Structural dynamics of the Streptomyces lividans K+ channel (SKC1): Oligomeric stoichiometry and stability
Structural dynamics of the Streptomyces lividans K+ channel (SKC1): Oligomeric stoichiometry and stability
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DOI：
10.1021/bi971018y
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发表时间：
1997-08-19
期刊：
影响因子：
2.9
通讯作者：
Perozo, E
中科院分区：
文献类型：
--
作者：
Cortes, DM;Perozo, E
SKC1是最近从变铅青链霉菌中发现的一种含两个跨膜(TM)片段的160个残基的钾通道。其高水平的表达、小的尺寸和易于纯化使SKC1成为高分辨率结构研究的理想候选者，我们通过评估其低聚行为、洗涤剂的稳定性、一般流体动力学性质和初步二级结构含量来开始对该通道的结构表征。连续的金属螯合层析和凝胶过滤层析易于表达和纯化到均一的目的蛋白。标准的十二烷基硫酸钠-PAGE和化学交联分析表明，即使在十二烷基硫酸钠存在的情况下，SKC1仍表现为紧密结合的四聚体。用凝胶移位实验评估其低聚状态，我们确定SKC1在大多数洗涤剂中以四聚体的形式稳定存在，并可以在非离子洗涤剂溶液中保持较长时间。四聚体在相对较高的温度下也是稳定的，低聚物到单体的转变发生在大约65℃。胶束络合物的斯托克斯半径为5 nm，通过十二烷基麦芽糖苷中SKC1的凝胶过滤色谱测定。CD谱对二级结构的初步估计表明，该通道主要以α-螺旋构象存在，其中50%以上为α-螺旋构象，近20%为β-折叠，10%为β-转折，约15%为未分配或随机卷曲。这些结果与围绕离子传导途径形成四聚体的一束α螺旋是电压依赖通道超家族成员的共同结构基序的想法是一致的。
SKC1, a 160-residue potassium channel with two putative transmembrane (TM) segments was recently identified from Streptomyces lividans. Its high levels of expression, small size, and ease of purification make SKC1 an ideal candidate for high-resolution structural studies, We have initiated the structural characterization of this channel by assessing its oligomeric behavior, stability in detergent, general hydrodynamic properties, and preliminary secondary structure content. SKC1 was readily expressed and purified to homogeneity by sequential metal-chelate and gel filtration chromatography. Standard SDS-PAGE, together with chemical cross-linking analysis indicated that SKC1 behaves as a tightly associated tetramer even in the presence of SDS. Using a gel shift assay to assess its oligomeric state, we determined that SKC1 is stable as a tetramer in most detergents and can be maintained in nonionic detergent solutions for extended periods of time. The tetramer is also stable at relatively high temperatures, with an oligomer-to-monomer transition occurring at approximately 65 degrees C. The Stokes radius of the micellar complex is 5 nm as determined from gel filtration chromatography of SKC1 in dodecyl maltoside. Preliminary estimations of secondary structure from CD spectroscopy showed that the channel exists mostly in a-helical conformation, with more than 50% alpha-helical, close to 20% beta-sheet, 10% beta-turn, and about 15% unassigned or random coil. These results are consistent with the idea that a bundle of alpha-helices forming a tetramer around the ion-conductive pathway is the common structural motif for members of the voltage-dependent channel superfamily.