Sarkar Susanta K, Andoy Nesha May, Benítez Jaime J, Chen Peng R, Kong Jason S, He Chuan, Chen Peng
Department of Chemistry and Chemical Biology, Baker Laboratory, Cornell University, Ithaca, New York 14853, USA.
J Am Chem Soc. 2007 Oct 17;129(41):12461-7. doi: 10.1021/ja072485y. Epub 2007 Sep 20.
Protein-DNA interactions are essential for gene maintenance, replication, and expression. Characterizing how proteins interact with and change the structure of DNA is crucial in elucidating the mechanisms of protein function. Here, we present a novel and generalizable method of using engineered DNA Holliday junctions (HJs) that contain specific protein-recognition sequences to report protein-DNA interactions in single-molecule FRET measurements, utilizing the intrinsic structural dynamics of HJs. Because the effects of protein binding are converted to the changes in the structure and dynamics of HJs, protein-DNA interactions that involve small structural changes of DNA can be studied. We apply this method to investigate how the MerR-family regulator PbrR691 interacts with DNA for transcriptional regulation. Both apo- and holo-PbrR691 bind the stacked conformers of the engineered HJ, change their structures, constrain their conformational distributions, alter the kinetics, and shift the equilibrium of their structural dynamics. The information obtained maps the potential energy surfaces of HJ before and after PbrR691 binding and reveals the protein actions that force DNA structural changes for transcriptional regulation. The ability of PbrR691 to bind both HJ conformers and still allow HJ structural dynamics also informs about its conformational flexibility that may have significance for its regulatory function. This method of using engineered HJs offers quantification of the changes both in structure and in dynamics of DNA upon protein binding and thus provides a new tool to elucidate the correlation of structure, dynamics, and function of DNA-binding proteins.
蛋白质与DNA的相互作用对于基因的维持、复制和表达至关重要。表征蛋白质如何与DNA相互作用并改变其结构，对于阐明蛋白质功能机制至关重要。在此，我们提出了一种新颖且可推广的方法，利用含有特定蛋白质识别序列的工程化DNA霍利迪连接体（HJs），在单分子荧光共振能量转移（FRET）测量中报告蛋白质与DNA的相互作用，利用HJs的内在结构动力学。由于蛋白质结合的效应转化为HJs结构和动力学的变化，因此可以研究涉及DNA小结构变化的蛋白质与DNA的相互作用。我们应用此方法来研究MerR家族调节因子PbrR691如何与DNA相互作用以进行转录调控。无辅基和有辅基的PbrR691均结合工程化HJ的堆叠构象体，改变其结构，限制其构象分布，改变其动力学，并改变其结构动力学的平衡。所获得的信息描绘了PbrR691结合前后HJ的势能面，并揭示了迫使DNA结构变化以进行转录调控的蛋白质作用。PbrR691结合两种HJ构象体并仍允许HJ结构动力学的能力，也说明了其构象灵活性，这可能对其调节功能具有重要意义。这种使用工程化HJs的方法提供了对蛋白质结合后DNA结构和动力学变化的定量分析，从而为阐明DNA结合蛋白的结构、动力学和功能之间的相关性提供了一种新工具。
