Oligomeric state of the Escherichia coli metal transporter YiiP
Oligomeric state of the Escherichia coli metal transporter YiiP
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DOI：
10.1074/jbc.m407044200
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发表时间：
2004-09-17
影响因子：
4.8
通讯作者：
Fu, D
中科院分区：
文献类型：
--
作者：
Wei, YN;Li, HL;Fu, D
YiiP 是在大肠杆菌质膜中发现的 32.9 kDa 金属转运蛋白（Chao, Y. 和 Fu, D. (2004) J. Biol. Chem. 279, 17173 - 17180）。在这里，我们报告了洗涤剂脂质胶束和膜中 YiiP 寡聚状态的测定。使用尺寸排阻色谱法和激光散射光度测定法直接测量用十二烷基、十一烷基、癸基或壬基-β-D-麦芽糖苷溶解的 YiiP 的分子质量，得到在较窄范围 (68.0 - 68.8 kDa) 内的 YiiP 均聚低聚物的质量分布，该分布等于实验误差内 YiiP 二聚体的预测质量。发现混合胶束中与 YiiP 相关的去垢剂-脂质质量从 135.5 kDa 增加到 232.6 kDa，与麦芽糖苷去垢剂的烷基链长度明显相关。将洗涤剂溶解的 YiiP 与 1-乙基-3-[3-二甲基氨基丙基]碳二亚胺盐酸盐 (EDC) 交联，以 EDC 浓度依赖性方式产生二聚交联产物。通过电子显微镜分析负染中的二维 YiiP 晶体来确定重构膜中纯化 YiiP 的寡聚状态。根据可测量的 25 埃光学衍射计算出的投影结构显示，在类似于 75 x 40 埃的分子边界内存在伪 2 重对称性，表明膜中存在 YiiP 二聚体。这些数据为去污剂-脂质胶束和重构的脂质双层中的 YiiP 二聚体关联提供了直接的结构证据。讨论了 YiiP 中二聚体关联的功能相关性。
YiiP is a 32.9-kDa metal transporter found in the plasma membrane of Escherichia coli (Chao, Y., and Fu, D. (2004) J. Biol. Chem. 279, 17173 - 17180). Here we report the determination of the YiiP oligomeric state in detergent-lipid micelles and in membranes. Molecular masses of YiiP solubilized with dodecyl-, undecyl-, decyl-, or nonyl-beta-D-maltoside were measured directly using size-exclusion chromatography coupled with laser light-scattering photometry, yielding a mass distribution of YiiP homo-oligomers within a narrow range (68.0 - 68.8 kDa) that equals the predicted mass of a YiiP dimer within experimental error. The detergent-lipid masses associated with YiiP in the mixed micelles were found to increase from 135.5 to 232.6 kDa, with an apparent correlation with the alkyl chain length of the maltoside detergents. Cross-linking the detergent-solubilized YiiP with 1-ethyl-3- [3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) resulted in a dimeric cross-linked product in an EDC concentration-dependent manner. The oligomeric state of the purified YiiP in reconstituted membranes was determined by electron microscopic analysis of two-dimensional YiiP crystals in negative stain. A projection structure calculated from measurable optical diffractions to 25 Angstrom revealed a pseudo-2-fold symmetry within a molecular boundary of similar to 75 x 40 Angstrom, indicative of the presence of YiiP dimers in membranes. These data provide direct structural evidence for a dimeric association of YiiP both in detergent-lipid micelles and in the reconstituted lipid bilayer. The functional relevance of the dimeric association in YiiP is discussed.