Transition of intrinsically unfolded α-synuclein into the fibrillar state characterized by NMR spectroscopy.

Proteins fold into appropriate configuration, called native structure, in order to achieve its cellular function. A protein with nonnative structure induces malfunction and causes the relevant disease. Such protein misfolding has been revealed as a common pathogenic process in many neurodegenerative diseases like Alzheimer s and Parkinson s disease. In Parkinson s disease (PD), a protein called α-synuclein (αS) is found a major component of Lewy body, a proteinaceous aggregate with amyloid fibril form of αS. Considering its character as an intrinsically unfolded protein, the overall change of conformation during PD is quite attractive for biophysicist to understand protein folding and misfolding, and the detailed information can lead to a therapeutic achievement for the treatment of PD.In this thesis, I have investigated the transition of an intrinsically unfolded αS into amyloid fibril with NMR spectroscopy and various biophysical methods. Natively unfolded proteins play key roles in normal and pathological biochemical processes. When confined in weakly aligning media, natively unfolded proteins such as αS display surprisingly variable NMR dipolar couplings as a function of position along the chain, suggesting the presence of residual secondary or tertiary structure. In Chapter 3, it is shown that that the variation of NMR dipolar couplings and heteronuclear relaxation rates in αS closely follows the variations of the bulkiness of amino acids along the polypeptide chain. The results demonstrate that the bulkiness of amino acids defines the local conformations and dynamics of αS and other natively unfolded proteins. Deviations from this random coil behavior can provide insight into residual secondary structure and long-range transient interactions in unfolded proteins. The transition from natively unfold conformation into amyloid fibril starts with a change in monomeric conformation. Previously it has been shown that αS adopts an autoinhibitory conformation in physiological condition. Changes in environmental factors like low pH, molecular crowding agents, high temperature, and/or high salt concentration accelerate αS aggregation. In these conditions, αS may transform into an aggregation-prone, partially folded intermediate, and such dimensional change at pH 3 was observed with CD, SAXS and fluorescence. As described in Chapter 4, NMR spectroscopy was applied to address such conformational change in atomic resolution. Chapter 5 describes the distinction between wt and A30P, a genetic mutant of αS, amyloid fibril core. Here we took the advantage of HR-MAS NMR spectroscopy to detect flexible regions in amyloid fibrils. Together with hydrogen/deuterium (H/D) exchange experiments, the arrangement of β-strands and loops in fibrillar core region is shown. Longer amyloid fibril core region for A30P is observed compared to wt amyloid fibril; the reason for the difference, however, should be addressed. Chapter 6 consists of a brief description about the conformation of the αS oligomer derived from amyloid fibril in supercooled aqueous solution. Electron microscopy (EM) and atomic force microscopy (AFM) reveals spherical conformation with variation in diameter and height. This study, combined with physiological investigation, would lead better understanding of the intermediates in amyloid fibril formation.