Guanidine hydrochloride denaturation of dopamine-induced α-synuclein oligomers: A small-angle X-ray scattering study

Abstract

Alpha-synuclein (α-syn) forms the amyloid-containing Lewy bodies found in the brain in Parkinson's disease. The neurotransmitter dopamine (DA) reacts with α-syn to form SDS-resistant soluble, non-amyloid, and melanin-containing oligomers. Their toxicity is debated, as is the nature of their structure and their relation to amyloid-forming conformers of α-syn. The small-angle X-ray scattering technique in combination with modeling by the ensemble optimization method showed that the un-reacted native protein populated three broad classes of conformer, while reaction with DA gave a restricted ensemble range suggesting that the rigid melanin molecule played an important part in their structure. We found that 6 M guanidine hydrochloride did not dissociate α-syn DA-reacted dimers and trimers, suggesting covalent linkages. The pathological significance of covalent association is that if they are non-toxic, the oligomers would act as a sink for toxic excess DA and α-syn; if toxic, their stability could enhance their toxicity. We argue it is essential, therefore, to resolve the question of whether they are toxic or not.

Alpha-synuclein oligomers and fibrils originate in two distinct conformer pools: a small angle X-ray scattering and ensemble optimisation modelling study

“…A highly structured compact conformation persisted over the pH range 2.5-10 and dimers were observed over the whole range. In our previously reported SAXS-EOM data for WT a-syn a tri-modal distribution of R g and D max was observed with less sharp separation of the peaks, 14 and there has been a recent report of SAXS-EOM measurements, in combination with NMR measurements, of WT a-syn showing that the protein adopts a single broad distribution of R g in 10 mM phosphate buffer. 31 The reported variations in conformer distribution make it clear that sample preparation conditions and measurement techniques influence the result.…”

“…We have shown previously that SAXS and ensemble optimisation modelling (EOM) of native a-syn can be used to study the distribution of conformers based on their R g and P r distribution. 14 Oxidation of the protein's four methionine residues markedly reduces the proportion of the most extended members of the ensemble of conformers. Since the native protein readily aggregates to form the fibrils associated with Parkinson's disease Lewy bodies, and the oxidised version is non-fibrillogenic, 12 it is plausible that the extended conformer is a key stage on the fibril formation pathway.…”

The N-Terminal Residues 43 to 60 Form the Interface for Dopamine Mediated α-Synuclein Dimerisation

“…[ 10 , 16 , 21 ] While the nature of the DA linkage to α-syn is still ill-defined, the DA:α-syn oligomers are highly stable and remain intact even in the presence of strong denaturants such as 6 M guanidine hydrochloride or urea. [ 22 ] Formation of DA:α-syn oligomers can be modulated by pH and lipids. [ 19 ] Lipids inhibit the formation of the DA-induced α-syn oligomers [ 23 ] while the DA metabolite 5,6-dihydroxylindole promotes soluble α-syn oligomer formation at physiological pH, but under alkaline conditions gives to insoluble oligomers.…”

“…Structural analysis using atomic force microscopy indicated the DA:α-syn oligomers possessed non-fibrillar morphologies. [ 12 , 24 ] Electrospray-ionisation-ion mobility spectrometry-mass spectrometry (ESI-IMS-MS) [ 25 ] and small angle X-ray scattering (SAXS) [ 22 , 26 ] showed DA binds to α-syn when the protein is in an extended conformation. Single-molecule photobleaching and substoichiometric fluorescent labeling determined that DA:α-syn aggregates form as two distinct species a minor assembly containing 15–19 monomers and a major assembly of 34–38 monomers.…”

Alpha‐Synuclein Dopaminylation Presented in Plasma of Both Healthy Subjects and Parkinson's Disease Patients

“…[ 29 ] This interaction is strong, and is not dissociated by 6 m guanidine hydrochloride. [ 30 ] Meanwhile, a non‐covalent interaction between DA and α‐syn has been identified [ 22,31 ] by molecular dynamics simulations. [ 21 ] Taken together, many studies have investigated modification of α‐syn by DA in vitro.…”