期刊
ANALYTICAL CHEMISTRY
卷 90, 期 5, 页码 3284-3290出版社
AMER CHEMICAL SOC
DOI: 10.1021/acs.analchem.7b04820
关键词
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资金
- European Research Council under the European Union's Seventh Framework Programme (FP7) through the ERC grant PhysProt [337969]
- Newman Foundation
- BBSRC
- Cambridge Centre for Misfolding Diseases
- Leverhulme Trust, Cambridge
- Magdalene College, Cambridge
- China Scholarship Council
- FEBS Long-term Fellowship
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 alpha-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 alpha-synuclein conformations, free alpha-synuclein and membrane-bound alpha-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.
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