Real-Time Measurement of a Weak Interaction of a Transcription Factor Motif with a Protein Hub at Single-Molecule Precision

被引:2
作者
Mayse, Lauren A. [1 ,2 ]
Wang, Yazheng [1 ,2 ]
Ahmad, Mohammad [1 ]
Movileanu, Liviu [1 ,2 ,3 ,4 ]
机构
[1] Syracuse Univ, Dept Phys, Syracuse, NY 13244 USA
[2] Syracuse Univ, Dept Biomed & Chem Engn, Syracuse, NY 13244 USA
[3] Syracuse Univ, Dept Biol, Syracuse, NY 13244 USA
[4] Syracuse Univ, BioInspired Inst, Syracuse, NY 13244 USA
基金
美国国家卫生研究院;
关键词
protein dynamics; single-molecule electrophysiology; protein engineering; protein-protein interactions; biosensors; nanopores; BIOLAYER INTERFEROMETRY; SET1; FAMILY; NANOPORE; RECOGNITION; ASSOCIATION; BARNASE; COMPLEX; QUANTIFICATION; BINDING;
D O I
10.1021/acsnano.4c04857
中图分类号
O6 [化学];
学科分类号
0703 ;
摘要
Transcription factors often interact with other protein cofactors, regulating gene expression. Direct detection of these brief events using existing technologies remains challenging due to their transient nature. In addition, intrinsically disordered domains, intranuclear location, and lack of cofactor-dependent active sites of transcription factors further complicate the quantitative analysis of these critical processes. Here, we create a genetically encoded label-free sensor to identify the interaction between a motif of the MYC transcription factor, a primary cancer driver, and WDR5, a chromatin-associated protein hub. Using an engineered nanopore equipped with this motif, WDR5 is probed through reversible captures and releases in a one-by-one and time-resolved fashion. Our single-molecule kinetic measurements indicate a weak-affinity interaction arising from a relatively slow complex association and a fast dissociation of WDR5 from the tethered motif. Further, we validate this subtle interaction by determinations in an ensemble using single nanodisc-wrapped nanopores immobilized on a biolayer interferometry sensor. This study also provides the proof-of-concept for a sensor that reveals unique recognition signatures of different protein binding sites. Our foundational work may be further developed to produce sensing elements for analytical proteomics and cancer nanomedicine.
引用
收藏
页码:20468 / 20481
页数:14
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