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

Abstract

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.

• Authors: Xiaofeng Ma, Manning Zhu, Jianyu Liu, Xu Li, Lihua Qu, Lin Liang, Wei Huang, Junli Wang, Ning Li, Jun-Hu Chen, Wenke Zhang, Zhongbo Yu
• Journal: Bioconjugate chemistry (Bioconjug Chem) Vol. 30 Issue 12 Pg. 2998-3006 (12 18 2019) ISSN: 1520-4812 [Electronic] United States
• PMID: 31714753 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Cross-Linking Reagents
• Histones
• KMT2A protein, human
• histone H3 trimethyl Lys4
• Myeloid-Lymphoid Leukemia Protein
• DNA
• EGLN3 protein, human
• Hypoxia-Inducible Factor-Proline Dioxygenases
• Histone-Lysine N-Methyltransferase

Topics

• Binding Sites
• Cross-Linking Reagents
• DNA (chemistry)
• Histone-Lysine N-Methyltransferase (metabolism)
• Histones (metabolism)
• Humans
• Hypoxia-Inducible Factor-Proline Dioxygenases
• Leukemia (etiology)
• Magnetics (methods)
• Methods
• Myeloid-Lymphoid Leukemia Protein (metabolism)
• Nanotechnology (methods)
• Protein Binding