Global conformation of the Escherichia coli replication factor DnaC protein in absence and presence of nucleotide cofactors
Global conformation of the Escherichia coli replication factor DnaC protein in absence and presence of nucleotide cofactors
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DOI：
10.1021/bi049377y
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发表时间：
2004-08-31
期刊：
影响因子：
2.9
通讯作者：
Bujalowski, W
中科院分区：
文献类型：
--
作者：
Galletto, R;Maillard, R;Bujalowski, W
全球的构象和寡聚状态的大肠杆菌复制因子DnaC蛋白的镁和核苷酸辅因子，ATP和ADP，以及它们的荧光类似物，MANT-ATP和MANT-ADP的存在和不存在下，已检查使用分析沉降速度和时间依赖性荧光各向异性技术。在溶液中，DnaC蛋白在很大的蛋白质浓度范围内仅以单体形式存在。sdegree（20，w）= 2.45 +/- 0.07 S的值表明蛋白质分子具有细长的形状。当被建模为旋转的长椭圆体时，水合DnaC蛋白具有4.0 +/-0.6的轴比，长轴a = 112埃，短轴B = 28埃。镁或核苷酸辅因子ATP或ADP的存在不影响蛋白质的整体构象及其单体状态。这些数据表明，最近发现的DnaC分子之间的协同相互作用，在与DnaB，解旋酶的复合物中，是由与解旋酶的结合诱导的，即，它们不是DnaC蛋白质的固有特性。DnaC-MANT-ATP和DnaC-MANT-ADP复合物的荧光各向异性衰减表明，该蛋白质在纳秒时间尺度上具有刚性的全局结构，几乎不受核苷酸辅因子的影响。然而，与ATP的复合物具有更灵活的结构，而与ADP的复合物更刚性，蛋白质分子呈现更细长的形状。镁只对与ATP类似物的复合物起控制作用。在不存在镁的情况下，ATP类似物牢固地保持在结合位点。在Mg 2+存在下，该固定位置被释放，并且允许类似物呈现灵活的构象状态。的DnaC蛋白的功能的结果的意义进行了讨论。
Global conformational and oligomeric states of the Escherichia coli replicative factor DnaC protein in the absence and presence of magnesium and nucleotide cofactors, ATP and ADP, and their fluorescent analogues, MANT-ATP and MANT-ADP, have been examined using analytical sedimentation velocity and time-dependent fluorescence anisotropy techniques. In solution, the DnaC protein exists exclusively as a monomer over a large protein concentration range. The value of sdegrees(20,w) = 2.45 +/- 0.07 S indicates that the protein molecule has an elongated shape. When modeled as a prolate ellipsoid of revolution, the hydrated DnaC protein has an axial ratio of 4.0 +/- 0.6 with long axis a = 112 Angstrom and the short axis b = 28 Angstrom, respectively. The presence of magnesium or nucleotide cofactors, ATP or ADP, does not affect the global conformation of the protein and its monomeric state. These data indicate that recently found cooperative interactions between the DnaC molecules, in the complex with the DnaB, helicase, are induced by the binding to the helicase, i.e., they are not the intrinsic property of the DnaC protein. Fluorescence anisotropy decays of the DnaC-MANT-ATP and DnaC-MANT-ADP complexes indicate that the protein has a rigid global structure on the nanosecond time scale, little affected by the nucleotide cofactors. Nevertheless, the complex with ATP has a more flexible structure, while the complex with ADP is more rigid, with the protein molecule assuming a more elongated shape. Magnesium exerts control only on the complex with the ATP analogue. In the absence of magnesium, the ATP analogue is firmly held in the binding site. In the presence of Mg2+, this fixed location is released and the analogue is allowed to assume a flexible conformational state. The significance of the results for the functioning of the DnaC protein is discussed.