Parkinson’s disease (PD) is a prevalent age-related neurodegenerative disease with an estimated 1.5% lifetime risk for developing the disease.1
PD patients exhibit resting tremors, bradykinesia, rigidity, and impaired balance caused by the loss of dopamine-producing cells in a midbrain region called the substantia nigra
The origin of dopaminergic cell death is unknown; however, biochemical, histological, and genetic studies have implicated a neuronal protein, α-synuclein (α-syn), in PD pathogenesis. For example, α-syn is the primary component of intracellular proteinacious aggregates called Lewy bodies and neurites found in PD patients.1
Age-related increases in α-syn concentration also are observed in nigral
Furthermore, genetic findings link early-onset PD to the gene triplication of α-syn and three missense α-syn mutations (A30P, E46K, A53T).3
Whether sporadic or genetic in origin, it is not simply the presence of α-syn that is associated with PD, but also its corresponding conformational state. While soluble α-syn is characterized as natively unfolded in vitro
it is the aggregated β-sheet form (amyloid fibrils) that is found in Lewy bodies and neurites.5,6
Protofibrillar (oligomeric) species have been implicated as pathogenic agents;7,8
however, the exact role of α-syn oligomers in cytotoxic and aggregation pathways is not clear. Oligomers have been shown to both accelerate9
and inhibit α-syn fibril formation.10
It is these features that consequently place PD in the category of a protein misfolding disease.6,11
Membrane interactions are of particular interest because α-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 of varying size and composition.15
The ability of α-syn to bind to membranes is anticipated by its amino acid sequence: of its 140 total residues, the first 89 residues contain seven imperfect eleven residue repeats (XKTKEGVXXXX) reminiscent of membrane binding apolipoproteins (apoPs).16,17
The hydrophobic central peptide fragment, or non-amyloid β component (NAC), is comprised of residues 61–95. While this region, first identified in amyloid plaques of Alzheimer’s disease patients,18
is involved in membrane association, it is implicated mainly as an initiation site for protein aggregation.19
In contrast, the C-terminal domain of α-syn is highly acidic (15 carboxylates) and generally does not associate with lipids; however, upon binding calcium ions (charge neutralization), even this region interacts with membranes.20
Notably, vesicle leakage assays reveal that protofibrillar α-syn can permeabilize membranes suggesting yet another possible mechanism for PD pathogenesis.8,21,22
The presence of anionic phospholipids or detergents indeed, have been shown to both stimulate23
amyloid formation. Because of the strong relationship between membranes and α-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.
Currently, two membrane-bound α-syn conformations have been derived from spectroscopic methods. Structures assigned from NMR spectroscopy on SDS-micelle-bound protein26–32
and site-directed spin labeling EPR on vesicle-bound α-syn30
are characterized by two N- and C-terminal, antiparallel, α-helices (residues 3–37 and 45–92) separated by a short linker (residues 38–44). However, several additional EPR33–35
and single molecule fluorescence36–38
studies show α-syn conformation is dominated by one continuous, extended α-helix when bound to small unilamellar vesicles (SUVs). A recent NMR study suggests that there are multiple distinct α-syn membrane binding modes that are dependent on α-syn-to-lipid stoichiometry.39
Despite numerous work on membrane composition and size effects on α-syn membrane binding properties, a consensus on the conditions required to promote α-syn into one, both, or other variants of the proposed membrane-bound structures remains elusive. It is not clear whether or not these conformers are modulated by membrane surface availability (i.e.
surface area available for protein binding), detergent or phospholipid headgroup composition, or simply solution conditions. Furthermore, given the ability for this protein to undergo large conformational rearrangements, it is even more likely that bound structures are not mutually exclusive and can interchange.
To develop a detailed understanding of how membranes influence α-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 prior work, we have exploited tryptophan40
as fluorescent amino acids of α-syn conformation and dynamics42,43
in solution. The emission properties of the indole side chain are exquisitely responsive to local environment and conformation providing remarkably useful probes of protein-lipid interactions.44–49
In this study, 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. Tryptophan was substituted at four different aromatic residues (F4W, Y39W, F94W, and Y125W) to report information on local polypeptide environment and conformational heterogeneity between SUV- and SDS-micelles-bound α-syn. Furthermore, insights into the role of surface coverage (total number of proteins bound and respective surface area occupied) were extracted from saturable equilibrium binding curves for all α-synucleins in the presence of SUVs.