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. Study holds ProTherm entries: 19226, 19227, 19228, 19229, 19230, 19231, 19232, 19233, 19234, 19235, 19236, 19237, 19238, 19239, 19240, 19241, 19242, 19243 Extra Details: two-state equilibrium; zinc; folding nucleus; free-energy profile
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:51 p.m.
|Number of data points||53|
|Proteins||Azurin ; Azurin|
|Assays/Quantities/Protocols||Experimental Assay: m ; Experimental Assay: dG_H2O ; Derived Quantity: ddG_H2O|
|Libraries||Mutations for sequence AECSVDIQGNDQMQFNTNAITVDKSCKQFTVNLSHPGNLPKNVMGHNWVLSTAADMQGVVTDGMASGLDKDYLKPDDSRVIAHTKLIGSGEKDSVTFDVSKLKEGEQYMFFCTFPGHSALMKGTLTLK|