During the past 20 years there has been a remarkable growth in the use of fluorescence in the biological sciences. Fluorescence is now a dominant methodology used extensively in biochemistry, biophysics, biotechnology, medical diagnostics, flow cytometry, DNA sequencing and genetic analysis to name a few. It is one of the most powerful methods to study protein folding, dynamics, assembly and interactions as well as membrane structure. α-Synuclein belongs to the class of intrinsically disordered proteins lacking of a well-folded structure under physiological conditions. The conversion of α-synuclein from a soluble monomer to an insoluble fibril may underlie the neurodegeneration associated with Parkinson’s disease (PD). Although the exact mechanism of α-synuclein toxicity is still unknown, it has been proposed that disturbs membrane structure, leading to increased membrane permeability and eventual cell death. This review highlights the significant role played by fluorescence techniques in unraveling the nature of interactions between α-synuclein and membranes and its implications in PD.
Fluorescence spectroscopy-based techniques using conventional fluorimeters have been extensively applied since the late 1960s to study different aspects of membrane-related phenomena, i.e., mainly relating to lipidlipid and lipid-protein (peptide) interactions. These types of studies encompass measurements of fluorescence excitation and emission spectra, fluorescence time decays (lifetimes) and fluorescence polarization (or anisotropy) using of a large variety of fluorescent probes [1,2]. The ability of α-synuclein to exchange between many different conformational states-disordered monomer, partially structured oligomer, α-helical membrane-bound monomer, and α-sheet fibrillar aggregate-may be central to its native function as well as to its role in Parkinson’s disease (PD). α-Synuclein is the primary component of Lewy bodies, dense cytoplasmic amyloid inclusions, which are associated with selective loss of dopaminergic neurons in the substantia nigra region of the brain. Although its precise role in the progression of PD remains unclear, point mutations (A30P, E46K and A53T) and multipliction of the α-synuclein gene linked to familial forms of PD established that it is crucial to disease development. Commonly used fluorescent techniques, include intrinsic and extrinsic fluorophores, fluorescent probes incorporated in the membrane, steady-state and lifetime measurements of fluorescence emision, fluorescence resonance energy transfer (FRET) and fluorescence microscopy, have been employed to monitor the kinetics of aggregation of α-synuclein, its distinct phospholipid vesicle and micelle interactions as well as its membrane-perturbation properties to contribute to our understanding of its native function and its role in Parkinson’s disease.
Fluorescence is a molecular phenomenon in which a substance absorbs light, then radiate part of this absorbed energy as light of another color, one of lower energy and thus longer wavelength. This process is known as excitation and emission. The shift from excitation wavelength to emission wavelength is called the Stokes shift. The ability of a fluorophore to absorb encountered light is known as the extinction coefficient. Once energy is absorbed, the fluorophore has some probability less than one of releasing this absorbed energy as light. This characteristic is called the Quantum yield (number of emitted photons relative to the number of absorbed photons) [3,4]. Substances with the largest quantum yields, approaching unity, display the brightest emissions. The lifetime is also important, as it determines the time available for the fluorophore to interact with or diffuse in its environment, and hence the information available from its emission. Together, these properties dictate the basic fluorescent properties (brightness and spectra) of an individual fluorescent dye. The power of fluorescence spectroscopy lies on its broad applicability. Almost all proteins have natural fluorophores, tyrosine and tryptophan residues, which allow study of changes in protein conformation. Also site-specific labeling with external fluorophores is easily achievable by mutagenesis and chemical modifications. Another advantage is that fluorescence spectroscopy requieres a small amount of material (pM-nM range) and has a high signal-to noise ratio [
Tryptophan (Trp) and tyrosine (Tyr) residues are naturally occurring fluorophores in proteins (
to Tyr or Trp, and Tyr to Trp, can influence the quantum yield of Trp and Tyr through energy transfer [
In many cases, the intrinsic fluorescence of the protein/ peptide does not exist or is not fluorescent in a convenient region of the UV visible spectrum. It is very common to create single Trp mutants or to attach covalently a fluorescent group to a cysteine residue. With this approach, it is posible to explore the environment around different regions of a protein, by different fluorescent methodologies, obtaining a large amount of information [
