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Different Structural Conformers of Monomeric α-Synuclein Identified after Lyophilizing and Freezing
[Image: see text] 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 n...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
American
Chemical
Society
2018
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6047843/ https://www.ncbi.nlm.nih.gov/pubmed/29750859 http://dx.doi.org/10.1021/acs.analchem.8b01264 |
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author | Stephens, Amberley D. Nespovitaya, Nadezhda Zacharopoulou, Maria Kaminski, Clemens F. Phillips, Jonathan J. Kaminski Schierle, Gabriele S. |
author_facet | Stephens, Amberley D. Nespovitaya, Nadezhda Zacharopoulou, Maria Kaminski, Clemens F. Phillips, Jonathan J. Kaminski Schierle, Gabriele S. |
author_sort | Stephens, Amberley D. |
collection | PubMed |
description | [Image: see text] 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. |
format | Online Article Text |
id | pubmed-6047843 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2018 |
publisher | American
Chemical
Society |
record_format | MEDLINE/PubMed |
spelling | pubmed-60478432018-07-17 Different Structural Conformers of Monomeric α-Synuclein Identified after Lyophilizing and Freezing Stephens, Amberley D. Nespovitaya, Nadezhda Zacharopoulou, Maria Kaminski, Clemens F. Phillips, Jonathan J. Kaminski Schierle, Gabriele S. Anal Chem [Image: see text] 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. American Chemical Society 2018-05-11 2018-06-05 /pmc/articles/PMC6047843/ /pubmed/29750859 http://dx.doi.org/10.1021/acs.analchem.8b01264 Text en Copyright © 2018 American Chemical Society This is an open access article published under a Creative Commons Attribution (CC-BY) License (http://pubs.acs.org/page/policy/authorchoice_ccby_termsofuse.html) , which permits unrestricted use, distribution and reproduction in any medium, provided the author and source are cited. |
spellingShingle | Stephens, Amberley D. Nespovitaya, Nadezhda Zacharopoulou, Maria Kaminski, Clemens F. Phillips, Jonathan J. Kaminski Schierle, Gabriele S. Different Structural Conformers of Monomeric α-Synuclein Identified after Lyophilizing and Freezing |
title | Different Structural Conformers of Monomeric α-Synuclein
Identified after Lyophilizing and Freezing |
title_full | Different Structural Conformers of Monomeric α-Synuclein
Identified after Lyophilizing and Freezing |
title_fullStr | Different Structural Conformers of Monomeric α-Synuclein
Identified after Lyophilizing and Freezing |
title_full_unstemmed | Different Structural Conformers of Monomeric α-Synuclein
Identified after Lyophilizing and Freezing |
title_short | Different Structural Conformers of Monomeric α-Synuclein
Identified after Lyophilizing and Freezing |
title_sort | different structural conformers of monomeric α-synuclein
identified after lyophilizing and freezing |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6047843/ https://www.ncbi.nlm.nih.gov/pubmed/29750859 http://dx.doi.org/10.1021/acs.analchem.8b01264 |
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