and the histone H3 methylated at K4, a critical epigenetic event in leukemia development. We found a rupture force of 12 pN for breaking the PPI, which is much higher than that required to peel DNA off from a nucleosome, 3 pN. We expect that our methods will make PPI measurements of mechanics and kinetics with great precision, facilitating PPI-related research, e.g., PPI-targeted drug discovery.

中文翻译：

单分子磁镊子在平行DNA电路中揭示了MLL1的PHD3-Bromo和H3K4me3之间的相互作用。

单分子力光谱法是直接测量蛋白质-蛋白质相互作用（PPI）的强大工具。PPI测量的高度特异性和精确性使得揭示分子间相互作用的详细机理成为可能。但是，由于特异性或非特异性相互作用而引起的蛋白质聚集是PPI检查中最具挑战性的问题之一。在这里，我们提出了一种使用单分子磁性镊子探测PPI的并行DNA电路的策略。与使用原子力显微镜的PPI检查相反，微球作为用于镊子的探针避免了悬臂的单探针问题。带负电荷的DNA作为连接子可避免PPI构建物中带有蛋白质连接子的严重聚集。目的蛋白中编码的非天然氨基酸扩展了双正交缀合的选择。我们展示了如何应用我们的策略来探测PHD3-Bromo和在K4甲基化的组蛋白H3之间的PPI，这是白血病发展中的关键表观遗传事件。我们发现破坏PPI的断裂力为12 pN，远高于从核小体上剥离DNA 3 pN所需的断裂力。我们希望我们的方法将以极高的精度进行力学和动力学的PPI测量，从而促进与PPI相关的研究，例如针对PPI的药物发现。这比从3 pN的核小体上剥离DNA所需要的要高得多。我们希望我们的方法将以极高的精度进行力学和动力学的PPI测量，从而促进与PPI相关的研究，例如针对PPI的药物发现。这比从3 pN的核小体上剥离DNA所需要的要高得多。我们希望我们的方法将以极高的精度进行力学和动力学的PPI测量，从而促进与PPI相关的研究，例如针对PPI的药物发现。

更新日期：2019-11-15

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