Domain location within the cystic fibrosis transmembrane conductance regulator protein investigated by electron microscopy and gold labelling.
Domain location within the cystic fibrosis transmembrane conductance regulator protein investigated by electron microscopy and gold labelling.
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通过电子显微镜和金标记研究囊性纤维化跨膜电导调节蛋白内的结构域位置。
DOI：
10.1016/j.bbamem.2010.08.012
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发表时间：
2011
期刊：
影响因子：
--
通讯作者：
Ford,RobertC
中科院分区：
文献类型：
--
作者：
Zhang,Liang;Aleksandrov,LubaA;Riordan,JohnR;Ford,RobertC
使用去污剂溶解的二聚复合物的电子显微镜研究了囊性纤维化跨膜电导调节蛋白（CFTR）的结构域组织。 Ni-NTA纳米金标记数据表明，在非磷酸化、无核苷酸状态下，C末端与细胞质ATP结合区密切相关，而部分调节域占据靠近脂膜细胞质表面的位置。整个第二核苷酸结合域 (NBD2) 的去除会导致 CFTR 结构的缺陷，该结构与单个 NBD 的大小和形状一致。数据表明，NBD2 比第一个核苷酸结合域 (NBD1) 更靠近 C2 对称轴，并且来自一个 CFTR 单体的 NBD2 也与来自相反单体的 NBD1 接触。这些数据表明，当前基于其他 ATP 结合盒蛋白的 CFTR 同源模型似乎是合理的，至少在低分辨率方面是合理的。我们还发现内部六组氨酸序列的 Ni-NTA 纳米金标记是定位单个蛋白质结构域的有价值的方法。
The domain organisation of the cystic fibrosis transmembrane conductance regulator (CFTR) protein was studied using electron microscopy of detergent-solubilised dimeric complexes. Ni-NTA nanogold labelling data suggest that in the nonphosphorylated, nucleotide-free state, the C-terminus is intimately associated with the cytoplasmic ATP-binding regions, whilst part of the regulatory domain occupies a position close to the cytoplasmic surface of the lipid membrane. Removal of the entire second nucleotide binding domain (NBD2) results in a deficit in the CFTR structure that is consistent with the size and shape of a single NBD. The data suggest that NBD2 lies closer to the C2 symmetry axis than the first nucleotide binding domain (NBD1) and that NBD2 from one CFTR monomer also contacts NBD1 from the opposing one. These data suggest that current homology models for CFTR based on other ATP-binding cassette proteins appear to be reasonable, at least to low resolution. We also find that Ni-NTA nanogold labelling of an internal hexa-Histidine sequence is a valuable approach to locate individual protein domains.