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

Amberley D Stephens,Clemens F Kaminski,Maria Zacharopoulou,Nadezhda Nespovitaya,Gabriele S Kaminski Schierle,Jonathan J Phillips

doi:10.1021/acs.analchem.8b01264

Amberley D Stephens, Clemens F Kaminski + Show 4 more

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https://doi.org/10.1021/acs.analchem.8b01264

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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 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.

Highlights

The study of protein misfolding and amyloid fibril formation is important in the field of neurodegeneration, including Parkinson’s, Alzheimer’s, Huntington’s, and prion diseases, as well as other misfolding diseases, such as antibody light chain amyloidosis and diabetes, where incorrectly folded proteins form insoluble β-sheets.[1−3] Understanding amyloid fibril formation is important in the field of nanomedicine, for instance, in the synthesis of structural tissue scaffolds, whereby artificial self-assembling amyloids can be utilized for their tensile strength and ease of functionalization.[4]
Analytical Chemistry oligomers.[13,31−35] We first examined whether lyophilizing or freezing aSyn had an influence on the variability of aggregation kinetics measured by thioflavin T (ThT) since ThT-based assays are frequently used to monitor amyloid fibril formation.[8,36−38]
We are slowly unpicking the biophysical mechanisms behind monomer to fibril formation, there are still a great number of variables, such as the effect of buffer solution and protein storage protocols that need to be taken into consideration and which may hamper our goal to reliably measure amyloid protein aggregation kinetics

Summary

Introduction

The study of protein misfolding and amyloid fibril formation is important in the field of neurodegeneration, including Parkinson’s, Alzheimer’s, Huntington’s, and prion diseases, as well as other misfolding diseases, such as antibody light chain amyloidosis and diabetes, where incorrectly folded proteins form insoluble β-sheets.[1−3] Understanding amyloid fibril formation is important in the field of nanomedicine, for instance, in the synthesis of structural tissue scaffolds, whereby artificial self-assembling amyloids can be utilized for their tensile strength and ease of functionalization.[4]. Monomeric aSyn is an intrinsically disordered (IDP) soluble protein formed of an N-terminus lipid binding domain (aa 1−60), a hydrophobic, so-called nonamyloid-component (NAC) region (aa 61−95), which increases the aggregation propensity of the protein, and a negatively charged C-terminus domain that modulates aggregation (aa 96−140).[11] Like many other amyloid proteins, so far, the mechanistic pathway from soluble unstructured aSyn to insoluble fibrils has not been fully elucidated To achieve this goal, one requires reliable and reproducible aggregation assays and an understanding of the conformational states of the protein at the start of the assay. The effect of protein storage on protein isolation steps storage ref homogenized and boiled IEX, RP-HPLC lyophilized cell lysate ammonium

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