PLoS One. 2016 Apr 20;11(4):e0151447. doi: 10.1371/journal.pone.0151447.

Dynamical Behavior of Human α-Synuclein Studied by Quasielastic Neutron Scattering.

Fujiwara S1, Araki K2, Matsuo T1, Yagi H3, Yamada T4, Shibata K5, Mochizuki H2.

  • 1Quantum Beam Science Center, Japan Atomic Energy Agency, Tokai, Ibaraki, Japan.
  • 2Department of Neurology, Osaka University Graduate School of Medicine, Suita, Osaka, Japan.
  • 3Center for Research on Green Sustainable Chemistry, Tottori University, Tottori, Japan.
  • 4Research Center for Neutron Science and Technology, CROSS-Tokai, Tokai, Ibaraki, Japan.
  • 5Neutron Science Section, J-PARC Center, Tokai, Ibaraki, Japan.

 

Abstract

α-synuclein (αSyn) is a protein consisting of 140 amino acid residues and is abundant in the presynaptic nerve terminals in the brain. Although its precise function is unknown, the filamentous aggregates (amyloid fibrils) of αSyn have been shown to be involved in the pathogenesis of Parkinson’s disease, which is a progressive neurodegenerative disorder. To understand the pathogenesis mechanism of this disease, the mechanism of the amyloid fibril formation of αSyn must be elucidated. Purified αSyn from bacterial expression is monomeric but intrinsically disordered in solution and forms amyloid fibrils under various conditions. As a first step toward elucidating the mechanism of the fibril formation of αSyn, we investigated dynamical behavior of the purified αSyn in the monomeric state and the fibril state using quasielastic neutron scattering (QENS). We prepared the solution sample of 9.5 mg/ml purified αSyn, and that of 46 mg/ml αSyn in the fibril state, both at pD 7.4 in D2O. The QENS experiments on these samples were performed using the near-backscattering spectrometer, BL02 (DNA), at the Materials and Life Science Facility at the Japan Accelerator Research Complex, Japan. Analysis of the QENS spectra obtained shows that diffusive global motions are observed in the monomeric state but largely suppressed in the fibril state. However, the amplitude of the side chain motion is shown to be larger in the fibril state than in the monomeric state. This implies that significant solvent space exists within the fibrils, which is attributed to the αSyn molecules within the fibrils having a distribution of conformations. The larger amplitude of the side chain motion in the fibril state than in the monomeric state implies that the fibril state is entropically favorable.

PMID: 27097022

 

Supplementary

a-synuclein (αSyn) is involved in the pathogenesis of Parkinson’s disease (PD), as filamentous aggregates of αSyn are often found to be a major component of the protein deposits in the brain of patients with PD. These aggregates (amyloid fibrils) and/or the intermediate structures toward the mature fibrils of αSyn are thought to be related to the pathogenesis of PD. How αSyn can be toxic is a subject of intense studies. Elucidating the mechanism of the amyloid fibril formation of αSyn is thus important for understanding the mechanism of the pathogenesis of PD.

 

Formation of amyloid fibrils in general involves partial unfolding of the proteins and the subsequent growth of the protofilaments and the mature fibrils (1). Involvement of the partial unfolding implies that the dynamics of the proteins plays an important role in the process of amyloid fibril formation. Investigating the protein dynamics during fibril formation is therefore important for elucidating the mechanism of this process.

 

Proteins show a hierarchy of the dynamics, from local fluctuations of the side chains and loop motions at ps-to-ns time scales through domain motions at ms scales to conformational changes of proteins at ms scales. Elucidating the role of the proteins dynamics requires a thorough understanding of this hierarchy. In particular, elucidation of the relationship between the fluctuations at the ps-to-ns scale and the conformational changes is important since it is well known that inhibiting the dynamics at the ps-to-ns scale suppresses the activity of the proteins (2).

 

When bacterially expressed in vitro, αSyn is intrinsically disordered in solution (3). This recombinant protein forms amyloid fibrils, depending on the solution conditions under which the proteins are dispersed. Elucidating possible variations in the dynamics of αSyn during the fibrillization process should provide insights into how the fibrils form. In this study, we investigated the dynamic properties of αSyn in the monomeric and fibril states, using the purified protein expressed in bacteria.

