Structure, conformational dynamics and formation of large amyloids: the case of alpha-synuclein fibrils

Nuclear magnetic resonance (NMR);magic-angle spinning (MAS);protein structure;alpha-synuclein (AS)

Comellas Canal, Gemma

关键词: Nuclear magnetic resonance (NMR);    magic-angle spinning (MAS);    protein structure;    alpha-synuclein (AS);   

Others  :  https://www.ideals.illinois.edu/bitstream/handle/2142/31996/ComellasCanal_Gemma.pdf?sequence=1&isAllowed=y

美国|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

In my thesis, I have focused on new methodology development combined with state-of-the-art solid-state nuclear magnetic resonance (NMR) experiments with scanning transmission electron microscopy (STEM) to obtain atomic level structural information of the alpha-synuclein (AS) fibrils and the mechanism of their formation; We first investigated the effect of protein deuteration and 1H decoupling optimization to maximize the resolution and sensitivity of biomolecular solid-state NMR; We then applied state-of-the-art solid-state NMR experiments to do a detailed structural characterization and conformational dynamics of AS fibrils using improved sample preparation and labeling schemes; These results show that the core of the fibrils extends for about 70 residues with a repeated secondary structure motif; Additionally, it demonstrates that the three mutation sites (A30P, E46K, A53T) are located in structured regions of the fibrils; Upon mutation, we have shown that the structure suffers major and minor perturbations by E46K and A53T, respectively; while the structure is unaltered by A30P; The fibril formation has also been investigated by capturing the transition from α-helical to β-sheet at the atomic level using solid-state NMR; Additionally, to investigate the AS fold, the mass-per-length (MPL) measurement of the fibrils has been obtained using STEM that together with solid-state NMR restraints have been used to propose possible models of how the fibrils arrange; Finally, initial results for solving 3D high-resolution structures of large proteins with new computational methods have been investigated.