Three-dimensional structure of a CIC-type CI Channel

CIC型CI通道的三维结构

• 批准号: 6437982
• 负责人: NIKOLAUS GRIGORIEFF
• 金额: $ 23.09万
• 依托单位: BRANDEIS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-02-01 至 2006-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6437982
• 关键词: Escherichia coli; cell membrane; chemical models; chloride channels; conformation; cryoelectron microscopy; cysteine; electron crystallography; image processing; membrane channels; membrane proteins; membrane structure; protein protein interaction; protein structure function; structural biology

DESCRIPTION (provided by applicant): C1- channels play a multitude of roles in biological membranes. The C1C family of C1- channels has been studied functionally for the past 20 years, and was identified molecularly only about a decade ago. CIC channels structure differs entirely from many other channels such as voltage-gated cation channels. Eukaryotic members have a molecular mass of approximately 100 kDa with a characteristic transmembrane topology including about 10 transmembrane segments. Accumulating evidence suggests that C1C pores are formed by a single subunit. However, their quaternary structure is homodimeric. These unique features predict an entirely new structure-function paradigm for this family of ion channels. My colleagues and I have succeeded in reconstituting the bacterial C1C homologue, EriC, into large, highly ordered two-dimensional crystals suitable for high-resolution electron cryo-microscopy. We propose to determine the three-dimensional structure of EriC at 3.7 A resolution or higher to visualize its secondary structure and the pore. To help determine the path of the polypeptide chain in unclear regions of the map, for example in the surface loops, cysteine mutagenesis combined with heavy atom labeling will be used. Cysteine mutagenesis and labeling will also be used to identify key functional groups in the structure. The location of the labels will be determined using difference Fourier maps. The work will produce the first model of a C1C-type ion channel, key to a more thorough understanding of C1C channel function.

描述（由申请人提供）：C1-通道在以下方面发挥多种作用： 生物膜。 C1C 家族的 C1- 通道已被研究 在过去的 20 年里，它的功能已被证实，并且仅在分子水平上被鉴定出大约 十年前。 CIC渠道结构与许多其他渠道完全不同 例如电压门控阳离子通道。真核生物成员具有分子质量 大约 100 kDa，具有特征跨膜拓扑结构，包括 约10个跨膜片段。越来越多的证据表明 C1C 孔 由单个亚基形成。然而，它们的四级结构是 同二聚体。这些独特的特征预示着全新的结构功能 该离子通道家族的范例。 我和我的同事成功重建了细菌 C1C 同系物，EriC，转化为大的、高度有序的二维晶体 用于高分辨率电子冷冻显微镜。我们建议确定 3.7 A 分辨率或更高的 EriC 三维结构可视化 它的二级结构和孔隙。帮助确定路径 地图上不清楚的区域（例如表面）中的多肽链 环，半胱氨酸诱变结合重原子标记将被使用。 半胱氨酸诱变和标记也将用于识别关键功能 结构中的组。标签的位置将使用确定 差分傅立叶图。这项工作将生产第一个 C1C 型模型 离子通道，是更深入了解 C1C 通道功能的关键。