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
SN - 0003-2700
VL - 90
SP - 3284
EP - 3290
JO - Analytical Chemistry
JF - Analytical Chemistry
IS - 5
ER -