Weak substrate binding to transport proteins studied by NMR.
Weak substrate binding to transport proteins studied by NMR.
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通过 NMR 研究发现与转运蛋白的弱底物结合。
DOI：
10.1016/s0006-3495(98)77722-7
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发表时间：
1998
影响因子：
3.4
通讯作者：
Anthony Watts
中科院分区：
文献类型：
--
作者：
P. Spooner;W. John O’Reilly;Steven W. Homans;N. Rutherford;Peter J.F. Henderson;Anthony Watts
糖底物的弱结合不能引起大肠杆菌L-岩藻糖-H+结合蛋白FucP的任何可量化的物理变化，并且该蛋白缺乏用于竞争结合分析的强结合配体。然而，使用依赖于底物固定化进行检测的核磁共振方法可以获得底物结合行为。从质子到碳自旋的交叉极化可以检测到13C标记底物的一部分，该部分与0.2Mol的功能转运系统在天然膜中过度表达有关。检测到的底物位于FucP结合部位，因为它的信号被未标记的底物L-岩藻糖和L-半乳糖减弱，但不受非转运性立体异构体d-岩藻糖的三到五倍摩尔过量的影响。FucP似乎同样很好地结合了底物的两个异构体。核磁共振方法旨在测量底物交换的速度，可以显示底物与载体中心(>；10−1s)交换缓慢，尽管其动力学不一定与蛋白质中的这个位置强烈耦合。弛豫测量支持这一观点，即与底物相互作用的波动将限制在该运输系统中的结合位置。
The weak binding of sugar substrates fails to induce any quantifiable physical changes in the l-fucose-H+symport protein, FucP, fromEscherichia coli, and this protein lacks any strongly binding ligands for competitive binding assays. Access to substrate binding behavior is however possible using NMR methods which rely on substrate immobiliza-tion for detection. Cross-polarization from proton to carbon spins could detect the portion of13C-labeled substrate associated with 0.2μmol of the functional transport system overexpressed in the native membranes. The detected substrate was shown to be in the FucP binding site because its signal was diminished by the unlabeled substrates l-fucose and l-galactose but was unaffected by a three- to fivefold molar excess of the non-transportable stereoisomer d-fucose. FucP appeared to bind both anomers of its substrates equally well. An NMR method, designed to measure the rate of substrate exchange, could show that substrate exchanged slowly with the carrier center (>10−1s), although its dynamics are not necessarily coupled strongly to this site within the protein. Relaxation measurements support this view that fluctuations in the interaction with substrate would be confined to the binding site in this transport system.
DOI：
10.1073/pnas.93.23.13367
发表时间：
1996-11-12
影响因子：
11.1
作者：
Loo, DDF;Zeuthen, T;Wright, EM
通讯作者：
Wright, EM