Figure 5
Indirect SPR analysis of PII-PipX complex formation in presence or absence of effector molecules. (A) His6-PipX (500 nM) was injected to a Ni-NTA loaded sensor chip in absence of PII (red line) or in presence of 100 nM PII (WT) (black dashed line) or 100 nM PII (I86N) (black line), without effectors. The injection phase of 200 s was followed by 400 s dissociation. (B) Dissociation from the sensor of His6-PipX (red line) alone or in presence of PII (WT) (black dashed line) or PII (I86N) (black line). The response signal at the end of the injection time (200 s) was normalized to 100%. (C,D) Dissociation assay as described in (B) for PipX-PII (I86N) (C) or PipX-PII (WT) (D) complexes in presence of different combinations of effector molecules: Without effectors (black line), in presence of 1 mM ADP (green line), 1 mM ATP (blue line) or 1 mM ATP/ 1 mM 2-OG (orange line). PipX without PII in absence of effectors was used as control (red line). (E) Response signal in % at time 600 s (400 s after the end of the injection) of injections of PipX complexed with PII (WT) or PII (I86N) in presence or absence of effectors. The remaining signal after dissociation at time 600 s is an indicator for the stability of the complex.
