TY - JOUR

T1 - Microfluidic Diffusion Platform for Characterizing the Sizes of Lipid Vesicles and the Thermodynamics of Protein-Lipid Interactions

AU - Gang, Hongze

AU - Galvagnion, Céline

AU - Meisl, Georg

AU - Müller, Thomas

AU - Pfammatter, Manuela

AU - Buell, Alexander K

AU - Levin, Aviad

AU - Dobson, Christopher M

AU - Mu, Bozhong

AU - Knowles, Tuomas P J

PY - 2018/3/6

Y1 - 2018/3/6

N2 - 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.

AB - 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.

KW - Diffusion

KW - Microfluidic Analytical Techniques/methods

KW - Particle Size

KW - Phosphatidylethanolamines/metabolism

KW - Phosphatidylserines/metabolism

KW - Protein Binding

KW - Unilamellar Liposomes/chemistry

KW - alpha-Synuclein/chemistry

U2 - 10.1021/acs.analchem.7b04820

DO - 10.1021/acs.analchem.7b04820

M3 - Journal article

C2 - 29313342

VL - 90

SP - 3284

EP - 3290

JO - Industrial And Engineering Chemistry Analytical Edition

JF - Industrial And Engineering Chemistry Analytical Edition

SN - 0003-2700

IS - 5

ER -