Synthetic NAC 71-82 Peptides Designed to Produce Fibrils with Different Protofilament Interface Contacts

Area of Science:

• Neuroscience
• Biochemistry
• Structural Biology

Background:

• Alpha-synucleinopathies are characterized by fibrillar inclusions in brain cells.
• The structural diversity of alpha-synuclein (α-synuclein) fibrils and its origins are not fully understood.
• Understanding fibril formation is crucial for developing therapeutic strategies for neurodegenerative diseases.

Purpose of the Study:

• To design synthetic peptides based on the α-synuclein NAC 71-82 fragment to investigate the generation of fibrillar polymorphs.
• To explore how peptide structure (single/double fragments, capped/non-capped ends) influences fibril morphology and properties.
• To elucidate the structural basis for the diversity observed in α-synuclein fibril formation.

Main Methods:

• Molecular dynamics simulations to design synthetic peptides.
• Protein concentration determination assays and circular dichroism spectroscopy to assess fibrillar yield and structure.
• Laser-tweezers Raman spectroscopy and Transmission Electron Microscopy (TEM) to characterize secondary structures and fibril morphology.
• Time-correlated single-photon counting to analyze Thioflavin T fluorescence lifetime profiles.

Main Results:

• Peptide models based on α-synuclein fragments were successfully designed and synthesized.
• Fibrils formed from double-fragment peptides exhibited lower β-sheet content, indicating less mature structures compared to single-fragment peptides.
• TEM analysis revealed distinct fibril morphologies, including short protofibrils, elongated twisted fibrils, and rod-like structures.
• Differences in Thioflavin T fluorescence lifetime profiles suggest morphological variations in the generated fibril samples.

Conclusions:

• Synthetic peptide models can effectively replicate morphological features of disease-relevant α-synuclein fibrils, such as twisted and rod-like polymorphs.
• Peptide design, including fragment number and capping, significantly influences fibril structure and maturation.
• This study highlights the potential of using rationally designed peptide models to study the complex mechanisms of α-synuclein fibrillization.