**3. Synucleinopathies**

Protein-conformation-dependent toxicity is an emerging theme in neurodegenerative disorders, including the synucleinopathies (Ulrih et al., 2008). The group of synucleinopathies comprises many neurodegenerative diseases, among which the best known and most common is PD, but it also includes Lewy body dementia, multiple system atrophy, neurodegeneration with brain iron accumulation type I, diffuse Lewy body disease, and Lewy body variant of Alzheimer's disease (Arawaka et al., 1998; Gai et al., 1998; Spillantini et al., 1997; Wakabayashi et al., 1997). These are all brain amyloidoses with the common characteristic of pathological intracellular inclusions of aggregates that have αsynuclein as the key component (Spillantini et al., 1997; Wakabayashi et al., 1997).

PD is characterized by the death of neurons that produce dopamine and are located in the *substantia nigra pars compacta* brain region (see below). This is accompanied by the appearance of Lewy bodies and Lewy neurites (Galvin et al., 1999). Lewy bodies are spherical protein inclusions that are found in the cytoplasm of surviving *substantia nigra* neurons, and they consist of a dense core surrounded by a halo of radiating fibrils of αsynuclein; they also contain a variety of other proteins. The fibrils seen in PD are structurally similar to those in the amyloid diseases, and they appear as linear rods of 5 nm to 10 nm diameter (Fink, 2006). PD affects more than 1% of the population over 65 years of age (Goedert, 2001), and typical symptoms include tremor, slow movements, fine motor difficulties, and loss of postural reflexes (Jankovic, 2008). The cause of PD remains unknown, but considerable evidence suggests a multifactorial etiology that involves genetic susceptibility and environmental factors (Fink, 2006). However, substantial evidence indicates that aggregation of α-synuclein is a critical step in the etiology of PD (Trojanowski & Lee, 2003).

Most cases of PD are of the late onset idiopathic type (Beyer, 2007). Evidence for an important role for α-synuclein in triggering PD also emerged when certain mutations were discovered that are associated with rare inherited autosomal dominant cases of PD. While, as indicated, familial early onset PD is caused by overexpression of α-synuclein due to duplication (Chartier-Harlin et al., 2004) or triplication (Singleton et al., 2003) of the αsynuclein gene locus, three specific point mutations have also been identified: A53T in a large kindred of Italian and Greek origin (Polymeropoulos et al., 1997); A30P in a German family (Kruger et al., 1998); and E46K in a Spanish family (Zarranz et al., 2004).

### **4. Alpha-synuclein**

88 Etiology and Pathophysiology of Parkinson's Disease

of a protein, lack specific tertiary structure and can be composed of an ensemble of conformations (Fink, 2005). Intrinsically unstructured proteins are frequently involved in important regulatory functions in the cell, and the lack of intrinsic structure is in many cases removed when the protein binds to its target molecule. Some functional advantages of these proteins might be an ability to bind to several different targets, the precise control of their binding thermodynamics, and their involvement in cell-cycle control and both transcriptional and translational regulation (Wright & Dyson, 1999). Gunasekaran et al. proposed that disordered proteins provide a simple solution to the need for large intermolecular interfaces while maintaining smaller proteins, and hence a smaller genome and a smaller cell size. For monomeric proteins with extensive intermolecular interfaces, such proteins would need to be 2-3-fold larger, and this would either increase intracellular crowding or enlarge the size of the cell by some 15% to 30%, owing to the increase in the

Protein-conformation-dependent toxicity is an emerging theme in neurodegenerative disorders, including the synucleinopathies (Ulrih et al., 2008). The group of synucleinopathies comprises many neurodegenerative diseases, among which the best known and most common is PD, but it also includes Lewy body dementia, multiple system atrophy, neurodegeneration with brain iron accumulation type I, diffuse Lewy body disease, and Lewy body variant of Alzheimer's disease (Arawaka et al., 1998; Gai et al., 1998; Spillantini et al., 1997; Wakabayashi et al., 1997). These are all brain amyloidoses with the common characteristic of pathological intracellular inclusions of aggregates that have α-

