Abstract Eukaryotic cells form biomolecular condensates to sense and adapt to their environment 1,2 . Poly(A)-binding protein (Pab1), a canonical stress granule marker 3,4 , condenses upon heat shock or starvation, promoting adaptation 5 . The molecular basis of condensation has remained elusive due to a dearth of techniques to probe structure directly in condensates. Here we apply hydrogen-deuterium exchange/mass spectrometry (HDX-MS) to investigate the molecular mechanism of Pab1’s condensation. We find that Pab1’s four RNA recognition motifs (RRMs) undergo different levels of partial unfolding upon condensation, and the changes are similar for thermal and pH stresses. Although structural heterogeneity is observed, the ability of MS to describe individual subpopulations allows us to identify which regions become partially unfolded and contribute to the condensate’s interaction network. Our data yield a clear molecular picture of Pab1’s stress-triggered condensation, which we term sequential activation , wherein each RRM becomes activated at a temperature where it partially unfolds and associates with other likewise activated RRMs to form the condensate. This model thus implies that sequential activation is dictated by the underlying free energy surface, an effect we refer to as thermodynamic specificity . Our study represents a methodological advance for elucidating the interactions that drive biomolecular condensation that we anticipate will be widely applicable. Furthermore, our findings demonstrate how condensation can use thermodynamic specificity to perform an acute response to multiple, stresses, a potentially general mechanism for stress-responsive proteins.

Sequential Activation and Local Unfolding Control Poly(A)-Binding Protein Condensation