Abstract
Parkinson's disease (PD) is pathologically characterized by the presence of Lewy bodies (LBs) in dopaminergic neurons of the substantia nigra. These intracellular inclusions are largely composed of misfolded α-synuclein (AS), a neuronal protein that is abundant in the vertebrate brain. Point mutations in AS are associated with rare, early-onset forms of PD, although aggregation of the wild-type (WT) protein is observed in the more common sporadic forms of the disease. Here, we employed multidimensional solid-state NMR experiments to assess A53T and E46K mutant fibrils, in comparison to our recent description of WT AS fibrils. We made de novo chemical shift assignments for the mutants, and used these chemical shifts to empirically determine secondary structures. We observe significant perturbations in secondary structure throughout the fibril core for the E46K fibril, while the A53T fibril exhibits more localized perturbations near the mutation site. Overall, these results demonstrate that the secondary structure of A53T has some small differences from the WT and the secondary structure of E46K has significant differences, which may alter the overall structural arrangement of the fibrils.
Highlights
Aggregation of the neuronal protein alpha-synuclein (AS) is strongly implicated in both sporadic and familial forms of Parkinson’s disease (PD), and with a variety of other neurodegenerative diseases collectively termed synucleinopathies
In summary, we have site- compared the structures of AS fibrils from the E46K and A53T early-onset PD mutants to the WT using magic angle spinning (MAS) solid-state nuclear magnetic resonance (SSNMR)
Our results demonstrate minor sitespecific perturbations near the mutation site, the 60 s and 80 s for A53T and large chemical shift perturbations within the entire core for E46K
Summary
Aggregation of the neuronal protein alpha-synuclein (AS) is strongly implicated in both sporadic and familial forms of Parkinson’s disease (PD), and with a variety of other neurodegenerative diseases collectively termed synucleinopathies. The mutant proteins differ in their fibrillation kinetics in vitro; A53T and E46K fibrillize more rapidly than WT [7,8], while A30P fibrillizes more slowly, preferentially adopting a protofibrillar intermediate state [9]. To better resolve the potential similarities and differences among fibrils comprised of mutant and WT AS, we have employed magic angle spinning (MAS) solid-state nuclear magnetic resonance (SSNMR) spectroscopy. This technique is uniquely suited to structural studies of amyloid proteins, such as fibrillar AS [10,11,12,13], which are insoluble and do not form Xray diffraction-quality crystals. We used MAS SSNMR to demonstrate that the A30P mutation does not affect the secondary structure of AS fibrils when compared sitespecifically with WT AS fibrils [19]
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