Snapshots of a dynamic folding nucleus in zinc-substituted Pseudomonas aeruginosa azurin.


Abstract

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

Submission Details

ID: d7nHzxjE4

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:51 p.m.

Version: 1

Publication Details
Wilson CJ;Wittung-Stafshede P,Biochemistry (2005) Snapshots of a dynamic folding nucleus in zinc-substituted Pseudomonas aeruginosa azurin. PMID:16042382
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
1AG0 1997-03-26T00:00:00+0000 2.4 STRUCTURE OF CYS 112 ASP AZURIN FROM PSEUDOMONAS AERUGINOSA
1AZN 1994-05-27T00:00:00+0000 2.6 CRYSTAL STRUCTURE OF THE AZURIN MUTANT PHE114ALA FROM PSEUDOMONAS AERUGINOSA AT 2.6 ANGSTROMS RESOLUTION
1AZR 1993-03-04T00:00:00+0000 2.4 CRYSTAL STRUCTURE OF PSEUDOMONAS AERUGINOSA ZINC AZURIN MUTANT ASP47ASP AT 2.4 ANGSTROMS RESOLUTION
1AZU 1980-08-04T00:00:00+0000 2.7 STRUCTURAL FEATURES OF AZURIN AT 2.7 ANGSTROMS RESOLUTION
1BEX 1998-05-18T00:00:00+0000 2.3 STRUCTURE OF RUTHENIUM-MODIFIED PSEUDOMONAS AERUGINOSA AZURIN
1CC3 1999-03-03T00:00:00+0000 1.65 PURPLE CUA CENTER
1E5Y 2000-08-04T00:00:00+0000 2.0 Azurin from Pseudomonas aeruginosa, reduced form, pH 5.5
1E5Z 2000-08-04T00:00:00+0000 2.0 Azurin from Pseudomonas aeruginosa, reduced form, pH 9.0
1E65 2000-08-08T00:00:00+0000 1.85 Azurin from Pseudomonas aeruginosa, apo form
1E67 2000-08-09T00:00:00+0000 2.14 Zn-Azurin from Pseudomonas aeruginosa

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
99.2 Azurin B3EWN9 AZUR_PSEAI
100.0 Azurin P00282 AZUR_PSEAE