If space is provided, bulky modification on the rim of azurin's beta-barrel results in folded protein.
Pseudomonas aeruginosa azurin is a blue-copper protein with a beta-barrel fold. Here we report that, at conditions where thermal unfolding of apo-azurin is reversible, the reaction occurs in a single step with a transition midpoint (T(m)) of 69 degrees C (pH 7). The active-site mutation His117Gly creates a cavity in the beta-barrel near the surface but does not perturb the overall fold (T(m) of 64 degrees C, pH 7). Oxidation of the active-site cysteine (Cysteine-112) in wild-type azurin, which occurs readily at higher temperatures, results in a modified protein that cannot adopt a native-like structure. In sharp contrast, Cysteine-112 oxidation in His117Gly azurin yields a modified apo-azurin that appears folded and displays cooperative, reversible unfolding (T(m) approximately 55 degrees C, pH 7). We conclude that azurin's beta-barrel is a rigid structural element that constrains the structure of its surface; a bulky modification can only be accommodated if complementary space is provided.
Pozdnyakova
Irina
I
Department of Chemistry, Tulane University, 6832 St. Charles Avenue, New Orleans, LA 70118-5698, USA.
Wittung-Stafshede
Pernilla
P
D001059:Apoproteins / Q000737:chemistry; D001400:Azurin / Q000737:chemistry* / Q000235:genetics; D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000378:metabolism; D009154:Mutation; D010084:Oxidation-Reduction; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011550:Pseudomonas aeruginosa; D013696:Temperature
D001059:Apoproteins; D001400:Azurin; D003545:Cysteine