A pH dependence study on the unfolding and refolding of apoazurin: comparison with Zn(II) azurin.
Azurin, a small blue copper protein from the bacterial species Pseudomonas aeruginosa, is mostly a beta-sheet protein arranged into a single domain. Previous folding studies have shown that the equilibrium denaturation of the holoprotein follows a two-state process; however, upon removal of the copper, the denaturation had been reported to follow a three-state process. The two unfolding transitions measured for apoazurin had been thought to arise from two different folding domains. However, in the present work, we found that the denaturation of apoazurin occurs over a single transition and we determined the folding free energy to be -27.8 +/- 2.4 kJ mol(-1). From this measurement along with measurements previously reported for the unfolding of the holoazurin, we were able to determine that Cu(II) and Cu(I) stabilize the native structure by 25.1 +/- 6.9 kJ/mol and 12.9 +/- 8.1 kJ/mol, respectively. It is our contention that the second transition displayed in the denaturation curves previously reported for apoazurin arise from protein heterogeneity-in particular, from the presence of Zn(II) azurin. We extended our investigation into the denaturation of Zn(II) azurin at pH 6.0 and 7.5. The equilibrium denaturation studies show that the zinc ion significantly stabilizes the native-state structure at pH 7.5 and very little at the lower pH. We attribute the decrease in the stabilizing effect of the zinc ion with decreasing pH to the protonation of two histidinyl side chains. When protonated the ligands, His 46 and His 117, are incapable of binding a metal ion. Further, comparing the denaturation curves of Zn(II) azurin measured by circular dichroism with those measured by fluorescence indicates that the denaturation of Zn(II) azurin is far less simple than the denaturation of apoazurin.
Hansen
John E
JE
Department of Chemistry, State University of West Georgia, Carrollton 30118, USA. jhansen@westga.edu
McBrayer
Mary Kate
MK
Robbins
Michael
M
Suh
Yong
Y
D001059:Apoproteins / Q000737:chemistry*; D001400:Azurin / Q000737:chemistry*; D002614:Chelating Agents / Q000494:pharmacology; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D004305:Dose-Response Relationship, Drug; D004492:Edetic Acid / Q000494:pharmacology; D019791:Guanidine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008024:Ligands; D008956:Models, Chemical; D011485:Protein Binding; D011489:Protein Denaturation; D017510:Protein Folding; D011550:Pseudomonas aeruginosa / Q000378:metabolism; D013053:Spectrophotometry; D013816:Thermodynamics; D013997:Time Factors; D014364:Tryptophan / Q000737:chemistry; D015032:Zinc / Q000737:chemistry*
D001059:Apoproteins; D002614:Chelating Agents; D008024:Ligands; C038778:apoazurin; D001400:Azurin; D014364:Tryptophan; D004492:Edetic Acid; D015032:Zinc; D019791:Guanidine