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
3FSA 2009-03-10 0.98 Pseudomonas aeruginosa Azurin with mutated metal-binding loop sequence (CAAHAAM); chemically reduced.
3FS9 2009-03-10 1.05 Pseudomonas aeruginosa Azurin with mutated metal-binding loop sequence (CAAHAAM)
3U25 2011-12-28 1.18 Crystal structure of P. aeruginoas azurin containing a Tyr-His hydrogen bonded pair
2FT6 2006-04-11 1.25 Structure of Cu(II)azurin with the metal-binding loop sequence 'CTFPGHSALM' replaced with 'CTPHPM'
2I7S 2006-12-05 1.35 Crystal structure of Re(phen)(CO)3 (Thr124His)(His83Gln) Azurin Cu(II) from Pseudomonas aeruginosa
3N2J 2011-04-06 1.35 Azurin H117G, oxidized form
2FNW 2006-10-03 1.4 Pseudomonas aeruginosa E2Q/H83Q/M109H-azurin RE(PHEN)(CO)3
2FT7 2006-04-11 1.4 Structure of Cu(I)azurin at pH 6, with the metal-binding loop sequence 'CTFPGHSALM' replaced with 'CTPHPM'
1JZG 2001-10-17 1.4 Pseudomonas aeruginosa Reduced Azurin (Cu1+) Ru(tpy)(phen)(His83)
3IBO 2009-12-08 1.45 Pseudomonas aeruginosa E2Q/H83Q/T126H-azurin RE(PHEN)(CO)3
4KOC 2014-05-14 1.46 Investigating the functional significance of the interlocked pair structural determinants in Pseudomonas aeruginosa azurin (V31I/V95I/Y108F)
4BWW 2014-06-11 1.48 Crystal structure of spin labelled azurin T21R1.
1XB3 2004-10-19 1.5 The D62C/K74C double mutant of Pseudomonas Aeruginosa Azurin
2I7O 2007-08-14 1.5 Structure of Re(4,7-dimethyl-phen)(Thr124His)(Lys122Trp)(His83Gln)AzCu(II), a Rhenium modified Azurin mutant
1JZF 2001-10-17 1.5 Pseudomonas aeruginosa Oxidized Azurin(Cu2+) Ru(tpy)(phen)(His83)
4JKN 2014-02-19 1.54 Mercury Metallated Pseudomonas aeruginosa Azurin at 1.54 A
4MFH 2014-01-15 1.54 Crystal Structure of M121G Azurin
2FT8 2006-04-11 1.55 Structure of Cu(I)azurin, pH8, with the metal-binding loop sequence 'CTFPGHSALM' replaced with 'CTPHPM'
2HX7 2007-01-30 1.55 Crystal structure of Cu(II) Azurin with the metal-binding loop sequence 'CTFPGHSALM' replaced with 'CSPHQGAGM'
2XV2 2010-12-29 1.6 Pseudomonas aeruginosa Azurin with mutated metal-binding loop sequence (CAAHAAM), chemically reduced, pH4.2
6GYI 2019-01-30 1.6 Azurin fom Pseudomonas aeruginosa treated with hydrosulfide
2GHZ 2006-07-04 1.6 Crystal structure of Azurin Phe114Pro mutant
2XV0 2010-12-29 1.6 Pseudomonas aeruginosa Azurin with mutated metal-binding loop sequence (CAAHAAM), chemically reduced, pH4.8
2HX8 2007-01-30 1.6 Crystal structure of Cu(I) Azurin with the metal-binding loop sequence 'CTFPGHSALM' replaced with 'CSPHQGAGM', at pH5
4HHG 2012-11-21 1.6 Crystal structure of the Pseudomonas aeruginosa azurin, RuH107NO YOH109
1JZE 2001-10-17 1.6 Pseudomonas aeruginosa Azurin Ru(bpy)2(im)(His83)
2FTA 2006-04-11 1.61 Structure of Cu(II)azurin with the metal-binding loop sequence 'CTFPGHSALM' replaced with 'CTPHPFM'
1JZI 2001-10-17 1.62 Pseudomonas aeruginosa Azurin Re(phen)(CO)3(His83)
4QKT 2014-08-13 1.64 Azurin mutant M121EM44K with copper
1CC3 1999-12-23 1.65 PURPLE CUA CENTER
5YT7 2018-08-08 1.66 crystal structure of circularly permutated Azurin 3
1JZH 2001-10-17 1.7 Pseudomonas aeruginosa Azurin Ru(tpy)(bpy)(His83)
2HX9 2007-01-30 1.7 Crystal structure of Cu(I) Azurin with the metal-binding loop sequence 'CTFPGHSALM' replaced with 'CSPHQGAGM', at pH4
