¹⁹F NMR studies of α‐synuclein‐membrane interactions

Abstract

a-Synuclein function is thought to be related to its membrane binding ability. Solution NMR studies have identified several a-synuclein-membrane interaction modes in small unilamellar vesicles (SUVs), but how membrane properties affect binding remains unclear. Here, we use 19 F NMR to study a-synuclein-membrane interactions by using 3-fluoro-L-tyrosine (3FY) and trifluoromethyl-Lphenylalanine (tfmF) labeled proteins. Our results indicate that the affinity is affected by both the head group and the acyl chain of the SUV. Negatively charged head groups have higher affinity, but different head groups with the same charge also affect binding. We show that the saturation of the acyl chain has a dramatic effect on the a-synuclein-membrane interactions by studying lipids with the same head group but different chains. Taken together, the data show that a-synuclein's N-terminal region is the most important determinate of SUV binding, but its C-terminal region also modulates the interactions. Our data support the existence of multiple tight phospholipid-binding modes, a result incompatible with the model that a-synuclein lies solely on the membrane surface.

Alpha-synuclein structure, functions, and interactions

“…Therefore, in vitro studies were established using membrane mimics, with different acyl head groups, different fatty acid chains, and various degrees of saturation of fatty acyl chains. [20] Liposomes are spherical lipid bilayers with the head groups located either inside or outside of the sphere and the fatty acid tails concealed within the space between these two layers. Liposomes differ in size and lipid composition, and they usually are classified as small unilamellar vesicles (SUVs) with a diameter 10–100 nm, large unilamellar vesicles (LUVs) with a diameter 100 nm - 1 μm, and giant unilamellar vesicles (GUVs) with a diameter >1 μm.…”

“…Taken together, these data indicate that α-syn has a preference for interaction with lipids and membranes containing unsaturated fatty acids with small anionic head groups and high curvature. [20]…”

Interaction of α-synuclein with vesicles that mimic mitochondrial membranes

“…The interaction of α-synuclein with vesicles of different lipid composition, phospholipid headgroups, sizes, and surfaces has been studied [19-30]. The protein has a higher affinity for small unilamellar vesicles than for large unilamellar vesicles (LUVs), and binds more strongly to vesicles containing anionic phospholipids [10, 19, 20, 31].…”

19F-Modified Proteins and 19F-Containing Ligands as Tools in Solution NMR Studies of Protein Interactions

“…By exploiting the large differences in resonance line width and signal intensity in the presence and absence of SUVs, the authors were able to quantify the fraction of membrane-bound protein. In addition, they were able to monitor the effect of lipid composition on the interaction between α-synuclein and the membrane, and found that both, the head group and the acyl chain of the SUV, affect the affinity (Wang, Li, & Pielak, 2010). In follow-up studies by 19 F NMR and fluorescence, interactions with large unilamellar vesicles, whose composition is similar to that of the inner and outer mitochondrial membrane, were probed.…”