Advanced surface passivation for high-sensitivity studies of biomolecular condensates.

Biomolecular condensates are cellular compartments that concentrate biomolecules without an encapsulating membrane. In recent years, significant advances have been made in the understanding of condensates through biochemical reconstitution and microscopic detection of these structures. Quantitative visualization and biochemical assays of biomolecular condensates rely on surface passivation to minimize background and artifacts due to condensate adhesion. However, the challenge of undesired interactions between condensates and glass surfaces, which can alter material properties and impair observational accuracy, remains a critical hurdle. Here, we introduce an efficient, broadly applicable, and simple passivation method employing self-assembly of the surfactant Pluronic F127 (PF127). The method greatly reduces nonspecific binding across a range of condensates systems for both phase-separated droplets and biomolecules in dilute phase. Additionally, by integrating PF127 passivation with the Biotin-NeutrAvidin system, we achieve controlled multipoint attachment of condensates to surfaces. This not only preserves condensate properties but also facilitates long-time fluorescence recovery after photobleaching imaging and high-precision single-molecule analyses. Using this method, we have explored the dynamics of polySIM molecules within polySUMO/polySIM condensates at the single-molecule level. Our observations suggest a potential heterogeneity in the distribution of available polySIM-binding sites within the condensates.

Surface Charge Can Modulate Phase Separation of Multidomain Proteins.

Phase separation has emerged as an important mechanism explaining the formation of certain biomolecular condensates. Biological phase separation is often driven by the multivalent interactions of modular protein domains. Beyond valency, the physical features of folded domains that promote phase separation are poorly understood. We used a model system─the small ubiquitin modifier (SUMO) and its peptide ligand, the SUMO interaction motif (SIM)─to examine how domain surface charge influences multivalency-driven phase separation. Phase separation of polySUMO and polySIM was altered by pH via a change in the protonation state of SUMO surface histidines. These effects were recapitulated by histidine mutations, which modulated SUMO solubility and polySUMO-polySIM phase separation in parallel and were quantitatively explained by atomistic modeling of weak interactions among proteins in the system. Thus, surface charge can tune the phase separation of multivalent proteins, suggesting a means of controlling phase separation biologically, evolutionarily, and therapeutically.

Advanced Surface Passivation for High-Sensitivity Studies of Biomolecular Condensates.

Biomolecular condensates are cellular compartments that concentrate biomolecules without an encapsulating membrane. In recent years, significant advances have been made in the understanding of condensates through biochemical reconstitution and microscopic detection of these structures. Quantitative visualization and biochemical assays of biomolecular condensates rely on surface passivation to minimize background and artifacts due to condensate adhesion. However, the challenge of undesired interactions between condensates and glass surfaces, which can alter material properties and impair observational accuracy, remains a critical hurdle. Here, we introduce an efficient, generically applicable, and simple passivation method employing self-assembly of the surfactant Pluronic F127 (PF127). The method greatly reduces nonspecific binding across a range of condensates systems for both phase-separated droplets and biomolecules in dilute phase. Additionally, by integrating PF127 passivation with the Biotin-NeutrAvidin system, we achieve controlled multi-point attachment of condensates to surfaces. This not only preserves condensate properties but also facilitates long-time FRAP imaging and high-precision single-molecule analyses. Using this method, we have explored the dynamics of polySIM molecules within polySUMO/polySIM condensates at the single-molecule level. Our observations suggest a potential heterogeneity in the distribution of available polySIM-binding sites within the condensates.