Different Structural Conformers of Monomeric α‑Synuclein
Identified after Lyophilizing and Freezing
Version 2 2018-07-13, 17:20
Version 1 2018-05-22, 17:48
Posted on 2018-07-13 - 17:20
Understanding the
mechanisms behind amyloid protein aggregation
in diseases, such as Parkinson’s and Alzheimer’s disease,
is often hampered by the reproducibility of in vitro assays. Yet,
understanding the basic mechanisms of protein misfolding is essential
for the development of novel therapeutic strategies. We show here,
that for the amyloid protein α-synuclein (aSyn), a protein involved
in Parkinson’s disease (PD), chromatographic buffers and storage
conditions can significantly interfere with the overall structure
of the protein and thus affect protein aggregation kinetics. We apply
several biophysical and biochemical methods, including size exclusion
chromatography (SEC), dynamic light scattering (DLS), and atomic force
microscopy (AFM), to characterize the high molecular weight conformers
formed during protein purification and storage. We further apply hydrogen/deuterium-exchange
mass spectrometry (HDX-MS) to characterize the monomeric form of aSyn
and reveal a thus far unknown structural component of aSyn at the
C-terminus of the protein. Furthermore, lyophilizing the protein greatly
affected the overall structure of this monomeric conformer. We conclude
from this study that structural polymorphism may occur under different
storage conditions, but knowing the structure of the majority of the
protein at the start of each experiment, as well as the factors that
may influence it, may pave the way to an improved understanding of
the mechanism leading to aSyn pathology in PD.
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Stephens, Amberley
D.; Nespovitaya, Nadezhda; Zacharopoulou, Maria; Kaminski, Clemens F.; Phillips, Jonathan J.; S. Kaminski Schierle, Gabriele (2018). Different Structural Conformers of Monomeric α‑Synuclein
Identified after Lyophilizing and Freezing. ACS Publications. Collection. https://doi.org/10.1021/acs.analchem.8b01264
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AUTHORS (6)
AS
Amberley
D. Stephens
NN
Nadezhda Nespovitaya
MZ
Maria Zacharopoulou
CK
Clemens F. Kaminski
JP
Jonathan J. Phillips
GS
Gabriele S. Kaminski Schierle