PD is characterized by the death of neurons that produce dopamine and are located in the *substantia nigra pars compacta* brain region (see below). This is accompanied by the appearance of Lewy bodies and Lewy neurites (Galvin et al., 1999). Lewy bodies are spherical protein inclusions that are found in the cytoplasm of surviving *substantia nigra* neurons, and they consist of a dense core surrounded by a halo of radiating fibrils of αsynuclein; they also contain a variety of other proteins. The fibrils seen in PD are structurally similar to those in the amyloid diseases, and they appear as linear rods of 5 nm to 10 nm diameter (Fink, 2006). PD affects more than 1% of the population over 65 years of age (Goedert, 2001), and typical symptoms include tremor, slow movements, fine motor difficulties, and loss of postural reflexes (Jankovic, 2008). The cause of PD remains unknown, but considerable evidence suggests a multifactorial etiology that involves genetic susceptibility and environmental factors (Fink, 2006). However, substantial evidence indicates that aggregation of α-synuclein is a critical step in the etiology of PD (Trojanowski

Most cases of PD are of the late onset idiopathic type (Beyer, 2007). Evidence for an important role for α-synuclein in triggering PD also emerged when certain mutations were discovered that are associated with rare inherited autosomal dominant cases of PD. While, as indicated, familial early onset PD is caused by overexpression of α-synuclein due to duplication (Chartier-Harlin et al., 2004) or triplication (Singleton et al., 2003) of the αsynuclein gene locus, three specific point mutations have also been identified: A53T in a large kindred of Italian and Greek origin (Polymeropoulos et al., 1997); A30P in a German

family (Kruger et al., 1998); and E46K in a Spanish family (Zarranz et al., 2004).

synuclein as the key component (Spillantini et al., 1997; Wakabayashi et al., 1997).

sequence length (Gunasekaran et al., 2003).

**3. Synucleinopathies** 

& Lee, 2003).

In brain homogenates, α-synuclein represents 0.5% to 1% of the total protein (Iwai et al., 1995). Northern blotting and *in-situ* hybridization in human and mice have show relatively high expression of α-synuclein in a restricted number of brain regions, one of which is the *substantia nigra* (Abeliovich et al., 2000; Lavedan, 1998). Here, α-synuclein is localized in the presynaptic terminals (George et al., 1995; Iwai et al., 1995), with about 15% found in the membrane fraction (Lee et al., 2002); after synaptosomal lysis, α-synuclein is in the soluble fraction (Iwai et al., 1995).

Although the normal physiological function of α-synuclein remains unknown, it appears to be involved in maintenance of the synaptic vesicle reserve pool of the brain (Davidson et al., 1998; Fortin et al., 2004; Iwai et al., 1995; Nuscher et al., 2004). However, other roles for αsynuclein have been considered: roles in lipid metabolism and transport (Scherzer et al., 2003; Sharon et al., 2001; Willingham et al., 2003), vesicle docking at the membrane (Larsen et al., 2006), exocytosis (Srivastava et al., 2007), lipid organisation (Madine et al., 2006) and prevention of oxidation of unsaturated lipids (Zhu et al., 2006). To date, no conclusive evidence showing the precise role of α-synuclein in cell physiology has been provided.

#### **4.1 Primary sequence**

Alpha-synuclein is a small (140 amino acid; 14 kDa) highly acidic protein (Figure 1), and it is intrinsically disordered under physiological conditions *in vitro* (Bisaglia et al., 2009; Fink, 2006). The first 89 residues are composed almost entirely of seven 11-amino-acid imperfect repeats, with a consensus sequence of KTKEGV (George et al., 1995). This strongly resembles sequence motifs found in exchangeable apolipoproteins, which are believed to constitute amphipathic helical lipid-binding domains (Segrest et al., 1992). This 11-residue periodicity is broken in one point by the insertion of four uncharged amino acids, separating units 4 and 5. There are no Cys or Trp residues in the α-synuclein sequence (George et al., 1995).