3UGE 2012-11-07 1.7 Silver Metallated Pseudomonas aeruginosa Azurin at 1.70 A
4K9J 2013-10-02 1.7 Structure of Re(CO)3(4,7-dimethyl-phen)(Thr126His)(Lys122Trp)(His83Glu)(Trp48Phe)(Tyr72Phe)(Tyr108Phe)AzCu(II), a Rhenium modified Azurin mutant
2GI0 2006-07-04 1.7 Crystal structure of Cu(I) Phe114Pro Azurin mutant
4KO6 2014-05-14 1.74 Investigating the functional significance of the interlocked pair structural determinants in Pseudomonas aeruginosa azurin (V31I/V95K/Y108F)
4KO5 2014-05-14 1.79 Investigating the functional significance of the interlocked pair structural determinants in Pseudomonas aeruginosa azurin (V31I/W48L/V95I/Y108F)
1GR7 2002-05-16 1.8 Crystal structure of the double mutant Cys3Ser/Ser100Pro from Pseudomonas Aeruginosa at 1.8 A resolution
3FT0 2009-03-10 1.8 Pseudomonas aeruginosa Azurin with mutated metal-binding loop sequence (CAAAAHAAAAM), chemically reduced
1I53 2001-10-17 1.8 RE(I)-TRICARBONLY DIIMINE (Q107H)) AZURIN
3JTB 2009-11-17 1.8 Cu(II) N47S/F114N variant of Pseudomonas Aeruginosa Azurin
1JZJ 2001-10-24 1.8 Pseudomonas aeruginosa Azurin Os(bpy)2(im)(His83)
1XB6 2004-10-19 1.82 The K24R mutant of Pseudomonas Aeruginosa Azurin
1E65 2000-08-16 1.85 Azurin from Pseudomonas aeruginosa, apo form
6MJS 2019-02-20 1.85 Azurin 122W/124W/126Re
4KOB 2014-05-14 1.87 Investigating the functional significance of the interlocked pair structural determinants in Pseudomonas aeruginosa azurin (V31I/V95I)
5I26 2016-04-13 1.89 Azurin T30R1, crystal form I
6MJT 2019-02-20 1.89 Azurin 122F/124W/126Re
4HIP 2012-11-21 1.9 Crystal structure of the Pseudomonas aeruginosa azurin, H126NO YOH109
5AZU 1994-01-31 1.9 CRYSTAL STRUCTURE ANALYSIS OF OXIDIZED PSEUDOMONAS AERUGINOSA AZURIN AT PH 5.5 AND PH 9.0. A PH-INDUCED CONFORMATIONAL TRANSITION INVOLVES A PEPTIDE BOND FLIP
3FQY 2009-11-10 1.9 Azurin C112D
1VLX 1997-03-12 1.9 STRUCTURE OF ELECTRON TRANSFER (COBALT-PROTEIN)
1R1C 2003-09-30 1.9 PSEUDOMONAS AERUGINOSA W48F/Y72F/H83Q/Y108W-AZURIN RE(PHEN)(CO)3(HIS107)
4AZU 1994-01-31 1.9 CRYSTAL STRUCTURE ANALYSIS OF OXIDIZED PSEUDOMONAS AERUGINOSA AZURIN AT PH 5.5 AND PH 9.0. A PH-INDUCED CONFORMATIONAL TRANSITION INVOLVES A PEPTIDE BOND FLIP
3FQ1 2009-11-10 1.9 Azurin C112D/M121I
2AZU 1993-07-15 1.9 X-RAY CRYSTAL STRUCTURE OF THE TWO SITE-SPECIFIC MUTANTS HIS35*GLN AND HIS35*LEU OF AZURIN FROM PSEUDOMONAS AERUGINOSA
3FQ2 2009-11-10 1.91 Azurin C112D/M121F
4WKX 2015-10-07 1.94 Reversible S-Nitrosylation in an Engineered Mutant of Pseudomonas aeruginosa Azurin with Red Copper Site
5I28 2016-04-13 1.95 Azurin T30R1, crystal form II
3FSZ 2009-03-10 2.0 Pseudomonas aeruginosa Azurin with mutated metal-binding loop sequence (CAAAAHAAAAM)
3FSW 2009-03-10 2.0 Pseudomonas aeruginosa Azurin with mutated metal-binding loop sequence (CAAAAHAAAM)
1E5Z 2000-08-19 2.0 Azurin from Pseudomonas aeruginosa, reduced form, pH 9.0
1E5Y 2000-08-19 2.0 Azurin from Pseudomonas aeruginosa, reduced form, pH 5.5
6IAV 2019-01-30 2.0 CO-AZURIN FROM PSEUDOMONAS AERUGINOSA TREATED WITH HYDROSULFIDE
1JVL 2002-01-04 2.0 Azurin dimer, covalently crosslinked through bis-maleimidomethylether
1EZL 2000-08-09 2.0 CRYSTAL STRUCTURE OF THE DISULPHIDE BOND-DEFICIENT AZURIN MUTANT C3A/C26A: HOW IMPORTANT IS THE S-S BOND FOR FOLDING AND STABILITY?
