Structure of the KcsA potassium channel from Streptomyces lividans:: A site-directed spin labeling study of the second transmembrane segment
Structure of the KcsA potassium channel from Streptomyces lividans:: A site-directed spin labeling study of the second transmembrane segment
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DOI：
10.1021/bi990856k
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发表时间：
1999-08-10
期刊：
影响因子：
2.9
通讯作者：
Hubbell, WL
中科院分区：
文献类型：
--
作者：
Gross, A;Columbus, L;Hubbell, WL
KcsA是原核生物的钾离子通道。本研究采用半胱氨酸扫描诱变和定点自旋标记，以调查的第二个跨膜段（残基82-120）在功能性四聚体通道重建的脂质双层的结构。自旋-自旋相互作用之间的氮氧侧链在接近4重对称轴的相关网站。为了帮助这些相互作用的定量分析，一个新的抗磁性类似物的氮氧侧链用于制备磁性稀释的样品具有恒定的结构。使用由自旋-自旋相互作用施加的约束，推导出该区段的堆积模型，其与最近报道的晶体结构非常一致[Doyle，D.，等人（1998）Science 280，69-77]。氮氧侧链的相对固定状态表明，该通道是刚性的电子顺磁共振时间尺度上。此外，在许多位置处的半胱氨酸的巯基反应性差表明通道不受允许掩埋的半胱氨酸反应的低频波动的影响。在预期位于孔中的位点处，侧链与O-2或乙二胺二乙酸镍（II）碰撞的可及性低。这种不可接近性，连同侧链在整个序列中的通常低的移动性，使得难以基于这些测量来检测孔的存在。然而，溶剂化孔的存在下，可以直接证明使用的极性参数推导出在低温下记录的EPR谱。这些测量也揭示了磷脂双层中极性梯度的存在。
KcsA is a prokaryotic potassium channel. The present study employs cysteine scanning mutagenesis and site-directed spin labeling to investigate the structure of the second transmembrane segment (residues 82-120) in functional tetrameric channels reconstituted in lipid bilayers. Spin-spin interactions are observed between nitroxide side chains at symmetry-related sites close to the 4-fold axis of symmetry. To aid in quantitative analysis of these interactions, a new diamagnetic analogue of the nitroxide side chain is used to prepare magnetically dilute samples with constant structure. Using constraints imposed by the spin-spin interactions, a packing model for this segment is deduced that is in excellent agreement with the recently reported crystal structure [Doyle, D., et al. (1998) Science 280, 69-77]. The relatively immobilized state of the nitroxide side chains suggests that the channel is rigid on the electron paramagnetic resonance time scale. Moreover, the poor sulfhydryl reactivity of the cysteine at many locations indicates that the channel is not subject to the low-frequency fluctuations that permit reaction of buried cysteines. At sites expected to be located in the pore, the accessibility of the side chains to collision with O-2 or nickel(II) ethylenediaminediacetate is low. This inaccessibility, together with the generally low mobility of the side chains throughout the sequence, makes it difficult to detect the presence of the pore based on these measurements. However, the presence of a solvated pore can be directly demonstrated using a polarity parameter deduced from the EPR spectra recorded at low temperature. These measurements also reveal the presence of a polarity gradient in the phospholipid bilayer.