Centre for Misfolding Diseases, Department of Chemistry , University of Cambridge , Lensfield Road , Cambridge , CB2 1EW , United Kingdom.
State Key Laboratory of Bioreactor Engineering and School of Chemistry and Molecular Engineering , East China University of Science and Technology , 130 Meilong Road , Shanghai , 200237 People's Republic of China.
Anal Chem. 2018 Mar 6;90(5):3284-3290. doi: 10.1021/acs.analchem.7b04820. Epub 2018 Feb 13.
Elucidation of the fundamental interactions of proteins with biological membranes under native conditions is crucial for understanding the molecular basis of their biological function and malfunction. Notably, the large surface to volume ratio of living cells provides a molecular landscape for significant interactions of cellular components with membranes, thereby potentially modulating their function. However, such interactions can be challenging to probe using conventional biophysical methods due to the heterogeneity of the species and processes involved. Here, we use direct measurements of micron scale molecular diffusivity to detect and quantify the interactions of α-synuclein, associated with the etiology of Parkinson's disease, with negatively charged lipid vesicles. We further demonstrate that this microfluidic approach enables the characterization of size distributions of different binary mixtures of vesicles, which are not readily accessible using conventional light scattering techniques. Finally, the size distributions of the two α-synuclein conformations, free α-synuclein and membrane-bound α-synuclein, were resolved under varying lipid:protein ratios, thus, allowing the determination of the dissociation constant and the binding stoichiometry associated with this protein-lipid system. The microfluidic diffusional sizing platform allows these measurements to be performed on a time scale of minutes using microlitre volumes, thus, establishing the basis for an approach for the study of molecular interactions of heterogeneous systems under native conditions.
阐明蛋白质在天然条件下与生物膜的基本相互作用对于理解其生物功能和功能障碍的分子基础至关重要。值得注意的是，活细胞的大表面积与体积比为细胞成分与膜的显著相互作用提供了分子基础，从而可能调节它们的功能。然而，由于涉及的物种和过程的异质性，使用传统的生物物理方法很难探测到这种相互作用。在这里，我们使用微米级分子扩散率的直接测量来检测和量化与帕金森病病因相关的α-突触核蛋白与带负电荷的脂质体的相互作用。我们进一步证明，这种微流控方法能够表征不同脂质体二元混合物的大小分布，而使用传统的光散射技术则不容易获得。最后，在不同的脂质：蛋白质比下，两种α-突触核蛋白构象（游离α-突触核蛋白和膜结合α-突触核蛋白）的大小分布得以分辨，从而能够确定与该蛋白-脂质系统相关的离解常数和结合化学计量。微流控扩散尺寸分析平台允许使用微升体积在分钟的时间尺度上进行这些测量，从而为在天然条件下研究异质系统的分子相互作用建立了一种方法的基础。
