Surface‐modified protein crowders influence mutant huntingtin exon 1 aggregation via crowding effects, crowder association, and crowder solution stability

Intracellular protein aggregation occurs in a highly crowded environment. The intracellular environment is highly heterogeneous, featuring diverse crowder protein surface chemistries along with varying crowder stability and solubility. It remains unclear how these aspects influence protein aggregation. Therefore, we assessed how a crowder protein and its surface properties impact aggregation. We utilize high concentrations of surface-modified proteins based on bovine serum albumin (BSA) to monitor how they influence the aggregation of mutant huntingtin exon 1, enabled by fluorescent proteins (mHttex1-VC) for förster resonance energy transfer (FRET). This system reveals three mechanisms through which bystander proteins direct mHttex1-VC aggregation: (1) monodisperse inert proteins appear to function as crowders, increasing the amount of fibrils and their length and width; (2) marginally soluble proteins strongly enhance mHttex1-VC aggregation and density through coaggregation; and (3) crowders that bind mHttex1-VC or folding-destabilized crowders reduce aggregation. The buffer conditions modulate the effects of the protein surface. Thus, in addition to macromolecular crowding effects, the crowder stickiness, solubility, and stability determine the aggregation of the test protein. We expect these effects to also play a role in cells.