1. Role of Membrane Interactions on the Mechanism of alpha-Synuclein Amyloid Formation Understanding the environmental factors affecting the aggregation of alpha-synuclein (alpha-syn) is of great importance because the accumulation and deposit of alpha-syn are intimately connected to Parkinsons disease (PD) etiology. Membrane interactions are of particular interest because alpha-syn localizes near synaptic vesicles and mitochondrial membranes in vivo. Specifically, the protein undergoes disordered-to-helical structural changes with the addition of membrane mimics such as SDS micelles and upon binding to anionic phospholipid vesicles (SUV) of varying size and composition. To develop a detailed understanding of how membranes influence alpha-syn conformation, site-specific probes of protein conformational heterogeneity and polypeptide-membrane interactions are necessary. Fluorescence spectroscopy is particularly suited for this application because of the availability of environmentally sensitive fluorophores and the ease of performing experiments near physiological temperatures and concentrations even down to a single molecule. In our studies, we have employed anionic SUVs and SDS micelles as membrane mimics to investigate membrane-induced conformational changes by fluorescence as well as circular dichroism (CD) spectroscopy. With this approach, we aim to determine the crucial protein-to-membrane conditions and key sites of interaction that promote protein aggregation and ultimately, monitor membrane-mediated amyloid formation processes. 1.1 Studies of N-terminal Peptides at the Membrane Interface To delineate possible key N-terminal polypeptide-membrane interaction sites, a single Trp-containing fluorescent variant of alpha-syn were employed (F4W). F4W has been demonstrated to be a particularly sensitive reporter of polypeptide environments and conformational changes in the presence of biomolecules such as lipids (Pfefferkorn CM, Lee JC, J. Phys. Chem. B 2010, 114, 4615-4622) or copper. Moreover, in our prior work, the F4W mutant exhibits nearly indistinguishable membrane binding properties as the wild-type protein. The minimal N-terminal amino acid sequence to preserve phospholipid vesicle binding was determined by investigating a series of synthetic W4 peptides and their spectroscopic properties: residues 1-4, 1-6, 1-10, and 1-15. CD and 1-dimensional NMR also were measured to assess the role of other (non-fluorescent) residues in secondary structural formation and membrane interactions, respectively. W4 interactions with the phospholipid acyl-chain region were directly probed using a series of brominated phospholipids as heavy-atom, collisional quenchers at different locations. 1.2 Promotion of Inter-alpha-Synuclein Interactions by the Membrane Surface There is a strong relationship between membranes and alpha-syn aggregation behavior, measurements of protein conformation and dynamics on the membrane surface are necessary to gain insight into how this protein converts from a benign to a pathogenic form. We have begun to investigate the specific relationship between amyloid formation and vesicle concentration. While our data suggest that alpha-syn proteins are not closely packed at saturation, it is plausible that changing the lipid-to-protein ratio could result in conformational rearrangement at the vesicle surface. It is noteworthy that the hydrophobic non-amyloid beta component region (61-95) has been proposed to be secluded from intermolecular contacts by the intramolecular N- and C-terminal interactions. Similarly, it is feasible for membrane stimulated aggregation to arise because as the local protein concentration increases at the surface, polypeptide conformational rearrangement ensues leading to exposure of potential protein-protein interaction sites promoting amyloid formation. Currently, experiments using bimolecular quenching and Forster energy transfer measurements are underway to identify and define conformational changes that ensue under these conditions that promote protein aggregation. 