Gross A, Columbus L, Hideg K, Altenbach C, Hubbell W L
Jules Stein Eye Institute, Department of Chemistry and Biochemistry, University of California, Los Angeles 90095-7008, USA.
Biochemistry. 1999 Aug 10;38(32):10324-35. doi: 10.1021/bi990856k.
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.
KcsA是一种原核钾通道。本研究采用半胱氨酸扫描诱变和定点自旋标记技术，来研究在脂质双分子层中重构的功能性四聚体通道中第二个跨膜片段（82 - 120位氨基酸残基）的结构。在靠近四重对称轴的对称相关位点的氮氧自由基侧链之间观察到了自旋 - 自旋相互作用。为了有助于对这些相互作用进行定量分析，使用一种新的氮氧自由基侧链抗磁性类似物来制备具有恒定结构的磁稀释样品。利用自旋 - 自旋相互作用施加的限制条件，推导出了该片段的堆积模型，该模型与最近报道的晶体结构[多伊尔，D.等人（1998年）《科学》280卷，69 - 77页]高度吻合。氮氧自由基侧链相对固定的状态表明，在电子顺磁共振时间尺度上通道是刚性的。此外，许多位置的半胱氨酸巯基反应性较差，这表明通道不会受到允许埋藏的半胱氨酸发生反应的低频波动的影响。在预期位于孔道中的位点，侧链与O₂或镍（II）乙二胺二乙酸发生碰撞的可及性较低。这种不可及性，连同侧链在整个序列中普遍较低的流动性，使得基于这些测量难以检测到孔道的存在。然而，使用从低温下记录的电子顺磁共振光谱推导的极性参数，可以直接证明溶剂化孔道的存在。这些测量还揭示了磷脂双分子层中极性梯度的存在。
