Sample preparation for structural and functional analyses of the STRA6 receptor for retinol-binding protein.
Sample preparation for structural and functional analyses of the STRA6 receptor for retinol-binding protein.
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DOI：
10.1016/bs.mie.2020.03.005
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发表时间：
2020
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STRA 6（由视黄酸6刺激）是一种75 kDa的多位跨膜蛋白，可促进细胞从视黄醇结合蛋白（RBP）摄取视黄醇。通过单粒子冷冻电子显微镜（cryo-EM）实现了在洗涤剂中纯化并在amphipol A8-35中重构的来自斑马鱼的STRA 6的结构表征。这提供了该蛋白质的第一个高分辨率快照，显示了紧密组装的同源二聚体的新拓扑结构，以及与钙调蛋白的意想不到的生理关联，以及对其潜在功能机制的见解。具体地说，一个大的疏水性空腔在中心的STRA 6连接到已知的网站与RBP的相互作用，建议视黄醇进入细胞的途径扩散到膜通过一个横向开口的空腔直接进入双层。此外，生产纯的和均匀的蛋白质的能力，允许以前无法实现的功能表征的STRA 6在重建系统。在这里，我们描述了详细的方法在昆虫细胞中表达的斑马鱼STRA 6，在洗涤剂中纯化和重组的amphipol结构表征的冷冻EM。此外，我们显示了重组的蛋白质在脂质体中的体外蛋白脂质体为基础的分析STRA 6介导的视黄醇摄取。最后，我们提出的方法和初步的cryo-EM数据，将STRA 6纳入脂质填充的纳米盘，一个接近天然的环境，研究膜蛋白的结构和功能。
STRA6 (Stimulated by Retinoic Acid 6) is a 75 kDa polytopic transmembrane protein that facilitates cellular retinol uptake from retinol-binding protein (RBP). Structural characterization of STRA6 from Danio rerio purified in detergent and reconstituted in amphipol A8–35 was achieved by single-particle cryo-electron microscopy (cryo-EM). This provided the first high-resolution snapshot of this protein, showing a novel topology of a tightly assembled homodimer, and an unexpected physiological association with calmodulin in addition to insights into its potential mechanism of function. Specifically, a large hydrophobic cavity in the center of STRA6 linked to the known site of interaction with RBP suggested a route of retinol entry into the cell by diffusion into the membrane through a lateral opening of the cavity directly into the bilayer. Moreover, the capability to produce pure and homogeneous protein has allowed previously unattainable functional characterization of STRA6 in a reconstituted system. Here, we describe detailed methods for Danio rerio STRA6 expression in insect cells, purification in detergent and reconstitution in amphipol for structural characterization by cryo-EM. Furthermore, we show reconstitution of the protein in liposomes for an in vitro proteoliposome-based assay of STRA6-mediated retinol uptake. Finally, we present methods and preliminary cryo-EM data for STRA6 incorporated in lipid-filled nanodiscs, a close to native milieu to study membrane protein structure and function.
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DOI：
10.1126/science.aad8266
发表时间：
2016-08-26
期刊：
Science (New York, N.Y.)
影响因子：
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作者：
Chen Y;Clarke OB;Kim J;Stowe S;Kim YK;Assur Z;Cavalier M;Godoy-Ruiz R;von Alpen DC;Manzini C;Blaner WS;Frank J;Quadro L;Weber DJ;Shapiro L;Hendrickson WA;Mancia F
通讯作者：
Mancia F
影响因子：
5.7
作者：
Etzkorn, Manuel;Raschle, Thomas;Wagner, Gerhard
通讯作者：
Wagner, Gerhard
DOI：
10.1016/0005-2736(88)90438-5
发表时间：
1988-10-06
期刊：
BIOCHIMICA ET BIOPHYSICA ACTA
影响因子：
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作者：
FEX, G;JOHANNESSON, G
通讯作者：
JOHANNESSON, G
DOI：
10.1007/978-1-60327-325-1_15
发表时间：
2010
期刊：
Methods in molecular biology (Clifton, N.J.)
影响因子：
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影响因子：
14.8
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