Crowder-Induced Protein Condensation: Role of the Polymer Concentration and Mesh Size in Crowded Systems

Condensation of macromolecules offers powerful tools for manipulating their functions. Macromolecular crowding induces condensation and modifies the phase behavior of the molecules. Despite their widespread application, previous studies have typically explored various concentration ranges of crowding agents to promote condensation incrementally. Here, we investigated condensation of three globular proteins, bovine serum albumin (BSA), bovine hemoglobin (Hb), and milk beta-lactoglobulin (β-lac), under the influence of poly(ethylene glycol) (PEG) as the crowding agent. Fixed protein concentrations and five different molecular weights of PEG ranging from 4000 to 35 000 g/mol were used. The concentration range triggering condensation was identified for these proteins. This study, to our knowledge, is the first to demonstrate that condensation was correlated closely with the transition from the dilute to the semidilute regime of PEG at the critical concentration (C*), regardless of the protein type. This concept was further elucidated by comparing the radius of gyration of proteins (RG,p) with the characteristic mesh size of the entangled network (ξ), revealing that condensation occurred when ξ < 2RG,p. Therefore, this study underscores the critical role of reaching C* in initiating phase separation of BSA, Hb, and β-lac, likely due to the hindered protein mobility within the polymer network when ξ < 2RG,p.