Fluorescence labeling methods influence the aggregation process of α-syn in vitro differently

Area of Science:

• Biophysics
• Neuroscience
• Materials Science

Background:

• Previous studies using correlative microscopy suggested suboptimal protein labeling strategies lead to heterogeneous aggregation pathways for insulin and beta-amyloid (Aβ).
• The failure of fluorescent labeling in a significant portion of observed fibrils for shorter peptides raised questions about the generalizability of these findings across different molecular systems.

Purpose of the Study:

• To investigate the aggregation process of alpha-synuclein (α-syn), a larger amyloidogenic peptide implicated in Parkinson's disease.
• To determine if suboptimal labeling strategies also produce artifacts in α-syn aggregation and to develop an improved labeling method.

Main Methods:

• Correlative Stimulated Emission Depletion (STED) microscopy and Atomic Force Microscopy (AFM) were employed.
• An unspecific labeling procedure was initially tested, followed by the development and application of a site-specific labeling method targeting a less aggregation-involved domain of α-syn.

Main Results:

• Unspecific labeling of α-syn reproduced the coexistence of labeled and unlabeled fibrils, similar to previous observations with shorter peptides.
• Site-specific labeling successfully ensured that all fibrillar aggregates of α-syn were fluorescent at a specific dye-to-protein ratio (1:22).

Conclusions:

• Labeling artifacts in protein aggregation studies can be avoided by carefully designing site-specific labeling strategies tailored to the molecular system.
• These findings for α-syn emphasize the importance of optimized labeling for accurate visualization of amyloidogenic peptide aggregation, crucial for understanding diseases like Parkinson's.