The overlooked impact of N-terminal acetylation on biomolecular condensation

Biomolecular condensation via phase separation of intrinsically disordered proteins is fundamental to cellular organization. Although post-translational modifications (PTMs) are known modulators of this process, the role of N-terminal acetylation (Nt-acetylation), a prevalent but irreversible PTM, remains largely unexplored. Here, we investigate how Nt-acetylation affects the phase behavior of Grh1, a Golgi-associated yeast protein prone to condensation. Using biophysical and imaging techniques including DIC and confocal microscopy, dynamic light scattering (DLS), differential scanning calorimetry (DSC), and hyperspectral imaging, we compared acetylated (AcGrh1) and non-acetylated forms of Grh1. Our results reveal that Nt-acetylation significantly alters condensate morphology, saturation concentration, responsiveness to pH and salt, and droplet material properties. AcGrh1 forms fewer, smaller, and more uniform droplets with reduced turbidity and electrostatic sensitivity. It shows higher saturation concentration and decreased thermal stability, indicating altered conformational dynamics and increased α-helicity under crowding. Notably, co-condensation assays show that AcGrh1 and Grh1 organize into core–shell architectures, suggesting changes in interfacial tension and immiscibility. These findings identify Nt-acetylation as a critical regulator of phase separation, acting through dimerization and structural modulation. Given its widespread occurrence and absence in most recombinant proteins, our study underscores the need to consider native PTMs in studies of biomolecular condensates to better reflect physiological conditions.