Membrane Interactions of α-Synuclein Probed by Neutrons and Photons

alpha-Synuclein (alpha-syn) is a key protein in the etiology of Parkinson's disease. In a disease state, alpha-syn accumulates as insoluble amyloid fibrils enriched in beta-sheet structure. However, in its functional state, alpha-syn adopts an amphipathic helix upon membrane association and plays a role in synaptic vesicle docking, fusion, and clustering. In this Account, we describe our contributions made in the past decade toward developing a molecular understanding of alpha-syn membrane interactions, which are crucial for function and have pathological implications. Three topics are covered: alpha-syn membrane binding probed by neutron reflectometry (NR), the effects of membrane on alpha-syn amyloid formation, and interactions of alpha-syn with cellular membranes. NR offers a unique perspective by providing direct measurements of protein penetration depth. By the use of segmentally deuterated alpha-syn generated through native chemical ligation, the spatial resolution of specific membrane-bound polypeptide regions was determined by NR. Additionally, we used NR to characterize the membrane-bound complex of alpha-syn and glucocerebrosidase, a lysosomal hydrolase whose mutations are a common genetic risk factor for Parkinson's disease. Although phosphatidylcholine (PC) is the most abundant lipid species in mammalian cells, interactions of PC with alpha-syn have been largely ignored because they are substantially weaker compared with the electrostatically driven binding of negatively charged lipids. We discovered that alpha-syn tubulates zwitterionic PC membranes, which is likely related to its involvement in synaptic vesicle fusion by stabilization of membrane curvature. Interestingly, PC lipid tubules inhibit amyloid formation, in contrast to anionic phosphatidylglycerol lipid tubules, which stimulate protein aggregation. We also found that membrane fluidity influences the propensity of alpha-synuclein amyloid formation. Most recently, we obtained direct evidence of binding of alpha-syn to exocytic sites on intact cellular membranes using a method called cellular unroofing. This method provides direct access to the cytosolic plasma membrane. Importantly, measurements of fluorescence lifetime distributions revealed that alpha-syn is more conformationally dynamic at the membrane interface than previously appreciated. This exquisite responsiveness to specific lipid composition and membrane topology is important for both its physiological and pathological functions. Collectively, our work has provided insights into the effects of the chemical nature of phospholipid headgroups on the interplay among membrane remodeling, protein structure, and alpha-syn amyloid formation.

Automated summary

The research highlights the importance of alpha-synuclein's interactions with cellular membranes for its physiological and pathological functions, revealing the effects of the chemical nature of phospholipid headgroups on the interplay among membrane remodeling, protein structure, and alpha-syn amyloid formation.