ATP and small amphiphilic molecules act as molecular matchmakers to fine-tune FET protein clusters.

FET (FUS-EWSR1-TAF15) family proteins form mesoscale clusters under physiological conditions at concentrations well below the threshold for phase separation. However, how ATP, an amphiphilic molecule and essential cellular metabolite, affects this clustering remains unclear. Here, I show that ATP modulates the size of subsaturation mesoscale clusters in a concentration-dependent manner. At low concentrations (1-2 mM), ATP promotes clustering by acting as a molecular crosslinker, leading to larger assemblies. At a moderate concentration (5 mM), clusters become smaller but remain stable, whereas at a higher concentration (10 mM), the cluster size is reduced. Other amphiphilic molecules, including sodium xylene sulfonate, sodium toluene sulfonate, and hexanediol, display comparable concentration-dependent effects. These observations cannot be explained solely by hydrotropic or kosmotropic mechanisms; instead, they arise from non-specific interactions between amphiphilic molecules and protein. Thus, the intrinsic chemical features of small molecules and FET proteins collectively govern mesoscale clustering at subsaturation concentrations.