Exploring the disordered state and folding of alpha-synuclein using single molecule fluorescence spectroscopy [electronic resource].

9781303299803

1 online resource (213 p.)

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Parkinson's disease is a prevalent neurodegenerative motor disorder characterized by deposits in neurons of amyloid fibril plaques called Lewy bodies. The protein alpha-synuclein is a key component in the development of Parkinson's disease where it is found as amyloid fibrils in Lewy bodies. Moreover, it is believed to directly mediate cell death leading to the disease. Alpha-synuclein is a small protein that is intrinsically disordered in solution but can adopt a variety of secondary and tertiary structures depending upon its environment or binding partner. Understanding how this disordered protein transitions to oligomeric states and fibrils found in disease is critical to understanding its role in the pathogenesis of Parkinson's disease. To gain detailed structural insight into the pathways that alpha-synuclein proceeds through from the disordered state to structured, potentially toxic oligomeric states, single molecule Forster resonance energy transfer was primarily used to measure the conformational state of alphasynuclein disordered in solution, bound to membranes, and in a variety of aggregation-prone states. By examining the conformational changes from the disordered state to an aggregation-prone state, mechanistic conclusions about the initiation of aggregation from these states were drawn. These in vitro biophysical studies are critical to assess how the in vivo state of the protein might be different and how the crowded intracellular environment might change the properties of alpha-synuclein. The biologically relevant form of alpha-synuclein is N-terminally acetylated, and the effect of this modification has not been examined in depth. In vitro examination of the N-terminally acetylated alpha-synuclein has shown that while the modification does not appear to change its solution conformational state substantially, it significantly strengthens alpha-synuclein interactions with lipid membranes. This interaction is thought to be relevant to the native function of alpha-synuclein. Finally by performing intracellular fluorescence correlation spectroscopy experiments the effect of the mammalian cytoplasmic environment on the general structural features of alpha-synuclein was assessed. The protein appears to maintain disorder in the cytoplasm, despite substantial macromolecular crowding and presence of potential binding partners. These findings suggest that in vitro studies on disordered alpha-synuclein indeed capture relevant conformational parameters of the protein. However, the biologically relevant N-terminal acetylation of alpha-synuclein significantly impacts the behavior of the protein, especially its membrane binding affinity, and should thus be considered a critical feature of alpha-synuclein and relevant to future studies of its biophysical properties.

Books / Online / Dissertations & Theses

English

July 25, 2014

Thesis (Ph.D.)--Yale University, 2013.

Yale University. Molecular Biophysics and Biochemistry.