Abstract

Eukaryotic cells form condensates to sense and adapt to their environment. Poly(A)-binding protein (Pab1), a canonical stress granule marker, condenses upon heat shock or starvation, promoting adaptation. The molecular basis of condensation has remained elusive due to a dearth of techniques to probe structure directly in condensates. We apply hydrogen–deute-rium exchange/mass spectrometry to investigate the mechanism of Pab1’s condensation. 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 populations allows us to identify which regions contribute to the condensate’s interaction network. Our data yield a 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. Subsequent association is dictated more by the underlying free energy surface than specific interactions, an effect we refer to as thermodynamic specificity. Our study represents an advance for elucidating the interactions that drive condensation. Furthermore, our findings demonstrate how condensation can use thermodynamic spec-ificity to perform an acute response to multiple stresses, a potentially general mechanism for stress- responsive proteins.