Single-Molecule Nanopositioning: Structural Transitions of a Helicase-DNA Complex during ATP Hydrolysis
Single-Molecule Nanopositioning: Structural Transitions of a Helicase-DNA Complex during ATP Hydrolysis
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DOI：
10.1016/j.bpj.2011.07.010
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发表时间：
2011-08-17
影响因子：
3.4
通讯作者：
Ha, Taekjip
中科院分区：
文献类型：
--
作者：
Balci, Hamza;Arslan, Sinan;Ha, Taekjip
用单分子FRET方法研究了大肠杆菌Rep解旋酶与部分双链DNA(PdDNA)结合时的构象变化。结晶学研究表明，与单链DNA结合的Rep可以以开放和封闭的构象存在，但2B亚区的取向不同。在不同残基标记的8个Rep突变体和连接处标记的pdDNA受体之间的FRET测量分别在四个核苷酸状态下进行。基于晶体结构的供体标记残基的位置，以及这些供体分子和DNA连接处的受体荧光团之间的FRET测量，被用来使用三角算法来预测DNA连接处最可能的位置。将这些预测的连接位置与晶体结构进行比较，以确定开放构象还是闭合构象与FRET数据更一致。我们的数据显示，在三磷酸腺苷的伽马态S和二磷酸腺苷中有两种截然不同的Rep-pdDNA构象，这是一个意想不到的发现。一级构象类似于在无核苷酸和ADP.PI状态下观察到的，二级构象是一种新的构象，其中双链DNA和2B亚区作为一个单位相对于蛋白质的其余部分移动13埃。在所有的核苷酸状态中发现的一次构象与晶体结构的闭合构象是一致的；二次构象是一种以前没有观察到的新构象。我们讨论了这一新观察到的构象的可能含义。
The conformational states of Escherichia coli Rep helicase undergoing ATP hydrolysis while bound to a partial-duplex DNA (pdDNA) were studied using single-molecule FRET. Crystallographic studies showed that Rep bound to single-stranded DNA can exist in open and closed conformations that differ in the orientation of the 2B subdomain. FRET measurements between eight Rep mutants donor-labeled at different residues and pdDNA acceptor-labeled at the junction were conducted at each of the four nucleotide states. The positions of donor-labeled residues, based on crystal structure, and FRET measurements between these donor molecules and the acceptor fluorophore at the DNA junction were used to predict the most likely position for the DNA junction using a triangulation algorithm. These predicted junction positions are compared with the crystal structure to determine whether the open or closed conformation is more consistent with the FRET data. Our data revealed that there are two distinct Rep-pdDNA conformations in the ATP gamma S and ADP states, an unexpected finding. The primary conformation is similar to that observed in nucleotide-free and ADP.Pi states, and the secondary conformation is a novel conformation where the duplex DNA and 2B subdomain moved as a unit by 13 angstrom relative to the rest of the protein. The primary conformation found in all nucleotide states is consistent with the closed conformation of the crystal structure however; the secondary conformation is a new conformation that has not been observed before. We discuss the possible implications of this newly observed conformation.