Supported lipid bilayers are widely used as model systems due to their robustness. Due to the solid support, the properties of supported lipid bilayers are different from those of freestanding bilayers. In this article, we examine whether different surface treatments affect the properties of supported lipid bilayers. It will be shown that depending on the treatment method, the diffusion of the lipids can be adjusted approximately threefold without altering the composition. Additionally, as the bilayer-support interaction decreases, it becomes easier to form coexisting liquid-ordered and liquid-disordered domains. The physical/chemical alterations that result from the different treatment methods will be discussed.
Alpha-synuclein is the major component of Lewy body inclusions found in the brains of patients with Parkinson's disease. Several studies indicate that alpha-synuclein binds to negatively charged phospholipid bilayers. We examined the binding of alpha-synuclein to membranes containing different amounts of negatively charged lipids using supported lipid bilayers, epifluorescence microscopy, fluorescence recovery after photobleaching, and bulk fluorescence techniques. The membranes contained phosphatidylcholine and phosphatidylglycerol. In the absence of protein, these lipids mix uniformly. Our results show that the propensity of alpha-synuclein to cluster on the membrane increases as the concentration of anionic lipid and/or protein increases. Regions on the lipid bilayer where alpha-synuclein is clustered are enriched in phosphatidylglycerol. We also observe divalent metal ions stimulate protein cluster formation, primarily by promoting lipid demixing. The importance of protein structure, lipid demixing, and divalent ions, as well as the physiological implications, will be discussed. Because membrane-bound alpha-synuclein assemblies may play a role in neurotoxicity, it is of interest to determine how membranes can be used to tune the propensity of alpha-synuclein to aggregate.
α-Synuclein is a presynaptic protein that binds to phospholipid membranes and is involved in the pathogenesis of Parkinson's disease (PD). In this paper we describe the effects of adding wild-type α-synuclein (WT) and three familial PD mutants (A53T, A30P and E46K) to membranes containing 15–35 mol % anionic lipid. Tubules were observed to form in the membranes to an extent that depended on the α-synuclein variant, the anionic lipid content and the protein concentration. For all four variants tubule formation decreased with increasing anionic lipid content. Tubules were more readily observed with A30P and E46K than with WT or A53T. The results are consistent with a model wherein the helical content of α-synuclein increases with increasing anionic lipid content, and α-synuclein conformers with low helical content have a high propensity to induce tubule formation. This work, combined with previous work from our laboratory (Pandey et al., Biophys. J. 2009),1 shows that for WT adsorption of the protein has deleterious effects on the membrane when the anionic lipid concentration is less than 30 mol % (tubule formation) or greater than 40 mol % (re-organization of the bilayer, clustering of protein).
The interaction of α-synuclein with phospholipid membranes has been examined using supported lipid bilayers and epi-fluorescence microscopy. The membranes contained phosphatidylcholine (PC) and phosphatidic acid (PA), which mix at physiological pH. Upon protein adsorption the lipids undergo fluid-fluid phase separation into PC-rich and PA-rich regions. The protein preferentially adsorbs to the PA-rich regions. The adsorption and subsequent aggregation of α-synuclein was probed by tuning several parameters: the charge on the lipids, the charge on the protein, and the screening environment. Conditions which promoted the greatest extent of adsorption resulted in structurally heterogeneous aggregates, while comparatively homogeneous aggregates were observed under conditions whereby adsorption did not occur as readily. Our observation that different alterations to the system lead to different degrees of aggregation and different aggregate structures poses a challenge for drug discovery. Namely, therapies aimed at neutralizing α-synuclein must target a broad range of potentially toxic, membrane-bound assemblies.
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