Presentation Information
[3SBT-05]Residue-specific kinetic analysis of α-synuclein amyloid fibrillation under flow by real-time Rheo-NMR
○Kenji Sugase1 (1. Kyoto Univ. (Japan))
Keywords:
Rheo-NMR,amyloid,kinetics,flow,LLPS
[Purpose]
Amyloid fibrillation is implicated in neurodegenerative diseases and proceeds through multistep pathways involving conformational changes, oligomer formation, fibril growth, and secondary nucleation, sometimes originating from liquid–liquid phase separation. Despite its importance, real-time, atomic-resolution observation of these processes has been difficult, and most studies rely on indirect probes.
[Method]
We developed a high-sensitivity Rheo-NMR instrument that enables NMR measurements under precisely controlled flow (Morimoto et al., Anal. Chem. 2017) and previously applied it to real-time analysis of SOD1 fibrillation (Iwakawa et al., J. Am. Chem. Soc. 2021). In this study, we applied Rheo-NMR to α-synuclein and continuously acquired 2D 1H–15N correlation spectra under flow to monitor fibrillation kinetics. Additional measurements were performed under liquid–liquid phase separation conditions induced by polyethylene glycol.
[Results]
Time-dependent decreases in NMR signal intensities followed sigmoidal kinetics consistent with a secondary nucleation-based fibrillation model. Fitting the data yielded residue-specific rate constants for primary and secondary nucleation. The NAC region (the aggregation-prone fibril core of α-synuclein) showed slower primary nucleation rates than the N- and C-terminal regions, while phase separation conditions markedly increased the primary nucleation rate within the NAC region.
[Consideration]
The observed kinetics agree with established models in which α-synuclein fibrillation proceeds via oligomer nucleation, fibril elongation, and secondary nucleation. Acceleration of NAC-region nucleation under phase separation suggests that local concentration and intermolecular interactions in dynamic solution environments strongly modulate early fibrillation steps.
[Conclusion]
Rheo-NMR enables residue-specific, quantitative kinetic analysis of amyloid fibrillation under flow and phase-separated conditions, providing direct insight into how dynamic fluid environments influence amyloid formation processes that are difficult to access with conventional methods.
Amyloid fibrillation is implicated in neurodegenerative diseases and proceeds through multistep pathways involving conformational changes, oligomer formation, fibril growth, and secondary nucleation, sometimes originating from liquid–liquid phase separation. Despite its importance, real-time, atomic-resolution observation of these processes has been difficult, and most studies rely on indirect probes.
[Method]
We developed a high-sensitivity Rheo-NMR instrument that enables NMR measurements under precisely controlled flow (Morimoto et al., Anal. Chem. 2017) and previously applied it to real-time analysis of SOD1 fibrillation (Iwakawa et al., J. Am. Chem. Soc. 2021). In this study, we applied Rheo-NMR to α-synuclein and continuously acquired 2D 1H–15N correlation spectra under flow to monitor fibrillation kinetics. Additional measurements were performed under liquid–liquid phase separation conditions induced by polyethylene glycol.
[Results]
Time-dependent decreases in NMR signal intensities followed sigmoidal kinetics consistent with a secondary nucleation-based fibrillation model. Fitting the data yielded residue-specific rate constants for primary and secondary nucleation. The NAC region (the aggregation-prone fibril core of α-synuclein) showed slower primary nucleation rates than the N- and C-terminal regions, while phase separation conditions markedly increased the primary nucleation rate within the NAC region.
[Consideration]
The observed kinetics agree with established models in which α-synuclein fibrillation proceeds via oligomer nucleation, fibril elongation, and secondary nucleation. Acceleration of NAC-region nucleation under phase separation suggests that local concentration and intermolecular interactions in dynamic solution environments strongly modulate early fibrillation steps.
[Conclusion]
Rheo-NMR enables residue-specific, quantitative kinetic analysis of amyloid fibrillation under flow and phase-separated conditions, providing direct insight into how dynamic fluid environments influence amyloid formation processes that are difficult to access with conventional methods.
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