HDX–MS finds that partial unfolding with sequential domain activation controls condensation of a cellular stress marker

Bimolecular condensation plays a role in many cellular processes. Despite considerable progress, a residue-level description of condensates has been lacking as obtaining high-resolution structural information is impeded by the condensation process itself. We overcame this issue by applying hydrogen–deuterium exchange/mass spectrometry (HDX–MS) to a canonical stress granule marker protein. We propose a sequential activation model where each domain is activated at different temperatures, executes partial unfolding, and associates only with other similarly activated domains to form the condensate, a mechanism we term thermodynamic specificity. The stress marker undergoes the same structural events upon pH- or heat-induced condensation, providing a unifying molecular portrait of stress response with the marker as a central sensor across different stresses.