Biomolecular condensates regulate essential cellular processes and are implicated in many diseases. Understanding how condensates form and function is key to uncovering how biomolecules organize into a functional cell. However, because condensates often form through non-stoichiometric interactions, classical stoichiometric models of molecular interactions are insufficient to explain the activities of condensates, motivating the development of the condensate microenvironment model. This model posits that condensates have properties emerging from the collective behavior of their constituent molecules, resulting in distinct physicochemical parameters within condensates that differ from the surrounding cellular environment. Notably, the emergent material properties, such as diffusivity and viscosity, are thought to influence condensate composition and function. However, current tools face four distinct but related challenges, limiting tests of the condensate microenvironment model: (1) difficulty in perturbing material properties; (2) limited ability to selectively target specific condensates; (3) difficulty capturing condensate dynamics in live cells; and (4) complexity in manipulating endogenous condensates. This thesis presents the development and application of a synthetic 17-amino-acid micropeptide, the killswitch, that perturbs the material properties of condensates in live cells. The killswitch works by immobilizing condensate components through phenylalanine-mediated self-association and can be targeted to diverse condensates using genetic or nanobody-based approaches. I first identified the molecular features that underlie killswitch function, then used the killswitch to perturb the material properties of nucleolar, oncogenic, and viral condensates in live cells. Immobilizing condensate components with the killswitch affected the abundance and mobility of untargeted condensate components, disrupting condensate function and leading to changes such as altered nucleolar protein composition and impaired adenoviral condensate organization. These results establish the killswitch as a powerful tool for perturbing condensate material properties and probing condensate microenvironments, enabling systematic investigation of how condensate dynamics, composition, and function are interrelated.
