- 标题
- 摘要
- 关键词
- 实验方案
- 产品
-
Different effects of alpha-Synuclein mutants on lipid binding and aggregation detected by single molecule fluorescence spectroscopy and ThT fluorescence-based measurements
摘要： Six alpha-Synuclein point mutations are currently known to be associated with familial parkinsonism: A30P, E46K, H50Q, G51D, A53E and A53T. We performed a comprehensive in vitro analysis to study the impact of all aSyn mutations on lipid binding and aggregation behavior. Markedly reduced lipid binding of A30P, moderately attenuated binding of G51D and only very slightly reduced binding for the other mutants were observed. A30P was particularly prone to form metal ion induced oligomers, whereas A53T exhibited only weak tendencies to form oligomers. In turn, fibril formation occurred rapidly in H50Q, G51D and A53T, but only slowly in A30P, suggesting mutants prone to form oligomers tend to form fibrils to a lesser extent. This was supported by the observation that fibril formation of wild type aSyn, A30P and A53T was impaired in the presence of ferric iron. Additionally, we found the aggregation kinetics of mixtures of A30P or A53T and wt aSyn to be determined by the faster aggregating aSyn variant. Our results implicate differential mechanisms playing a role in aSyn pathology on the molecular level. This might contribute to a better understanding of Parkinson′s disease pathogenesis and provide potential links to develop prevention strategies and disease-modifying therapy.
关键词： synucleinopathy,Alpha-synuclein (α‐Synuclein),Parkinson′s disease,protein‐lipid interaction,protein aggregation,mutant
更新于2025-09-23 15:23:52
-
Polymorph-specific distribution of binding sites determines thioflavin-T fluorescence intensity in α-synuclein fibrils
摘要： Thioflavin-T (ThT) is the most commonly used fluorescent dye for following amyloid formation semi-quantitatively in vitro, specifically probing the fibrillar cross-b-sheet content. In recent years, structural polymorphism of amyloid fibrils has been shown to be an important aspect of amyloid formation, both in vitro and in neurodegenerative diseases. Therefore, understanding ThT–amyloid interactions in the context of structural polymorphism of amyloids is necessary for correct interpretation of ThT fluorescence data. Here we study the influence of fibril morphology on ThT fluorescence and ThT binding sites, with two morphologically distinct but chemically identical a-synuclein polymorphs. In ThT fluorescence assays the two polymorphs show type-specific fluorescence intensity behaviour although their b-sheet content has been shown to be similar. Further, fluorescence lifetime measurements of fibril-bound ThT reveal the presence of at least two qualitatively different ThT binding sites on the polymorphs. The relative distributions of the binding sites on the fibril surfaces appear to be morphology dependent, thus determining the observed polymorph-specific ThT fluorescence intensities. These results, highlighting the role of fibril morphology in ThT-based amyloid studies, underline the relevance of polymorphs in ThT–amyloid interaction and can explain the variability often observed in ThT amyloid binding assays.
关键词： thioflavin-T,polymorphism,Amyloid,atomic force microscopy,a-synuclein
更新于2025-09-04 15:30:14