The structure of α-synuclein can be divided into three regions (Figure 1). The N-terminal domain (residues 1-60) is positively charged and contains five of the imperfect repeats (Fink, 2006; George et al., 1995). The sequence 61-95 is the most hydrophobic portion of the protein, and this was originally described as the "non-amyloid-beta component" (NAC) of Alzheimer´s disease plaques (Takeda et al., 1998). Several studies have defined this region as responsible for α-synuclein aggregation and β-sheet formation (Bodles et al., 2001; Giasson et al., 2001). The homologous β-synuclein, which is distinct from α-synuclein by the absence of the central hydrophobic sequence, is much less prone to self-aggregation. The interaction between β-synuclein and α-synuclein has been argued to inhibit aggregation (Park & Lansbury, 2003). The highly acidic C-terminal domain of α-synuclein is rich in Pro and acidic residues, with a predominance of Glu residues over Asp (George et al., 1995). This domain contains three of the four Tyr residues, at positions 125, 133 and 136; the fourth Tyr residue is at position 39. It has been shown that monomeric α-synuclein has a more compact structure than expected for a completely unfolded polypeptide, and this compactness has been linked to its inhibition of fibril formation due to burial of the hydrophobic NAC domain (Bertoncini et al., 2005; Dedmon et al., 2005). In addition, it has been shown that the 1–102 and 1–110 C-terminal-truncated α-synuclein fragments, but not that of 1–120, are efficient in the promotion of α-synuclein aggregation. The negatively charged 104, 105, 114 and 115 residues in the C-terminus have been suggested to be responsible for reduced αsynuclein aggregation and a lack of seeding of wild-type α-synuclein (Murray et al., 2003).

Alpha-Synuclein Interactions with Membranes 91

which is probably mediated by Met116, Val118, Tyr125 and Met127. Within the C-terminal domain, residues 120-130 contact residues 105-115, and the region around residue 120 also interacts with the N-terminus around residue 20. These long-range interactions that stabilize the monomeric conformations of α-synuclein also inhibit its oligomerization and aggregation. The autoinhibitory conformations fluctuate in the range of nanoseconds to microseconds (Bertoncini et al., 2005). Consistent with this, small-angle X-ray scattering analysis has shown that the radius of gyration, which is used to describe the dimensions of polypeptide chain, is ~40 Å with native α-synuclein, which is much larger than that predicted for a folded globular protein of 140 residues (15 Å), although it is significantly

Using an atomic-force-microscopy-based single-molecule mechanical unfolding methodology, Sandal et al. (2008) studied the α-synuclein conformation equilibrium under various conditions. Their method allowed the measuring of the force required for unfolding a single protein molecule. It was thus possible to detect conformers with a lifetime that was longer than 10-3 s, which due to their longevity, might be the most biologically relevant structures. In 10 mM TRIS/HCl buffer solution at pH 7.5, the α-synuclein secondary

*In-vitro* studies of recombinant α-synuclein have demonstrated that purified α-synuclein forms fibril aggregates that resemble those found in Lewy bodies (Serpell et al., 2000). In contrast to its helical secondary structure in the presence of lipids, α-synuclein monomers form soluble oligomers (sometimes referred to as protofibrils) that can undergo a conformational change from disordered to predominantly beta secondary structure. These oligomers can assemble and form insoluble fibrils, which are found in inclusion bodies,

Extensive data suggest that the first step of fibrillogenesis is the formation of a partially folded intermediate that promotes self-association of α-synuclein and formation of various oligomeric species (Uversky et al., 2001). Factors that increase the concentrations of these intermediates will favor aggregation (Fink, 2006). Protein aggregation and the kinetics of fibril formation typically appear sigmoidal, and they are usually attributed to a nucleated polymerization process in which the initial lag phase corresponds to the requirement for the formation of critical nuclei; the subsequent exponential growth phase corresponds to fibril elongation, and the final plateau is ascribed to the exhaustion of the soluble monomers and

All three of the above-mentioned PD-related point mutations have been shown to accelerate α-synuclein aggregation *in vitro* (Uversky, 2007). The A53T and A30P point mutations both accelerate oligomer formation, although only A53T readily forms large amyloid fibrils (Conway et al., 2000). E46K appears to be even more effective in the promotion of aggregate

As fibril formation of native α-synuclein occurs in most cases of synucleinopathies, most studies have deal with the mechanisms that trigger this process. Both physical and chemical factors have been demonstrated to affect this aggregation process (Lundvig et al., 2008). As mentioned above, it is believed that interactions between the C-terminus and the central portion of α-synuclein can prevent or minimize its aggregation/fibril formation. As the majority of hydrophobic interactions in the C-terminal of α-synuclein arise through its three

smaller than that of a fully unfolded random coil (52 Å) (Uversky et al., 2001).

structure contains a random coil (38.2%) and β-structure (7.3%) (Sandal et al., 2008).

together with other proteins (Conway et al., 2000; Fink, 2006; Wood et al., 1999).

formation in cultured cells than these other two mutations (Pandey et al., 2006).

