Intramembrane proteases typically cleave multiple substrates within their transmembrane domains (TMDs). Because substrate TMDs lack a consensus sequence around their scissile sites, it remains unclear how the enzyme discriminates substrates from nonsubstrates at the level of their TMDs. Here, we compare the previously well investigated TMDs of γ-secretase substrates C99 and Notch1 in terms of helix flexibility. Our results reveal that the low-stability site neigboring a functionally relevant diglycine hinge of C99 has an equivalent in the Notch1 TMD. This suggests that the tetra-alanine motif of Notch1 also functions as a hinge which may facilitate its cleavage.