Modulation of odorant-binding protein fibrillogenesis in crowded environments.

Amyloid aggregation of proteins with β-barrel topology is increasingly being considered in relation to pathological processes in humans. Nevertheless, the influence of the densely packed intracellular and extracellular environments (macromolecular crowding conditions) on the fibrillogenesis of these highly structured proteins remains insufficiently studied. Addressing this gap, we focused our analysis on odorant-binding proteins (OBPs), as recent studies suggest the involvement of their amyloidogenesis in olfactory dysfunction, particularly in neurodegenerative diseases. Within the bovine OBP (bOBP) model, a stage-specific and polymer-dependent effect of crowding conditions on β-barrel protein fibrillogenesis was demonstrated using various crowding agents, polyethylene glycol (PEG-8k and PEG-12k) and dextran (Dex-15-25k and Dex-70k), and a comprehensive range of physicochemical methods. Specifically, the addition of PEGs shortened and even eliminated the lag phase of fibrillogenesis by accelerating primary nucleation. Concurrently, all crowding agents attenuated the processes of the exponential phase of fibrillogenesis. These effects of crowding agents were attributed to: 1) inhibition of global structural transformations in the β-barrel, and 2) enhanced clustering of formed aggregates, which suppresses secondary processes like fibril fragmentation. The combined action of crowding agents reduced mature amyloid formation, promoted the accumulation of highly hydrophobic and cytotoxic prefibrillar precursors, and generated clusters of structurally heterogeneous aggregates. Our findings reveal that macromolecular crowding influences the fibrillogenesis trajectory and the properties of amyloid aggregates formed from β-barrel proteins, which must be considered when analyzing the biological consequences of these processes in vivo.