Department of Chemistry and Chemical Engineering, Chalmers University of Technology , SE-41296 Gothenburg, Sweden.
Materials Physics and Application Division, MPA-11, Los Alamos National Laboratory , Los Alamos, New Mexico 87545, United States.
Nano Lett. 2017 Jan 11;17(1):476-485. doi: 10.1021/acs.nanolett.6b04493. Epub 2016 Nov 29.
Proteins are key components in a multitude of biological processes, of which the functions carried out by transmembrane (membrane-spanning) proteins are especially demanding for investigations. This is because this class of protein needs to be incorporated into a lipid bilayer representing its native environment, and in addition, many experimental conditions also require a solid support for stabilization and analytical purposes. The solid support substrate may, however, limit the protein functionality due to protein-material interactions and a lack of physical space. We have in this work tailored the pore size and pore ordering of a mesoporous silica thin film to match the native cell-membrane arrangement of the transmembrane protein human aquaporin 4 (hAQP4). Using neutron reflectivity (NR), we provide evidence of how substrate pores host the bulky water-soluble domain of hAQP4, which is shown to extend 7.2 nm into the pores of the substrate. Complementary surface analytical tools, including quartz crystal microbalance with dissipation monitoring (QCM-D) and fluorescence microscopy, revealed successful protein-containing supported lipid bilayer (pSLB) formation on mesoporous silica substrates, whereas pSLB formation was hampered on nonporous silica. Additionally, electron microscopy (TEM and SEM), light scattering (DLS and stopped-flow), and small-angle X-ray scattering (SAXS) were employed to provide a comprehensive characterization of this novel hybrid organic-inorganic interface, the tailoring of which is likely to be generally applicable to improve the function and stability of a broad range of membrane proteins containing water-soluble domains.
蛋白质是众多生物过程中的关键组成部分，其中跨膜（膜贯穿）蛋白质的功能尤其需要深入研究。这是因为这类蛋白质需要整合到代表其天然环境的脂质双层中，此外，许多实验条件还需要固体支撑物来进行稳定和分析。然而，固体支撑物的基底可能会由于蛋白质-材料相互作用和缺乏物理空间而限制蛋白质的功能。在这项工作中，我们对介孔二氧化硅薄膜的孔径和孔序进行了调整，以匹配跨膜蛋白人水通道蛋白 4（hAQP4）的天然细胞膜排列。使用中子反射（NR），我们提供了证据表明底物孔如何容纳大体积的水溶性 hAQP4 结构域，该结构域显示延伸 7.2nm 进入基底的孔中。互补的表面分析工具，包括石英晶体微天平（QCM-D）和荧光显微镜，揭示了在介孔二氧化硅基底上成功形成含有蛋白质的支撑脂质双层（pSLB），而在无孔二氧化硅上则阻碍了 pSLB 的形成。此外，电子显微镜（TEM 和 SEM）、光散射（DLS 和停流）和小角 X 射线散射（SAXS）被用来提供这种新型有机-无机杂化界面的综合特性，这种界面的调整可能具有普遍适用性，可以改善含有水溶性结构域的广泛膜蛋白的功能和稳定性。
