Different Structural Conformers of Monomeric α-Synuclein Identified after Lyophilizing and Freezing

Analytical Chemistry
Amberley D StephensGabriele S Kaminski Schierle

Abstract

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 differ...Continue Reading

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Citations

Dec 5, 2019·Communications Biology·Anna Y BelorusovaEva-Lotte Lindstedt
Jun 6, 2020·Nature Communications·Amberley D StephensGabriele S Kaminski Schierle
Aug 23, 2020·Biomolecules·Line K SkaanningBirthe B Kragelund
Jan 12, 2020·Communications Biology·Anna Y BelorusovaEva-Lotte Lindstedt
Jun 10, 2021·Methods : a Companion to Methods in Enzymology·Takahiro Watanabe-Nakayama, Kenjiro Ono
Sep 23, 2020·ACS Chemical Neuroscience·Amberley D StephensGabriele S Kaminski Schierle
Nov 26, 2020·Biochemistry·Amberley D StephensGabriele S Kaminski Schierle
Aug 14, 2020·Journal of Medicinal Chemistry·Anna BelorusovaNatacha Rochel
Sep 11, 2021·Chemical Reviews·Hannah M BrittKonstantinos Thalassinos

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Methods Mentioned

BETA
ion exchange chromatography
size
dynamic
atomic force microscopy
Size Exclusion Chromatography
Assay
electrophoresis
light scattering
X-ray
AFM

Software Mentioned

NanoScope Analysis
ProteinLynx Global Server
MatLab
ICY
DynamX
FIJI
Excel
ProteinLynx Global Server - PLGS

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