Lipids are amphiphilic in nature and consist of a hydrophobic chain and a hydrophilic head group region. Accordingly, there are in principle two regions within the molecule to which fluorescent dyes can be covalently coupled. The intramolecular localization of the dye as well as the chemical nature of the dye itself are highly relevant, as they will determine the biophysical properties of the final fluorescent lipid analog as such, which in turn are relevant for the assay in which the probes will be eventually used. The majority of the fluorescent dyes that have been used in lipid derivatization, especially those emitting in the visible light region, are hydrophilic. When they are attached in the chain region, the dye will change the hydrophilic/hydrophobic balance of the lipid molecule [
also be labeled by covalent attachment of probes to the lipids. This is useful with more water-soluble probes like fluorescein or rhodamine. The probes can be forced to localize in the membrane by attachment to long acyl chains or to the phospholipids themselves. Depending on chemical structure, the fluorescent group can be positioned either on the fatty acid side chains (Fluorenyl-PC) or at the membrane-water interface (TexasRed-PE) (
Parkinson’s disease (PD) is the second most common neurodegenerative disease, affecting 1% - 2% of the population over age 65. It involves loss of dopaminergic neurons in the substantia nigra, which leads to decreased dopamine levels in the striatum, causing symptom such as tremors, rigidity in muscles and bradykinesia [11-13]. While the exact cause of PD is not known, aggregation of the presynaptic protein α-synuclein is believed to be a critical role in the etiology of the disease. There is a growing number of cellular toxicity studies showing that oligomers have a higher cytotoxicity compared to the fibrillar form of the proteins [14-17], suggesting that soluble amyloid oligomers may be the cause of cellular toxicity instead of the fibrillar aggregates [15,18-21]. One conceivable pathway to cellular toxicity would be the permeabilization of cellular membranes by oligomers. However, the mechanisms by which oligomers cause bilayer permeabilization still remain unclear. On the one hand it has been hypothesized that α-synuclein oligomers are able to form pore-like structures mediating the permeation of small molecules [20- 23] whereas on the other hand membrane thinning by the interaction with the oligomeric protein, which increases the permeability of the phospholipid bilayers has been postulated. In the case of α-synuclein, the first reports on membrane permeabilization by pore-like, annular shaped oligomers are from the Lansbury group in the early 2000s [22,23]. While the AFM images obtained by this group suggest pore-like mechanism, quantitative biophysical information on the structures and composition of these oligomeric aggregation intermediates is lacking, for this reason several research groups have been employed various techniques including fluorescence spectroscopy to understand the mechanism by which α-synuclein is implicated in PD. Native alpha synuclein is a small protein, 14 kDa, which analysis of the primary structure shows three different regions: the N-terminal region consisting of five degenerate 11-mer repeats KTKEGV of overall positive charge, the hydrophobic central part (known as the NAC region) and the C terminal 40 residues which are highly enriched in negatively charged amino acids (
Monitoring intrinsic Tyr fluorescence of α-synuclein during fibril formation or interaction with lipid vesicles revealed changes in the shape and intensity of the Tyr emission spectrum which is manifested by a shift in the emission maximum from 305 to 340 nm. Th estructural transition occurs in the N-terminal and central parts of the protein, whereas the C-terminal remains unfolded.
Fink et al., (2003) characterized the binding of α- synuclein to vesicles of different compositions by measuring the change in intrinsic fluorescence emanating from the four Tyr residues in the protein as a function of lipid concentration. They observed that α-synuclein can bind to the surface of PC vesicles, but not as strong as with PA/PC or PG/PC. This suggest that electrostatic interactions play an important role in the binding of protein to lipid, however the binding was also observed at very high ionic strengths, showing that additional forces, presumably hydrophobic, must also contribute significantly to the interaction of protein and the lipid. It is the hydrophobic interactions, because of the helical conformation, that lead to the critical penetration of the bilayer. Using NBDand Laurdan-labeled vesicles they also demonstrated that soluble monomeric α-synuclein and fibrillar α-synuclein can insert into the bilayer to a depth of several angstroms, and affect membrane properties. With a dye release assay they could observe the ability of, its protofibrils and fibrils to disrupt lipid membranes. The results clearly show that fibrillar α-synuclein has a much higer ability to disrupt membrane bilayers than soluble monomeric α-synuclein, and the order of effectiveness in disruption is PA > PG > PC, which is consistent with the membrane affinity [
