High-throughput methods for studying protein self-assembly.

Abstract

Biomolecular condensation has emerged as a central mechanism of cellular organization, regulating fundamental processes from transcription to stress responses. Its dysregulation - often involving transitions from dynamic condensates to more solid or aggregated states - has been linked to human disease and thus represents a growing therapeutic opportunity. Yet, despite substantial progress, precisely relating protein sequence to condensate behavior, function, and dysregulation remains a largely unresolved challenge. Recent advances in high-throughput approaches are beginning to address this gap. By combining large-scale mutagenesis to fitness, fluorescence- or imaging-based selections, and deep sequencing, these methods enable systematic interrogation of different types of protein self-assembly across vast sequence spaces. However, most of the currently available assays measure indirect readouts such as solubility, stability, or cellular fitness and differ in the way they capture different parameters of the self-assembly process. Here, we review emerging high-throughput strategies to study protein condensation and aggregation at scale, emphasizing what they truly measure, their limitations, and how the cross-talk among these complementary approaches can provide a more accurate and mechanistic mapping of sequence-to-assembly relationships.