 

We employed neutron scattering for this purpose. Neutron scattering provides a unique tool to directly measure the dynamics of proteins at ps-to-ns time scales and ångstrom length scales (4). In particular, incoherent quasielastic neutron scattering (QENS) provides information on the average motion of the entire protein. This technique has been applied to many protein systems, including the photosynthesis systems, bacteriorhodopsin, hemoglobin, tau-protein, and amyloid b peptide (see, for example, the references cited in this paper). We carried out the QENS experiments of the solution samples of αSyn in the monomeric and fibril states, and compared the dynamic properties of αSyn in these states. The QENS experiments were carried out using the near-backscattering spectrometer, BL02 (DNA), at the Materials and Life Science Facility at the Japan Accelerator Research Complex, which is one of the powerful neutron sources in the world.

 

QENS is unique and powerful because this technique provides information on both the rates and amplitudes of the internal motions of the proteins. Moreover, for solution samples, the information on the global motions of the entire proteins and the local motions such as the side chain motions within the proteins can be obtained. Figure 1 shows the diffusion coefficients (Dapp) of αSyn in the monomeric and fibril states obtained from analysis of the QENS spectra. It is shown clearly that diffusive global motions are observed in the monomeric state but largely suppressed in the fibril state. Note that Dapp has significant differences to the translational diffusion coefficients evaluated from dynamic light scattering, suggesting that Dapp obtained from QENS contains contributions from rotational diffusion and segmental motions within the protein.

 

 

fig1

Figure 1.  Dapp of αSyn in the monomeric (■) and the fibril () states evaluated from the QENS spectra. The translational diffusion coefficients of αSyn in the monomeric state determined by dynamic light scattering () are also shown.

 

Analysis of the spectra also shows that the residence time of the local motions within the protein is larger in the fibril state than in the monomeric state, indicating that the rates of the atomic motions within the protein is faster in the monomeric state than in the fibril state. Information on the amplitudes of these local motions was also obtained. Figure 2 shows the radius of the confined sphere in which the atoms undergo diffusive motions. Surprisingly, this radius was found to be larger in the fibril state than in the monomeric state. This is contrary to the expectation that aggregation reduces the motions of the molecules in the aggregates. This indicates that the amplitudes of the local motions are larger in the fibril state than in the monomeric state.

 

 

fig2

Figure 2. Radii of the confined spheres of αSyn in the monomeric (■) and the fibril () states.

 

This implies that significant solvent space exists within the fibrils, which is attributable to the αSyn molecules within the fibrils having a distribution of conformations. The larger amplitudes of the local motions in the fibril state imply that the degree of the conformational freedom is larger in the fibril state than in the monomeric state, i.e., the fibril state is entropically favorable. This then implies that the fibril formation is entropically driven. This has an implication that once a potential barrier is overcome, fibril formation proceeds naturally. The rate-limiting step of the fibril formation would then be the kinetics involved to overcome the potential barrier.

 

As polymerized proteins formed by a “normal” mechanism such as F-actin, which is formed by helical association of the actin molecules and plays important roles in cell motility, shows reduced dynamics (5,6), ordered structures usually have lower flexibility. The enhanced dynamics in amyloid fibrils contradicts this usually accepted paradigm. Since amyloid fibrils form in a number of proteins that are not related to each other structurally or functionally at all (7), this “abnormal” dynamics may well be relevant to the general mechanism of amyloid fibril formation.

 

References:

(1)     Dobson CM. Protein folding and misfolding. Nature. 2003; 426: 884-890.

(2)     Zaccai G. How soft is a protein? A protein dynamics force constant measured by neutron scattering. Science. 2000; 288: 1604-1607.

(3)     Weinreb PH, Zhen W, Poon AW, Conway KA, Lansbury PT. NACP, a protein implicated in Alzheimer’s disease and learning, is natively unfolded. Biochemistry. 1996; 35: 13709-13715.

(4)     Smith JC. Protein dynamics: comparison of simulations with inelastic neutron scattering experiments. Q Rev Biophys. 1991; 24: 227-291.

(5)     Fujiwara S, Plazanet M, Matsumoto F, Oda T. Differences in internal dynamics of actin under different structural dtates detected by neutron scattering. Biophys J. 2008; 94: 4880-4889.

(6)     Fujiwara S, Plazanet M, Matsumoto F, Oda T. Internal motions of actin characterized by quasielastic neutron scattering. Eur Biophys J. 2011; 40: 661-671.

(7)     Greenwald J, Riek R. Biology of amyloid: Structure, function, and regulation. Structure. 2010; 18: 1244-1260.

 

Contact:

Satoru Fujiwara, Ph.D.

Quantum Beam Science Research Directorate

National Institutes for Quantum and Radiological Science and Technology

2-4 Shirakata, Tokai, Naka-Gun, Ibaraki, 319-1106, Japan.

E-mail address: fujiwara.satoru@qst.go.jp

 

 

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