**5. Fibril formation** 

intermediates (Ulrih et al., 2008).

Fig. 1. Top: Amino-acid sequence of human α-synuclein. The imperfect 11-mer repeats are as indicated, with the predominant KTKEGV consensus sequences underlined. The locations of the three point mutations that have been linked to early-onset PD (A30T, E46K, A53T) are shown in bold type, and the four Tyr residues are shaded. Bottom: The α-synuclein sequence can be divided into three regions: the N-terminus adopts an α-helix upon binding to lipids, the hydrophobic NAC domain can form β-sheet structure, and the negatively charged C-terminus is unstructured.

#### **4.2 Alpha-synuclein structure under physiological conditions**

Weinreb et al. were the first to attempt to define the secondary structure of α-synuclein. Sedimentation of α-synuclein under physiological conditions is slower than for globular proteins of a similar molecular weight, suggesting an elongated structure of the native protein. Circular dichroism has demonstrated the lack of α-synuclein secondary structure in solution: 68% as random coils and less than 2% as helical content. The reminder of the protein is β-sheet, although it is difficult to quantify the β-sheet structure by circular dichroism. Fourier-transform infrared spectroscopy has confirmed that native α-synuclein is unstructured. The conformational properties of α-synuclein were not changed by heat denaturation and were independent of α-synuclein concentration, salt concentration, chemical denaturants and pH. These features prompted the conclusion that under physiological conditions, α-synuclein exists as a mixture of rapidly equilibrating extended conformers, and that it is representative of a class of natively unfolded proteins (Weinreb et al., 1996). With a slightly different isolation protocol, circular dichroism showed 9% α-helix, 35% β-sheet, and 56% random coil structure in solution (Narayanan & Scarlata, 2001).

Dedmon et al. (2005) used paramagnetic relaxation enhancement and nuclear magnetic resonance (NMR) to show interactions between different parts of the α-synuclein molecule. Some α-synuclein mutants were prepared, with the insertion of nitroxide-labeled cysteine residues, which allowed the observation of short-life-time interactions. Partial condensation of α-synuclein is driven by long-range contacts between residues 30-100 in the center of the molecule, and residues 120-140 in the C-terminal tail. It appears that this interaction can shield the NAC region (residues 61-95) from aggregation, which is the most hydrophobic part of α-synuclein (Dedmon et al., 2005). Bertoncini et al. used a similar methodology to show that the most important interaction is a hydrophobic cluster that comprises the Cterminal part of the NAC region (residues 85-95) and the C-terminus (residues 110-130),

Fig. 1. Top: Amino-acid sequence of human α-synuclein. The imperfect 11-mer repeats are as indicated, with the predominant KTKEGV consensus sequences underlined. The locations of the three point mutations that have been linked to early-onset PD (A30T, E46K, A53T) are shown in bold type, and the four Tyr residues are shaded. Bottom: The α-synuclein

sequence can be divided into three regions: the N-terminus adopts an α-helix upon binding to lipids, the hydrophobic NAC domain can form β-sheet structure, and the negatively

Weinreb et al. were the first to attempt to define the secondary structure of α-synuclein. Sedimentation of α-synuclein under physiological conditions is slower than for globular proteins of a similar molecular weight, suggesting an elongated structure of the native protein. Circular dichroism has demonstrated the lack of α-synuclein secondary structure in solution: 68% as random coils and less than 2% as helical content. The reminder of the protein is β-sheet, although it is difficult to quantify the β-sheet structure by circular dichroism. Fourier-transform infrared spectroscopy has confirmed that native α-synuclein is unstructured. The conformational properties of α-synuclein were not changed by heat denaturation and were independent of α-synuclein concentration, salt concentration, chemical denaturants and pH. These features prompted the conclusion that under physiological conditions, α-synuclein exists as a mixture of rapidly equilibrating extended conformers, and that it is representative of a class of natively unfolded proteins (Weinreb et al., 1996). With a slightly different isolation protocol, circular dichroism showed 9% α-helix, 35% β-sheet, and 56% random coil structure in solution (Narayanan & Scarlata, 2001). Dedmon et al. (2005) used paramagnetic relaxation enhancement and nuclear magnetic resonance (NMR) to show interactions between different parts of the α-synuclein molecule. Some α-synuclein mutants were prepared, with the insertion of nitroxide-labeled cysteine residues, which allowed the observation of short-life-time interactions. Partial condensation of α-synuclein is driven by long-range contacts between residues 30-100 in the center of the molecule, and residues 120-140 in the C-terminal tail. It appears that this interaction can shield the NAC region (residues 61-95) from aggregation, which is the most hydrophobic part of α-synuclein (Dedmon et al., 2005). Bertoncini et al. used a similar methodology to show that the most important interaction is a hydrophobic cluster that comprises the Cterminal part of the NAC region (residues 85-95) and the C-terminus (residues 110-130),