Because the λmax of tyrosine is insensitive to its environment several tryptophan mutants have been constructed as fluorescence probes. Incorporation of Trp into α-synuclein permits the use of a variety of probes, includig solvent accessibility probes such as intrinsic fluorescence emission and acrylamide quenching, to analyze the presence of oligomers and the kinetics of their formation via fluorescence anisotropy and changes in compactness via FRET [9,10]. Investigations of Y39W and Y125W mutants show that both tryptophans are exposed to solvent in monomeric unfolded α-synuclein. During fibril formation Trp39 becomes partially buried in the core of the fibrils as revealed by a blue shift in λmax (340 nm), whereas Trp125 remains solvent exposed. Interestingly, at acidic pH the emission maxima of Y39W and Y125W are 345 and 345.5 nm, indicating the hydrophobic collapse of α-synuclein under acidic conditions, presumably due to loss of the negative charges in the Cterminal region, and consistent with previous studies showing a partially folded intermediate at acidic pH [
FRET measurements were used to study the structure and dynamics of α-synuclein in three different conditions: at physiological pH 7.4, acidic pH 4.4 and in the presence of SDS micelles [
Membrane permeabilization by α-synuclein oligomers may play a major role in the pathological mechanism of PD, potentially due to the impairment of membranous cellular structures such as mitochondria and synaptic vesicles [33,34]. The first decisive step for membrane permeabilization is the recruitment of oligomers to the lipid bilayer. This process dependent on the presence of negatively charged lipids being in a liquid disordered phase, while the headgroup is only of minor importance [35,36]. This behavior, which is comparable to monomeric α-synuclein suggests that mainly electrostatic interactions between the positively charged core of the oligomers and negatively charged lipid headgroups cause membrane binding [37,38]. In order to elucidate the mechanisms by which the α-synuclein oligomers permeabilize lipid bilayers, Subramaniam group tested different lipid mixtures in leakage experiments either based on the release of calcein from large unilamellar vesicles (LUVs) containing self-quenching concentrations of the probe or on measuring the influx of dithionite into LUVs labeled with 1-palmitoyl-2-6-[(7-nitro-2-1,3-benzoxadiazol-4-yl) amino] hexanoyl-phosphatidylcholine (C6-NBD-PC). While for LUVs prepared from palmitoyl-oleoyl-phosphatidylcholine (POPC) no leakage occurred, a strong disruption of the vesicles could be observed for LUVs consisting of the negatively charged lipids di-oleoylphosphatidylserine (DOPS), palmitoyl-oleoyl-phosphatidylglycerol (POPG) or soy phosphatidylinositol (PI). They also observed a slight influence of the lipid headgroup [
To investigate and visualize directly the lipidand domain-specific association of α-synuclein with membranes, Herrmann et al. (2008) studied the binding of fluorescently labeled α-synuclein to giant unilamellar vesicles (GUVs) of different composition labeled with 1 mol% of the green fluorescent lipid analogue 1-palmitoyl-2-[6-[(7- nitro-2-1,3-benzox-adiazol-4-yl)amino]hexanoyl]-sn-glycero-3-phosphocholine (C6-NBD-PC), which is known to localize preferentially to the liquid-disorder (Ld) phase [
In order to gain more insight into the mechanism of lipid bilayer disruption by α-synuclein oligomer species, Subramaniam group have used confocal fluorescence microscopy to observe the oligomer induced membrane permeability of giant unilamellar vesicles. They prepared Rhodamine labeled POPG GUVs encapsulating the dye 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS). To reduce the signal from un-encapsulated HPTS they added the quencher p-Xilene-bis, N-pyridinium bromide (DPX) to the solution outside the GUVs. When oligomeric α-synuclein was added to the imaging chamber, the fluorescence from the vesicle interior was lost. The images show that the leakage proces is fast and that the vesicles appear morphologically unchanged. Results from DLS experiments confirm that LUVs stay largely intact upon oligomer interaction [
Fluorescence spectroscopy can be applied to a wide range of problems in the chemical and biological sciences. The measurements can provide information on a wide range of molecular process, including the interactions of solvent molecules with fluorophores, rotational diffusion of biomolecules, distances between sites on biomolecules, conformational changes, and binding interactions. The usefulness of fluorescence is being expanded by advances in technolgy for celular imaging and single-molecule detection. These advances in fluorescence technology are decreasing the cost and complexity of previously complex instruments. Fluorescence spectroscopy will continue to contribute to rapid advances in biology, biotechnology and nanotechnology. With regard to alpha-synuclein, fluorescence techniques have allowed characterization of the interaction of this protein and its various aggregated states with membranes of different types and show that oligomeric and fibrillar forms of the protein cause substantial perturbation of membranes and likely promote membrane permeability leading to cytotoxicity. In addition, elucidating the underlying mechanisms by which the α-synuclein oligomers are able to penetrate the phospholipid bilayer will give valuable insights into their mode of action and presumably facilitate the development of a possible intervention strategy.
A.G.H. would like to acknowledge the support of PAICYT (CN416-10) Universidad Autonoma de Nuevo Leon. The authors would also like to acknowledge the contribution of a large number of authors who have contributed to the elucidation of alpha-synuclein interaction with membranes.