Perozo E, Cuello L G, Cortes D M, Liu Y S, Sompornpisut P
Department of Molecular Physiology and Biological Physics, Center for Structural Biology, University of Virginia, Charlottesville 22908, USA.
Novartis Found Symp. 2002;245:146-58; discussion 158-64, 165-8. doi: 10.1002/0470868759.ch10.
The fundamental processes that underlie ion channel function are permeation/ selectivity and gating. In an effort to understand ion channel gating, we have used an approach that combines reporter-group spectroscopic techniques (spin labelling/ electron paramagnetic resonance, EPR) and electrophysiological methods with classical biochemical and molecular biological procedures. As an ideal test channel, we have focused our attention on the K+ channel from Streptomyces lividans, KcsA. Through site-directed spin labelling, cysteine chemistry was used to introduce nitroxide radicals into specific sites within KcsA with high reactivity and specificity. EPR spectroscopy analysis of the spin labelled mutants yields two types of structural information: (1) mobility and solvent accessibility of the attached nitroxide through collisional relaxation methods and (2) distances between pairs of nitroxides through dipole-dipole interactions. Using this approach, we analysed the correlation between KcsA crystal structure and the EPR data, extend it to derive low-resolution folds of full-length KcsA and apply it in the determination of the molecular rearrangements responsible for pH-dependent gating.
离子通道功能的基本过程包括通透/选择性和门控。为了理解离子通道门控，我们采用了一种将报告基团光谱技术（自旋标记/电子顺磁共振，EPR）、电生理方法与经典生化和分子生物学程序相结合的方法。作为一个理想的测试通道，我们将注意力集中在来自淡紫链霉菌的K+通道KcsA上。通过定点自旋标记，利用半胱氨酸化学方法将氮氧自由基以高反应性和特异性引入KcsA内的特定位点。对自旋标记突变体的EPR光谱分析产生两种类型的结构信息：（1）通过碰撞弛豫方法得到附着氮氧自由基的流动性和溶剂可及性；（2）通过偶极-偶极相互作用得到成对氮氧自由基之间的距离。利用这种方法，我们分析了KcsA晶体结构与EPR数据之间的相关性，将其扩展以推导全长KcsA的低分辨率折叠结构，并将其应用于确定与pH依赖性门控相关的分子重排。
