Deep-blue emitting fibers: tracking aggregation of a hydrophobin through intrinsic fluorescence

Amyloid fibrils are highly ordered protein aggregates typically characterized by a cross-β sheet structure. Class I hydrophobin Vmh2, produced by the fungus Pleurotus ostreatus, exhibits a unique ability to self-assemble into giant fibrillar structures and to emit deep-blue fluorescence upon UV excitation, despite lacking aromatic amino acids. This work presents the first in-depth characterization of such fluorescence in hydrophobins, using it as an intrinsic, label-free probe to study aggregation behaviour. The influence of various environmental factors-including protein concentration, pH, and temperature-on this process was examined. Vmh2 deep-blue fluorescence increased over time, displaying a sigmoidal curve typical of amyloid proteins, and was strongly dependent on protein concentration. pH variations significantly affected fluorescence intensity, with strong quenching observed at low pH. Remarkably, fluorescence was reversible through alternating acid and base treatments. Temperature also had a critical influence: while high temperatures (60-80 °C) accelerated aggregation, the most intense fluorescence was recorded at 40 °C, indicating this condition may favour a more ordered hierarchical fibril structure. This work underscores the potential of using intrinsic optical properties of protein fibrils for real-time monitoring of self-assembly processes without extrinsic labelling.