1.3 Assessing the Phospholipid Lateral Reorganization in a Bilayer upon alpha-Synuclein Binding While much research efforts have been geared towards the understanding of the protein conformational dynamics upon alpha-syn-membrane interaction, a central question that remains to be addressed is how the protein association influences the phospholipid bilayer structure and properties. Particularly, we sought to examine lateral reorganization processes of the phospholipids by using a fluorescence assay in which we measure fluorophore-labeled phospholipid ((1-oleoyl-2-12-(7-nitro-2-1,3-benzoxadiazol-4-yl)amino lauroyl-sn-glycero-3-phosphate) (NBD-PA)) self-quenching in large unilamellar vesicles (LUVs). We performed titrations of alpha-syn into a series of LUVs containing 50% anionic lipids (phosphatidylacid (PA), phosphatidylserine (PS), and phosphatidylinositol (PI), respectively) incorporated with a small amount of NBD-PA (0.2%) and found that in all cases, the protein laterally sequesters PA upon membrane binding, suggesting perturbation of the bilayer. Our results also showed that alpha-syn has preferences for PI and PA compared to PS. To gain further insights, we will employ fluorescence correlation spectroscopy (FCS) to evaluate the lateral diffusion times of specific phospholipids as well as to characterize the bilayer morphology by fluorescence microscopic studies of giant unilamellar vesicles. 2. Residue-specific Fluorescent Probes of alpha-Synuclein: Detection of Early Events at the N- and C-termini during Fibril Assembly To unravel the role of specific residues during the fibril assembly process, we prepared single-Cys mutants in the disordered (G7C and Y136C) and proximal (V26C and L100C) fibril core sites and derivatized them with environment sensitive dansyl (Dns) fluorophores (Yap TL, Pfefferkorn, Lee JC, Biochemistry 2011, 50, 1963-1965). Our study has provided site-specific information on the role of the alpha-syn N- and C-termini in amyloid formation. Kinetics obtained for Dns fluorophores in the disordered (7 and 136) regions precede proximal (26 and 100) amyloid core sites. Both seeded and spontaneous aggregation kinetics suggest that residues 7 and 136 exhibit local conformational and environmental changes prior to, whereas changes for residues 26 and 100 occur concomitantly with, macroscopic fibril formation. Our results support the hypothesis that local structural reorganization at the N- and C-termini are necessary for alpha-syn to break the conformational constraints from either intra- or inter-polypeptide electrostatics attraction and thus, favor the formation of parallel, in-register, beta-sheet fibrils.

Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
National Heart, Lung, and Blood Institute
Zip Code
O'Leary, Emma I; Jiang, Zhiping; Strub, Marie-Paule et al. (2018) Effects of phosphatidylcholine membrane fluidity on the conformation and aggregation of N-terminally acetylated ?-synuclein. J Biol Chem 293:11195-11205
McGlinchey, Ryan P; Lee, Jennifer C (2018) Why Study Functional Amyloids? Lessons from the Repeat Domain of Pmel17. J Mol Biol :
Jiang, Zhiping; Flynn, Jessica D; Teague Jr, Walter E et al. (2018) Stimulation of ?-synuclein amyloid formation by phosphatidylglycerol micellar tubules. Biochim Biophys Acta Biomembr :
Flynn, Jessica D; Lee, Jennifer C (2017) Physical Chemistry in Biomedical Research: From Cuvettes toward Cellular Insights. J Phys Chem Lett 8:1943-1945
Jiang, Zhiping; Heinrich, Frank; McGlinchey, Ryan P et al. (2017) Segmental Deuteration of ?-Synuclein for Neutron Reflectometry on Tethered Bilayers. J Phys Chem Lett 8:29-34
de Messieres, Michel; Ng, Abby; Duarte, Cornelio J et al. (2016) Single-Particle Tracking of Human Lipoproteins. Anal Chem 88:596-9
Brisbois, Chase A; Lee, Jennifer C (2016) Apolipoprotein C-III Nanodiscs Studied by Site-Specific Tryptophan Fluorescence. Biochemistry 55:4939-48
McGlinchey, Ryan P; Lee, Jennifer C (2015) Cysteine cathepsins are essential in lysosomal degradation of ?-synuclein. Proc Natl Acad Sci U S A 112:9322-7
Pfefferkorn, Candace M; Walker 3rd, Robert L; He, Yi et al. (2015) Tryptophan probes reveal residue-specific phospholipid interactions of apolipoprotein C-III. Biochim Biophys Acta 1848:2821-8
Rostovtseva, Tatiana K; Gurnev, Philip A; Protchenko, Olga et al. (2015) ?-Synuclein Shows High Affinity Interaction with Voltage-dependent Anion Channel, Suggesting Mechanisms of Mitochondrial Regulation and Toxicity in Parkinson Disease. J Biol Chem 290:18467-77

Showing the most recent 10 out of 24 publications