Fig. 4.
Cargo destabilizes the closed and activated states during SRP·SR interaction. (A) Equilibrium titration of the SRP·SR complex assembled in GppNHp with (■) and without (●) RNC using acrylodan-labeled SRP C235. Nonlinear fits of data gave Kd values of 10 ± 2 nM (without RNC) and 40 ± 4 nM (with RNC). (B) Relative fluorescence changes of acrylodan-labeled SR C356 in the presence and absence of cargo, obtained using 50 nM SRP and 15 nM labeled SR with 100 μM GppNHp. An accurate Kd value could not be determined with this probe because of the large amount of cargo-loaded SRP that would be required to saturate labeled SR C356. (C) Equilibrium constants of the GTP-independent (Kd−G) and GTP-dependent (Kd+G) SRP·SR complexes with or without RNC. The equilibrium for rearrangement (Krel) were calculated from Krel = Kd-G/Kd+G. (D) Thermodynamic analysis of the interaction of cargo with SRP at different conformational stages during the SRP–SR interaction.