4HHW 2012-11-21 2.0 Crystal structure of the Pseudomonas aeruginosa azurin, H124NO YOH122
1XB8 2004-10-19 2.0 Zn substituted form of D62C/K74C double mutant of Pseudomonas Aeruginosa Azurin
4QLW 2014-08-13 2.0 Azurin mutant M121E with iron
6MJR 2019-02-20 2.01 Azurin 122W/124F/126Re
4KO9 2014-05-14 2.05 Investigating the functional significance of the interlocked pair structural determinants in Pseudomonas aeruginosa azurin (V95I/Y108F)
4KO7 2014-05-14 2.07 Investigating the functional significance of the interlocked pair structural determinants in Pseudomonas aeruginosa azurin (V31I/W48F/V95I)
3AZU 1993-07-15 2.1 X-RAY CRYSTAL STRUCTURE OF THE TWO SITE-SPECIFIC MUTANTS HIS35GLN AND HIS35LEU OF AZURIN FROM PSEUDOMONAS AERUGINOSA
3FPY 2009-11-10 2.1 Azurin C112D/M121L
3NP3 2010-10-13 2.1 C112D/M121E Pseudomonas Aeruginosa Azurin
3JT2 2009-11-17 2.1 Cu(II) N47S/M121L variant of Pseudomonas Aeruginosa azurin
1E67 2000-08-16 2.14 Zn-Azurin from Pseudomonas aeruginosa
1NZR 1995-02-27 2.2 CRYSTAL STRUCTURE OF THE AZURIN MUTANT NICKEL-TRP48MET FROM PSEUDOMONAS AERUGINOSA AT 2.2 ANGSTROMS RESOLUTION
2TSA 1996-11-08 2.2 AZURIN MUTANT M121A
1ILS 1996-03-08 2.2 X-RAY CRYSTAL STRUCTURE THE TWO SITE-SPECIFIC MUTANTS ILE7SER AND PHE110SER OF AZURIN FROM PSEUDOMONAS AERUGINOSA
4HZ1 2012-12-12 2.2 Crystal Structure of Pseudomonas aeruginosa azurin with iron(II) at the copper-binding site.
2HXA 2007-01-30 2.21 Crystal structure of Cu(I) Azurin with the metal-binding loop sequence 'CTFPGHSALM' replaced with 'CSPHQGAGM', at pH3.5
2IDF 2008-03-18 2.25 P. aeruginosa azurin N42C/M64E double mutant, BMME-linked dimer
3NP4 2010-10-13 2.25 C112D/M121E Pseudomonas aeruginosa Azurin
2XV3 2010-12-29 2.3 Pseudomonas aeruginosa Azurin with mutated metal-binding loop sequence (CAAAAHAAAAM), chemically reduced, pH5.3
1ILU 1996-03-08 2.3 X-RAY CRYSTAL STRUCTURE THE TWO SITE-SPECIFIC MUTANTS ILE7SER AND PHE110SER OF AZURIN FROM PSEUDOMONAS AERUGINOSA
1BEX 1998-10-14 2.3 STRUCTURE OF RUTHENIUM-MODIFIED PSEUDOMONAS AERUGINOSA AZURIN
2TSB 1996-11-08 2.3 AZURIN MUTANT M121A-AZIDE
2OJ1 2007-11-27 2.3 Disulfide-linked dimer of azurin N42C/M64E double mutant
1ETJ 1997-04-21 2.3 AZURIN MUTANT WITH MET 121 REPLACED BY GLU
3FSV 2009-03-10 2.3 Pseudomonas aeruginosa Azurin with mutated metal-binding loop sequence (CAAAHAAAM)
3IN0 2009-10-27 2.35 Crystal structure of the F114P/M121Q variant of Pseudomonas aeruginosa azurin in the Cu(II) state
1AZR 1993-07-15 2.4 CRYSTAL STRUCTURE OF PSEUDOMONAS AERUGINOSA ZINC AZURIN MUTANT ASP47ASP AT 2.4 ANGSTROMS RESOLUTION
5SYD 2017-08-16 2.4 Circularly permutated azurin (cpAz) based on P. aeruginosa azurin sequence
1AG0 1997-10-29 2.4 STRUCTURE OF CYS 112 ASP AZURIN FROM PSEUDOMONAS AERUGINOSA
3OQR 2010-10-13 2.4 C112D/M121E Azurin, pH 10.0
3IN2 2009-11-17 2.6 Crystal structure of the N47S/M121L variant of Pseudomonas aeruginosa azurin in the Cu(II) state
1AZN 1994-10-15 2.6 CRYSTAL STRUCTURE OF THE AZURIN MUTANT PHE114ALA FROM PSEUDOMONAS AERUGINOSA AT 2.6 ANGSTROMS RESOLUTION
1AZU 1980-09-26 2.7 STRUCTURAL FEATURES OF AZURIN AT 2.7 ANGSTROMS RESOLUTION
1JVO 2002-01-04 2.75 Azurin dimer, crosslinked via disulfide bridge
2IWE 2007-06-12 2.83 Structure of a cavity mutant (H117G) of Pseudomonas aeruginosa azurin

Relevant UniProtKB Entries

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