Chemical shift assignments for F-plasmid TraI (381-569).

Chemical shift assignments for F-plasmid TraI (381-569).
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DOI:
10.1007/s12104-010-9269-y
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发表时间:
2011-04
影响因子:
0.9
通讯作者:
Schildbach JF
Schildbach JF
中科院分区:
生物学4区
文献类型:
--
作者:
Wright NT;Majumdar A;Schildbach JF

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TraI,F质粒编码的切口酶,是一种1,756个氨基酸的蛋白质,对于F质粒DNA从一种细菌接合转移到另一种细菌是必需的。虽然F TraI的N-和C-末端结构域的晶体结构已经确定,但蛋白质的中心结构域在结构上尚未探索。已知这些中间结构域(在残基306和1,500之间)通过高度进行性解旋酶活性结合单链DNA(ssDNA)和解旋DNA。在这个中心区域中,更多的C-末端部分(~900-1500)似乎与E. coliRecBCD复合物。然而,更多的N-末端部分(306-900)显示出与其他蛋白质有限的序列相似性。在试图定义这个中间区域的折叠结构域的结构和辨别其功能,我们已经分离出的TraI的稳定区域有限的蛋白水解。这些区域之一,TraI(381-569),被确定和编码它的基因构建体被工程化。表达、纯化蛋白质,并对其序列特异性化学位移进行分配。
TraI, the F plasmid-encoded nickase, is a 1,756 amino acid protein essential for conjugative transfer of F plasmid DNA from one bacterium to another. While crystal structures of N- and C-terminal domains of F TraI have been determined, central domains of the protein are structurally unexplored. These middle domains (between residues 306 and 1,500) are known to both bind single-stranded DNA (ssDNA) and unwind DNA through a highly processive helicase activity. Of this central region, the more C-terminal portion (~900–1500) appears related to helicase RecD of the E. coli RecBCD complex. The more N-terminal portion (306–900), however, shows limited sequence similarity to other proteins. In an attempt to define the structure of well-folded domains of this middle region and discern their function, we have isolated stable regions of TraI following limited proteolysis. One of these regions, TraI (381–569), was identified and a genetic construct encoding it was engineered. The protein was expressed, purified, and the sequence-specific chemical shifts for it were assigned.
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