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

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.


Table of Contents
. Analysis of aSyn purity by coomassie stained SDS-PAGE gel and analytical RP-HPLC Figure S2. Different aggregation kinetics measured for frozen and lyophilised aSyn Figure S3. Fitted curves calculated using the Finke Watzky equation to average ThT fluorescence data from lyophilised and frozen aSyn Figure S4. DLS experiments displayed as percentage intensity versus size show a higher percentage of HMW conformers after lyophilising than after and freezing. Figure S5. AFM reveals different aSyn HMW conformers after lyophilising and freezing. Figure S6. Comparison of the relative deuterium uptake (Da) for aSyn (resides 77 -89) lyophilised and frozen samples over exposure time at pH 4 and pH 7 reveals differences between monomeric lyophilised and frozen aSyn. Figure S7. Coverage map of alpha-synuclein peptides from HDX-MS. Figure S8. Difference in deuterium uptake between the aSyn frozen and aSyn lyophilised samples (Da). Table S1. Amino acid verification of purified human aSyn. Table S2. The remaining aSyn monomer concentration taken at the end of the ThT-based aggregation assay and determined by analytical SEC is reduced in lyophilised samples but displays an increased variance between wells. Table S3. Goodness of fit represented by R-square and root mean square error (RMSE) calculated from fitted curves of lyophilised and frozen aSyn samples. Table S4. DLS analysis of size distribution and % intensity of aSyn reveals a higher degree of HMW conformers in lyophilised versus frozen samples. Figure S1. Analysis of aSyn purity by coomassie stained SDS-PAGE gel and analytical RP-HPLC (A) Additional HIC increases the purity of aSyn as shown on a 4-12% Bis-Tris gel. Protein sample after IEX contained 78.3% aSyn and 94.7% aSyn after HIC as determined by densitometry using Fiji image analysis software. (B) 50 uL of aSyn from purification batch 1 (purple) and batch 2 (green) were analysed by analytical RP-HPLC on a Discovery Bio Wide Pore C18-5 column and eluted using a gradient of 5% acetonitrile + 0.1% trifluoroacetic acid (TFA) to 95% acetonitrile + 0.1% TFA with H 2 O + 0.1% TFA over 40 minutes at 1 ml/min. Percentage of aSyn in purification batch 1 was 85.2% and 90.7% in purification batch 2 based on absorbance at 280 nm.

Figure S2. Different aggregation kinetics measured for frozen and lyophilised aSyn
Mean ThT fluorescence intensity including standard deviation is shown for aSyn subjected to freezing (blue) and lyophilisation (red). 100 µM aSyn was incubated in a 96 well plate with continuous orbital agitation at 300 rpm for 120 hours. Data represent 3 experiments with 6 well replicates for each condition. Kinetic data analysis is shown in Table 2 Table S2.

B Frozen
A Lyophilised Figure S4. DLS experiments displayed as percentage intensity versus size show a higher percentage of HMW conformers after lyophilising than after and freezing. Percentage intensity of the 100 µM aSyn sample is plotted against diameter of sample detected (d.nm). Data of aSyn lyophilised in NaP buffer and reconstituted in H 2 O (A) and aSyn frozen in NaP buffer (B) display the presence monomeric protein (4 -8 d.nm) and HMW conformers (~200 nm) and (~5000 nm). A higher percentage intensity of HMW conformers is seen in the lyophilised compared to the frozen sample. One representative set of data from a total of two repeats including seven readings each. Data is also represented in Table S3 Figure S5. AFM reveals different aSyn HMW conformers after lyophilising and freezing. aSyn was imaged on freshly cleaved mica and representative images are shown of HMW conformers in the (A.) lyophilised sample and (B.) frozen sample. HMW conformers in the lyophilised sample were more heterogenous in shape and size (C.) Size range of HMW conformers from using lyophilising (red) and freezing (blue) protocols are shown as a percentage of total HMW conformers, calculated from 30 images in each sample. HMW conformers formed after lyophilising had a larger surface area.

Figure S6. Coverage map of alpha-synuclein peptides from HDX-MS.
Peptide mapping of aSyn yielded a total of 89 peptides, 100% coverage of the sequence and a redundancy coefficient of 13.49. The redundancy coefficient is an average number of peptides for which each residue was identified.

Figure S7. Comparison of the relative deuterium uptake (Da) for aSyn (resides 77 -89) lyophilised and frozen samples over exposure time at pH 4 and pH 7 reveals differences between monomeric lyophilised and frozen aSyn.
Shown is the relative mass uptake for the lyophilised and frozen samples over 0.5 to 50 minutes, for the NAC region peptide aa 77-89. At pH 4.00 (A.) the kinetics are slow enough to observe differences in deuterium uptake between the lyophilised and the frozen sample. At pH 7.00 (B.), the kinetics of exchange are too fast to observe a significant difference. This effect is observed throughout the protein sequence. B A S-8 Figure S8. Difference in deuterium uptake between the aSyn frozen and aSyn lyophilised samples (Da). Shown is the difference (frozen-lyophilised) in mean deuteration level per amino acid residue, averaged across the five time points (0.5 minutes, 0.75 minutes, 1 minute, 5 minutes and 50 minutes), as calculated from equation 1. A positive value of the plotted difference seen throughout aSyn shows that the lyophilised sample is less prone to exchange through out the sequence, due to increased solvent protection and/or increased hydrogen bonding. Values shown are the average of all five time points sampled from two experiments. Error bars indicate 1 s.d. S-9

AAA Results
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