Fueling protein-DNA interactions inside porous nanocontainers
Fueling protein-DNA interactions inside porous nanocontainers
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DOI：
10.1073/pnas.0610673104
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发表时间：
2007-07-31
影响因子：
11.1
通讯作者：
Ha, Taekjip
中科院分区：
文献类型：
--
作者：
Cisse, Ibrahim;Okumus, Burak;Ha, Taekjip
囊泡包封为许多可溶性蛋白质和核酸提供了生物相关的环境，并且为单分子荧光测定提供了最佳的固定介质。此外，将生物分子限制在小体积内为独特的实验配置开辟了新的途径。然而，囊泡的不渗透性，甚至对离子和其他小分子如ATP，阻碍了更广泛的应用。因此，我们开发了将孔诱导成囊泡的方法。多孔囊泡，然后用于调节大肠杆菌RecA蛋白和ssDNA之间的相互作用，通过改变囊泡外核苷酸。观察到DNA上相同RecA细丝的重复结合和解离，其再结合速率比没有限制的情况下大两个数量级，表明RecA细丝的先前未报道的成核途径。这种方法提供了一种生物友好和简单的替代表面拴系，是理想的瞬态和弱相互作用的生物复合物的研究。
Vesicle encapsulation offers a biologically relevant environment for many soluble proteins and nucleic acids and an optimal immobilization medium for single-molecule fluorescence assays. Furthermore, the confinement of biomolecules within small volumes opens up new avenues to unique experimental configurations. Nevertheless, the vesicles' impermeability, even toward ions and other small molecules such as ATP, hinders more general applications. We therefore developed methods to induce pores into vesicles. Porous vesicles were then used to modulate the interaction between Escherichia coli RecA proteins and ssDNA by changing the extravesicular nucleotides. Repetitive binding and dissociation of the same RecA filament on the DNA was observed with a rebinding rate two orders of magnitude greater than in the absence of confinement, suggesting a previously unreported nucleation pathway for RecA filament. This method provides a biofriendly and simple alternative to surface tethering that is ideal for the study of transient and weakly interacting biological complexes.