Snapshots of a dynamic folding nucleus in zinc-substituted Pseudomonas aeruginosa azurin.
Zinc-substituted Pseudomonas aeruginosa azurin folds in two-state equilibrium and kinetic reactions. In the unfolded state, the zinc ion remains bound to the unfolded polypeptide via two native-state ligands (His117 and Cys112). The significantly curved Chevron plot for zinc-substituted azurin was earlier ascribed to movement of the folding-transition state. At low concentrations of denaturant, the transition state occurs early in the folding reaction (low Tanford beta-value), whereas at high-denaturant concentration, it moves closer to the native structure (high Tanford beta-value). Here, we use this movement to track the formation and growth of zinc-substituted azurin's folding nucleus with atomic resolution using protein engineering. The average phi (phi) value for 17 positions (covering all secondary-structure elements) goes from 0.25 in 0 M GuHCl (beta approximately 0.46) to 0.76 in 4 M GuHCl (beta approximately 0.86); a phi-value of 1 or 0 indicates native-like or unfolded-like interactions, respectively. Analysis of individual phi-values reveals a delocalized nucleus where structure condenses around a leading density centered on Leu50 in the core. The diffuse moving transition state for zinc-substituted azurin is in sharp contrast to the fixed polarized folding nucleus observed for apo-azurin. The dramatic difference in apparent kinetic behavior for the two forms of azurin can be rationalized as a minor alteration on a common free-energy profile that exhibits a broad activation barrier.
Wilson
Corey J
CJ
Department of Biochemistry and Cell Biology, Keck Center for Structural Computational Biology, Houston, Texas 77251, USA.
Wittung-Stafshede
Pernilla
P
D019943:Amino Acid Substitution / Q000235:genetics; D001059:Apoproteins / Q000737:chemistry; D001400:Azurin / Q000737:chemistry* / Q000235:genetics; D019791:Guanidine / Q000737:chemistry; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011550:Pseudomonas aeruginosa / Q000737:chemistry* / Q000235:genetics; D013816:Thermodynamics; D015032:Zinc / Q000737:chemistry*
D001059:Apoproteins; D001400:Azurin; D015032:Zinc; D019791:Guanidine