**4.2 Alpha-synuclein structure under physiological conditions** 

charged C-terminus is unstructured.

which is probably mediated by Met116, Val118, Tyr125 and Met127. Within the C-terminal domain, residues 120-130 contact residues 105-115, and the region around residue 120 also interacts with the N-terminus around residue 20. These long-range interactions that stabilize the monomeric conformations of α-synuclein also inhibit its oligomerization and aggregation. The autoinhibitory conformations fluctuate in the range of nanoseconds to microseconds (Bertoncini et al., 2005). Consistent with this, small-angle X-ray scattering analysis has shown that the radius of gyration, which is used to describe the dimensions of polypeptide chain, is ~40 Å with native α-synuclein, which is much larger than that predicted for a folded globular protein of 140 residues (15 Å), although it is significantly smaller than that of a fully unfolded random coil (52 Å) (Uversky et al., 2001).

Using an atomic-force-microscopy-based single-molecule mechanical unfolding methodology, Sandal et al. (2008) studied the α-synuclein conformation equilibrium under various conditions. Their method allowed the measuring of the force required for unfolding a single protein molecule. It was thus possible to detect conformers with a lifetime that was longer than 10-3 s, which due to their longevity, might be the most biologically relevant structures. In 10 mM TRIS/HCl buffer solution at pH 7.5, the α-synuclein secondary structure contains a random coil (38.2%) and β-structure (7.3%) (Sandal et al., 2008).

### **5. Fibril formation**

*In-vitro* studies of recombinant α-synuclein have demonstrated that purified α-synuclein forms fibril aggregates that resemble those found in Lewy bodies (Serpell et al., 2000). In contrast to its helical secondary structure in the presence of lipids, α-synuclein monomers form soluble oligomers (sometimes referred to as protofibrils) that can undergo a conformational change from disordered to predominantly beta secondary structure. These oligomers can assemble and form insoluble fibrils, which are found in inclusion bodies, together with other proteins (Conway et al., 2000; Fink, 2006; Wood et al., 1999).

Extensive data suggest that the first step of fibrillogenesis is the formation of a partially folded intermediate that promotes self-association of α-synuclein and formation of various oligomeric species (Uversky et al., 2001). Factors that increase the concentrations of these intermediates will favor aggregation (Fink, 2006). Protein aggregation and the kinetics of fibril formation typically appear sigmoidal, and they are usually attributed to a nucleated polymerization process in which the initial lag phase corresponds to the requirement for the formation of critical nuclei; the subsequent exponential growth phase corresponds to fibril elongation, and the final plateau is ascribed to the exhaustion of the soluble monomers and intermediates (Ulrih et al., 2008).

All three of the above-mentioned PD-related point mutations have been shown to accelerate α-synuclein aggregation *in vitro* (Uversky, 2007). The A53T and A30P point mutations both accelerate oligomer formation, although only A53T readily forms large amyloid fibrils (Conway et al., 2000). E46K appears to be even more effective in the promotion of aggregate formation in cultured cells than these other two mutations (Pandey et al., 2006).

As fibril formation of native α-synuclein occurs in most cases of synucleinopathies, most studies have deal with the mechanisms that trigger this process. Both physical and chemical factors have been demonstrated to affect this aggregation process (Lundvig et al., 2008).

