Modeling Detergent Organization around Aquaporin-0 Using Small-Angle X-ray Scattering
Modeling Detergent Organization around Aquaporin-0 Using Small-Angle X-ray Scattering
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DOI：
10.1021/ja301667n
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发表时间：
2012-06-20
影响因子：
15
通讯作者：
Mangenot, Stephanie
中科院分区：
文献类型：
--
作者：
Berthaud, Alice;Manzi, John;Mangenot, Stephanie
膜蛋白在水溶液中的溶解需要两亲分子如去污剂的存在。然后，蛋白质的跨膜区域被这些分子形成的冠包围，确保亲水性外表面。这种冠的存在，强烈阻碍了结构研究的溶解膜蛋白的小角X-射线散射（SAXS），经常用于监测可溶性蛋白质的构象变化的技术。通过尺寸排阻色谱，SAXS和折射率的在线组合，我们已经确定了一个精确的几何模型的正十二烷基β-n-麦芽吡喃糖苷冠周围的水通道蛋白-0，最丰富的膜蛋白的眼睛透镜。SAXS数据与围绕蛋白质晶体结构的椭圆形环形中成形的去污剂冠很好地拟合，类似于最近报道的纳米盘的椭圆形（Skar-Gislinge等人，J. Am. 2010，132，13713-13722）。根据曲线拟合方案确定的圆环厚度与脂双层的厚度非常一致，而根据圆环体积推导出的洗涤剂分子数量与根据折射法和质量分析在同一样本上获得的洗涤剂分子数量相当。SAXS前向散射。首次测量了蛋白质周围去污剂的部分比容。本协议是未来的膜蛋白在溶液中的构象研究的关键一步。
Solubilization of integral membrane proteins in aqueous solutions requires the presence of amphiphilic molecules like detergents. The transmembrane region of the proteins is then surrounded by a corona formed by these molecules, ensuring a hydrophilic outer surface. The presence of this corona has strongly hampered structural studies of solubilized membrane proteins by small-angle X-ray scattering (SAXS), a technique frequently used to monitor conformational changes of soluble proteins. Through the online combination of size exclusion chromatography, SAXS, and refractometry, we have determined a precise geometrical model of the n-dodecyl beta-n-maltopyranoside corona surrounding aquaporin-0, the most abundant membrane protein of the eye lens. The SAXS data were well-fitted by a detergent corona shaped in an elliptical toroid around the crystal structure of the protein, similar to the elliptical shape recently reported for nanodiscs (Skar-Gislinge et al. J. Am. Chem. Soc.2010, 132, 13713-13722). The torus thickness determined from the curve-fitting protocol is in excellent agreement with the thickness of a lipid bilayer, while the number of detergent molecules deduced from the volume of the torus compares well with those obtained on the same sample from refractometry and mass analysis based on SAXS forward scattering. For the first time, the partial specific volume of the detergent surrounding a protein was measured. The present protocol is a crucial step toward future conformational studies of membrane proteins in solution.