Too close for comfort: Self-crowding transforms protein structure and stability beyond volume exclusion

Proteins operate in crowded physiological environments, yet their conformational and oligomeric states are largely inferred from experiments performed under dilute buffer conditions. Here, we show that for lysozyme (LYS) and bovine serum albumin (BSA), self-crowding at physiologically relevant concentrations alters protein structure and assembly, leading to well-defined dense protein states. Circular dichroism reveals a pronounced yet reversible shift from $\alpha$ -helices to $\beta$ -sheets and turns in both proteins, indicating structural changes that are stable but distinct from amyloid aggregation. Small-angle X-ray scattering shows that LYS exhibits net attractive interprotein interactions, whereas BSA displays dominant repulsions that destabilize its dimeric state. At low concentrations, BSA acts as a self-hydrotrope, stabilizing monomers through weak attractions, while at higher concentrations self-crowding promotes dimer dissociation through protein interface destabilization and solution reorganization. Together, these findings demonstrate that protein self-crowding drives reversible restructuring of protein conformation and interactions, challenging classical volume-exclusion models of macromolecular crowding.