PROJECT SUMMARY Biology is undergoing a revolution, where biomolecular condensates are upending our understanding in much of molecular, cell, and developmental biology. These condensates, formed through phase separation, mediate crucial cellular functions and are linked with neurodegeneration and cancer. Yet we are only starting to gain a glimpse of physical understanding of the properties and complex behaviors of biomolecular condensates. A main goal of this project is to bridge the widening gap in understanding between the biology and physics of phase separation. Building on the strong foundations laid in the previous funding period, we will integrate theoretical, computational, and experimental approaches to tackle the enormous challenges in interpreting microscale biological phenomena in terms of the conformations and interactions of the constituent macromolecules. Questions to be addressed include: (1) computing the effects of point mutations and posttranslational modifications on phase equilibrium; (2) quantifying nonspecific interactions in condensates; (3) measuring molecular and material properties of condensates; and (4) modeling condensate aging. Some of the challenges arise from intrinsically disordered regions ubiquitously present in macromolecules that drive phase separation. We will therefore carry out comprehensive studies into the conformational and dynamic properties of intrinsically disordered proteins and their pathways when forming specific complexes with partners. While the latter specific, bimolecular interactions represent the traditional paradigm of molecular recognition, phase separation defines a paradigm of molecular recognition at the microscale, mediated by weak, nonspecific interactions among an ensemble of molecules. A third paradigm of molecular recognition is semi-specific association of proteins with membranes, where the conformation and pose of the bound protein and the composition of the proximal lipids are all highly dynamic. We will deeply probe the semi-specific association of WASP and synaptogamins with acidic membranes to fill in critical gaps in their functional mechanisms. The planned research will not only advance fundamental understanding in biology but also yield new opportunities for designing therapies.