Studies of thermally induced denaturation of azurin and azurin derivatives by differential scanning calorimetry: evidence for copper selectivity.
Azurin, a blue copper protein from Pseudomonas aeruginosa, and several derivatives of azurin have been studied by differential scanning calorimetry. Two well-separated, irreversible transitions are observed in a scan of apoazurin under a variety of conditions, and they are assigned to distinct steps in the denaturation process. No specific structural component can be assigned to the lower temperature transition, but a "flap" structure which is found near the metal binding site may be involved. Circular dichroic spectra suggest that melting of the beta-sheet structure, the main structural motif in the native protein, occurs during the second transition. With the exceptions of the Ni(II) and p-(hydroxymercuri)benzoate derivatives, the transitions are superposed in the metalated forms, and the enthalpies of denaturation are more endothermic. By comparison with other first-row divalent transition ions and especially Zn(II), the Cu(II) derivative exhibits the most endothermic denaturation process. Along with other data, this suggests that the binding energy is greater for Cu(II). It is postulated that the selectivity for copper over zinc arises because of the irregular binding geometry offered by the folded protein. Denaturation of the Hg(II) derivative is even more endothermic, confirming that the type 1 binding site has a very great affinity for Hg(II). Finally, when substoichiometric amounts of Hg(II) are added to the apoprotein, there is evidence that a novel mercury-bridged dimer of azurin forms.
D001400:Azurin / Q000031:analogs & derivatives / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism*; D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D003300:Copper / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D010944:Plants / Q000378:metabolism; D010970:Plastocyanin / Q000378:metabolism; D011487:Protein Conformation; D011489:Protein Denaturation; D011550:Pseudomonas aeruginosa / Q000378:metabolism
D001426:Bacterial Proteins; D001400:Azurin; D003300:Copper; D010970:Plastocyanin