Deciphering Amyloid Fibril Formation through Protein Concentration by Optical Trapping.

Optical trapping, combined with time-lapse transmission and fluorescence imaging, enables precise real-time observation of protein concentration dynamics during amyloid fibril formation. Condensates form at the laser focus within 30 min and grow to ∼1.2 μm in diameter regardless of pD by optical trapping of apoferritin (Fer8). Initial trapping efficiency is higher at pD 8.4 (stable 24-mer) than under acidic conditions (pD 1.5 and 2.0; subunit dimer), while acidic solutions show a pronounced second-phase concentration increase after ∼72 min, ultimately far exceeding the concentration in pD 8.4. However, the second-phase surge is absent at pD 3.0, despite evidence of amyloid formation (ThT fluorescence and transmission electron microscopy (TEM)), likely due to slower elongation presumably owing to higher protein stability than at lower pD. Quantitative analysis of Fer8 subunit concentration reveals a critical concentration (0.53-0.63 mM) for the rapid ThT fluorescence increase onset for all conditions at pD 1.5-3.0. This moderate concentration, combined with secondary structure observations, suggests that optical trapping facilitates specific alignment of Fer8 molecules beyond simple concentration. These findings highlight optical trapping's power to dissect the pD-dependent interplay between protein structure, nucleation, and amyloid fibril elongation, providing insights into the early stages of amyloid fibril formation.