A spectroscopic and calorimetric investigation on the thermal stability of the Cys3Ala/Cys26Ala azurin mutant.


Abstract

The disulfide bond connecting Cys-3 and Cys-26 in wild type azurin has been removed to study the contribution of the -SS- bond to the high thermal resistance previously registered for this protein (. J. Phys. Chem. 99:14864-14870). Site-directed mutagenesis was used to replace both cysteines for alanines. The characterization of the Cys-3Ala/Cys-26Ala azurin mutant has been carried out by means of electron paramagnetic resonance spectroscopy at 77 K, UV-VIS optical absorption, fluorescence emission and circular dichroism at room temperature. The results show that the spectral features of the Cys-3Ala/Cys-26Ala azurin resemble those of the wild type azurin, indicating that the double mutation does not affect either the formation of the protein's overall structure or the assembly of the metal-binding site. The thermal unfolding of the Cys-3Ala/Cys-26Ala azurin has been followed by differential scanning calorimetry, optical absorption variation at lambda(max) = 625 nm, and fluorescence emission using 295 nm as excitation wavelength. The analysis of the data shows that the thermal transition from the native to the denaturated state of the modified azurin follows the same multistep unfolding pathway as observed in wild type azurin. However, the removal of the disulfide bridge results in a dramatic reduction of the thermodynamic stability of the protein. In fact, the transition temperatures registered by the different techniques are down-shifted by about 20 degrees C with respect to wild type azurin. Moreover, the Gibbs free energy value is about half of that found for the native azurin. These results suggest that the disulfide bridge is a structural element that significantly contributes to the high stability of wild type azurin. Study holds ProTherm entries: 14595, 14596, 14597, 14598, 14599, 14600, 14601 Extra Details: SS bond; high thermal resistance; Cys-3Ala/Cys-26Ala azurin mutant; multistep unfolding thermodynamic stability

Submission Details

ID: 4ciacPDt

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:45 p.m.

