Measurement of binding affinity between LidA and GTP-bound or GDP-bound Rab1 by ITC.

<p>(A) Raw ITC data. Top panel: twenty injections of GTP-bound Rab1(Q70L) solutions were titrated into LidA(188-580) solution in ITC cell. The area of each injection peak corresponds to the total heat released for that injection. Bottom panel: the binding isotherm for GTP-bound Rab1(Q70L) and LidA(188-580) interaction, the integrated heat is plotted against the stoichiometry of 1∶1, data fitting revealed a binding affinity of 7.5 nM. (B) Top panel: twenty injections of GDP-bound Rab1(S25N) solutions were titrated into LidA(188-580) solution in ITC cell. The area of each injection peak corresponds to the total heat released for that injection. Bottom panel: the binding isotherm for GDP-bound Rab1(S25N) and LidA(188-580) interaction, the integrated heat is plotted against the stoichiometry of 1∶1, data fitting revealed a binding affinity of 7.6 nM. Both titrations were performed in the absence of added Mg²⁺ and GDP/GTP.