Crowder titrations enable the quantification of driving forces for macromolecular phase separation

Protein-specific crowding accelerates aging in phase-separated droplets

“…These observations lead us to deduce that the system exhibits a competitive behavior, characterized by a struggle between the two species to occupy distinct spaces, resulting in fully segregative phase separation. Recently, it has been shown that titration of such “depletant crowders” can be used to quantify polymer-solvent interaction [54].…”

“…We accomplish this by fitting the experimental binodal concentrations against a free energy model that combines Flory-Huggins theory [67] with statistical associating fluid theory (SAFT) [68]. This type of hybrid model has shown to be an excellent choice for interpreting and predicting biomolecular phase behavior [11], [69], [54] as it discriminates between non-specific interactions, captured in a general way by the term "solvation", and specific association between defined sites located along the polymer backbone. In defining the model, we consider the solution of BSA, PEG and buffer as a ternary mixture, of which the species are respectively referred to as components A, B, and S (the latter being from "solvent").…”

Optical characterization of molecular interaction strength in protein condensates

“…Uncertainties for the calculation of the density, dry mass density, and related quantities (i.e., protein volume fraction, longitudinal modulus) arise from the application of dextran as a crowding agent in some of the conditions studied (see Section S 1.6 for further details). It was found that in the absence of specific interactions and with high molecular weight crowders, depletion effects dominate and the crowder molecules are excluded from the condensates [48]. In light of these findings, the underlying assumption for our calculation of the density was that the crowder (dextran T500) is largely excluded from the protein condensates, and the values for α and θ were chosen accordingly.…”

Biomolecular condensates form spatially inhomogeneous network fluids

“…Previous work has shown that the homotypic interactions within NPM1, uncovered in the presence of crowders, can also affect both the phase behavior and the mesoscopic structure of condensates formed with SURF6N 73, 74 . A new method that leveraged the Edmond-Ogston formalism 119 , allows for the intrinsic strengths of homotypic interactions to be uncovered using crowder titrations 47 . Knowledge of the strengths of homotypic interactions, and the relative interplay with heterotypic interactions, will allow for simulations of binary and higher-order mixtures that mimic nucleolar GCs.…”

“…Here, the complexation of polyelectrolytes is driven by a combination of enthalpically favorable associations and entropically favored release of counterions 38, 41, 43, 45 . If the complexes are higher-order clusters of undefined stoichiometry, then we arrive at the Ogston limit where size and hydration details lower the solubility and the complexes undergo a segregative transition to generate coexisting dilute and dense phases 21, 46, 47 . The dilute phase will comprise dissociated polyions, complexed polyions that form higher-order oligomers 48, 49 and pre-percolation clusters 50, 51 , all of which must be electroneutral and hence will involve different degrees of ion associations.…”