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p.Y278S,p.Y283S,p.F291S,p.F309S,p.Y319S,p.Y264S,p.Y335S,p.Y341S,p.F95S,p.F197S,p.F207S,p.F215S,p.Y222S,p.F228S,p.Y250S,p.F244S,p.Y257S,p.Y275S,p.Y235S,p.Y250S,p.Y271S,p.Y288S,p.Y274S,p.Y301S,p.Y324S

Of the 25 mutants, six were found to substantially impede binding to hydrogel droplets formed from mCherry fused to the wild type LC domain of hnRNPA2. Five of the six tyrosine- or phenylalanine-to-serine mutations that substantially impede hydrogel binding occur within the region of the LC domain that is protected from NAI modification in the fibrous state (Y278S, Y283S, F291S, F309S and Y319S). The sixth mutant that significantly impeded in hydrogel binding, Y264S, occurs on the amino terminal side of the NAI protected region within a span where we failed to find acetylated side chains – a dead zone in the footprint (residues 258–282). We tentatively conclude that these six residues are particularly important for polymerization of hnRNPA2, and that polymerization causes NAI protection.The remaining 19 mutants fell into two categories with respect to hydrogel binding. Twelve mutants bound to hydrogels in a manner indistinguishable from wild type hnRNPA2. Two of these mutants, Y335S and Y341S, were located in the very C-terminal region of the LC domain, concordant with a small region that was fully accessible to NAI modification irrespective of whether the protein was in a polymeric or denatured state. Seven of these phenotype-void mutants, F95S, F197S, F207S, F215S, Y222S, F228S and Y250S were located in the amino terminal region of the LC domain that was widely accessible to NAI modification irrespective of structural state. The remaining three mutations that had no discernible effect on hydrogel binding, F244S, Y257S, Y275S, were all localized in the dead zone of the NAI footprint. Finally, seven mutants, including Y235S, Y250S, Y271S, Y288S, Y274S, Y301S and Y324S, mildly affected binding to mCherry:hnRNPA2 hydrogels. These seven mutants mapped randomly across the LC domain of hnRNPA2. We conclude that tyrosine- and phenylalanine-to-serine mutations in NAI protected regions impede hydrogel binding, whereas those in NAI accessible regions do not impede hydrogel binding. This conclusion favors functional significance of the NAI footprint.