Functional characterization of the dimerization domain of the ferric uptake regulator (Fur) of Pseudomonas aeruginosa

摘要

pThe functional properties of the recombinant C-terminal dimerization domain of the iPseudomonas aeruginosa/i Fur (ferric uptake regulator) protein expressed in and purified from iEscherichia coli/i have been evaluated. Sedimentation velocity measurements demonstrate that this domain is dimeric, and the UV CD spectrum is consistent with a secondary structure similar to that observed for the corresponding region of the crystallographically characterized wild-type protein. The thermal stability of the domain as determined by CD spectroscopy decreases significantly as pH is increased and increases significantly as metal ions are added. Potentiometric titrations (pH 6.5) establish that the domain possesses a high-affinity and a low-affinity binding site for metal ions. The high-affinity (sensory) binding site demonstrates association constants (iK/isubA/sub) of 10(±7)×10sup6/sup, 5.7(±3)×10sup6/sup, 2.0(±2)×10sup6/sup and 2.0(±3)×10sup4/sup Msup?1/sup for Nisup2+/sup, Znsup2+/sup, Cosup2+/sup and Mnsup2+/sup respectively, while the low-affinity (structural) site exhibits association constants of 1.3(±2)×10sup6/sup, 3.2(±2)×10sup4/sup, 1.76(±1)×10sup5/sup and 1.5(±2)×10sup3/sup Msup?1/sup respectively for the same metal ions (pH 6.5, 300 mM NaCl, 25 °C). The stability of metal ion binding to the sensory site follows the Irving–Williams order, while metal ion binding to the partial sensory site present in the domain does not. Fluorescence experiments indicate that the quenching resulting from binding of Cosup2+/sup is reversed by subsequent titration with Znsup2+/sup. We conclude that the domain is a reasonable model for many properties of the full-length protein and is amenable to some analyses that the limited solubility of the full-length protein prevents./p