As mentioned above, it is believed that interactions between the C-terminus and the central portion of α-synuclein can prevent or minimize its aggregation/fibril formation. As the majority of hydrophobic interactions in the C-terminal of α-synuclein arise through its three

Alpha-Synuclein Interactions with Membranes 93

Data that have documented the tendency of α-synuclein to colocalize with synaptic vesicles *in vivo* (Maroteaux et al., 1988) and the presence of the 11-residue repeated domains in a pattern similar to that found in the apolipoproteins (George et al., 1995) sparked a series of studies to determine the α-synuclein lipid-binding ability. Alpha-synuclein interactions with membranes have been found to be one of the most contentious areas regarding this protein (Fink, 2006), as there have been numerous reports on sometimes completely contradicting results, and as there might be major differences between the situation *in vivo* and *in vitro*. Also, membranes have been reported to both accelerate (Lee et al., 2002) and inhibit (Narayanan & Scarlata, 2001; Zhu & Fink, 2003) α-synuclein fibril formation, so this probably reflects the varying conditions used in the different studies (Zhu & Fink, 2003). All three of these α-synuclein mutations occur within the N-terminus, which is responsible for its membrane binding, hence suggesting an effect on membrane interactions (Fortin et al., 2010). The A30P α-synuclein mutation, and to a lesser extent that of A53T, disrupts the helical structure of α-synuclein (Bussell & Eliezer, 2001), although it does not significantly affect the structure of membrane-associated α-synuclein (Bussell & Eliezer, 2004). The E46K α-synuclein mutant binds to negatively charged vesicles with a higher protein/lipid ratio than does wild-type α-synuclein (Choi et al., 2004), while A30P affects the localization, and

presumably the membrane binding, of α-synuclein *in vivo* (Fortin et al., 2010).

It is generally accepted that α-synuclein preferentially interacts with small unilamellar vesicles (SUVs) containing negatively charged head groups (Davidson et al., 1998; Jo et al., 2000) or interfacial packing defects (Kamp & Beyer, 2006; Nuscher et al., 2004), and that upon SUV binding, α-synuclein undergoes a conformational transition from an intrinsically disordered state to an α-helical structure (Davidson et al., 1998; Jo et al., 2000; Nuscher et al., 2004). Various combinations of charged and uncharged lipids have been used in these studies. These negatively charged acidic phospholipids include phosphatidylglycerol (PG), phosphatidic acid (PA), phosphatidylserine (PS) and phosphatidylinositol (PI), while the uncharged, neutral lipids commonly used include phosphatidylcholine (PC) and

The interactions of α-synuclein with membranes have been shown to affect the properties of both the protein and the membranes, and both electrostatic and hydrophobic interactions are important in the protein-bilayer association (Zhu et al., 2003). There are several factors that are believed to have central roles in modulation of the binding equilibrium of αsynuclein to membranes, including chemical properties of the membranes (Davidson et al., 1998; Jo et al., 2000), ionic strength of the solution (Davidson et al., 1998; Zhu et al., 2003), vesicle size, or more precisely, the curvature of the phospholipid surface (Davidson et al., 1998; Narayanan & Scarlata, 2001; Rhoades et al., 2006), and mass ratio of α-synuclein to the lipids (Zhu & Fink, 2003). Here, an overview of some of the more important findings

Davidson et al. were the first to demonstrate that α-synuclein binds only to acidic phospholipids and preferentially to vesicles with smaller diameters. Circular dichroism spectroscopy was used to determine the effects of this lipid binding on the secondary structure of α-synuclein. In buffer solution, α-synuclein is mainly unstructured, with less than 3% of the structure as α-helix. Incubation of α-synuclein with vesicles made of a

**6.2 Lipid and membrane selectivity** 

**6.2.1 Membrane interactions** *in vitro* 

phosphatidylethanolamine (PE) (Valenzuela, 2007).

regarding the lipid specificities of α-synuclein are given.

Tyr, we created Tyr to Ala mutants to examine the importance of these Tyr residues in fibril formation of α-synuclein *in vitro*. This was completely inhibited in the timescale over which measurements were made (70 hours) when the three C-terminal Tyr were replaced with Ala. In addition, substitution of Tyr133 by Ala also inhibitted fibril formation, whereas the individual Y125A and Y136A mutants showed limited inhibition. Replacement of Tyr39 by Ala also resulted in substantial inhibition of fibril formation. Structural analysis showed that the Y133A α-synuclein mutant has a substantially different conformation, as it is rich in αhelical secondary structure, as compared with wild-type α-synuclein and its other mutants. However, no formation of any tertiary structure was seen, as judged from the near-UV circular-dichroism spectra. These observations suggest that the long-range intramolecular interactions between the N-terminal and C-terminal of α-synuclein are crucial for the process of fibril formation (Ulrih et al., 2008).