Version: 1

Publication Details
Guzzi R;Sportelli L;La Rosa C;Milardi D;Grasso D;Verbeet MP;Canters GW,Biophys. J. (1999) A spectroscopic and calorimetric investigation on the thermal stability of the Cys3Ala/Cys26Ala azurin mutant. PMID:10423449
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'
3N2J 2011-04-06 1.35 Azurin H117G, oxidized form
2I7S 2006-12-05 1.35 Crystal structure of Re(phen)(CO)3 (Thr124His)(His83Gln) Azurin Cu(II) from Pseudomonas aeruginosa
2FNW 2006-10-03 1.4 Pseudomonas aeruginosa E2Q/H83Q/M109H-azurin RE(PHEN)(CO)3
1JZG 2001-10-17 1.4 Pseudomonas aeruginosa Reduced Azurin (Cu1+) Ru(tpy)(phen)(His83)
2FT7 2006-04-11 1.4 Structure of Cu(I)azurin at pH 6, with the metal-binding loop sequence 'CTFPGHSALM' replaced with 'CTPHPM'
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.
1JZF 2001-10-17 1.5 Pseudomonas aeruginosa Oxidized Azurin(Cu2+) Ru(tpy)(phen)(His83)
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
4MFH 2014-01-15 1.54 Crystal Structure of M121G Azurin
4JKN 2014-02-19 1.54 Mercury Metallated Pseudomonas aeruginosa Azurin at 1.54 A
2HX7 2007-01-30 1.55 Crystal structure of Cu(II) Azurin with the metal-binding loop sequence 'CTFPGHSALM' replaced with 'CSPHQGAGM'
2FT8 2006-04-11 1.55 Structure of Cu(I)azurin, pH8, with the metal-binding loop sequence 'CTFPGHSALM' replaced with 'CTPHPM'
2XV2 2010-12-29 1.6 Pseudomonas aeruginosa Azurin with mutated metal-binding loop sequence (CAAHAAM), chemically reduced, pH4.2
2GHZ 2006-07-04 1.6 Crystal structure of Azurin Phe114Pro mutant
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)
2XV0 2010-12-29 1.6 Pseudomonas aeruginosa Azurin with mutated metal-binding loop sequence (CAAHAAM), chemically reduced, pH4.8
6GYI 2019-01-30 1.6 Azurin fom Pseudomonas aeruginosa treated with hydrosulfide
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
2GI0 2006-07-04 1.7 Crystal structure of Cu(I) Phe114Pro Azurin mutant
1JZH 2001-10-17 1.7 Pseudomonas aeruginosa Azurin Ru(tpy)(bpy)(His83)
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
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
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)
3JTB 2009-11-17 1.8 Cu(II) N47S/F114N variant of Pseudomonas Aeruginosa Azurin
1GR7 2002-05-16 1.8 Crystal structure of the double mutant Cys3Ser/Ser100Pro from Pseudomonas Aeruginosa at 1.8 A resolution
1I53 2001-10-17 1.8 RE(I)-TRICARBONLY DIIMINE (Q107H)) AZURIN
3FT0 2009-03-10 1.8 Pseudomonas aeruginosa Azurin with mutated metal-binding loop sequence (CAAAAHAAAAM), chemically reduced
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
6MJS 2019-02-20 1.85 Azurin 122W/124W/126Re
1E65 2000-08-16 1.85 Azurin from Pseudomonas aeruginosa, apo form
4KOB 2014-05-14 1.87 Investigating the functional significance of the interlocked pair structural determinants in Pseudomonas aeruginosa azurin (V31I/V95I)
6MJT 2019-02-20 1.89 Azurin 122F/124W/126Re
5I26 2016-04-13 1.89 Azurin T30R1, crystal form I
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
1VLX 1997-03-12 1.9 STRUCTURE OF ELECTRON TRANSFER (COBALT-PROTEIN)
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
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
1R1C 2003-09-30 1.9 PSEUDOMONAS AERUGINOSA W48F/Y72F/H83Q/Y108W-AZURIN RE(PHEN)(CO)3(HIS107)
3FQ1 2009-11-10 1.9 Azurin C112D/M121I
3FQY 2009-11-10 1.9 Azurin C112D
4HIP 2012-11-21 1.9 Crystal structure of the Pseudomonas aeruginosa azurin, H126NO YOH109
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
4HHW 2012-11-21 2.0 Crystal structure of the Pseudomonas aeruginosa azurin, H124NO YOH122
1E5Y 2000-08-19 2.0 Azurin from Pseudomonas aeruginosa, reduced form, pH 5.5
1XB8 2004-10-19 2.0 Zn substituted form of D62C/K74C double mutant of Pseudomonas Aeruginosa Azurin
6IAV 2019-01-30 2.0 CO-AZURIN FROM PSEUDOMONAS AERUGINOSA TREATED WITH HYDROSULFIDE
1E5Z 2000-08-19 2.0 Azurin from Pseudomonas aeruginosa, reduced form, pH 9.0
3FSZ 2009-03-10 2.0 Pseudomonas aeruginosa Azurin with mutated metal-binding loop sequence (CAAAAHAAAAM)
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?
4QLW 2014-08-13 2.0 Azurin mutant M121E with iron
1JVL 2002-01-04 2.0 Azurin dimer, covalently crosslinked through bis-maleimidomethylether
3FSW 2009-03-10 2.0 Pseudomonas aeruginosa Azurin with mutated metal-binding loop sequence (CAAAAHAAAM)
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)
3FPY 2009-11-10 2.1 Azurin C112D/M121L
3JT2 2009-11-17 2.1 Cu(II) N47S/M121L variant of Pseudomonas Aeruginosa azurin
3AZU 1993-07-15 2.1 X-RAY CRYSTAL STRUCTURE OF THE TWO SITE-SPECIFIC MUTANTS HIS35GLN AND HIS35LEU OF AZURIN FROM PSEUDOMONAS AERUGINOSA
3NP3 2010-10-13 2.1 C112D/M121E 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
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
2TSB 1996-11-08 2.3 AZURIN MUTANT M121A-AZIDE
1ILU 1996-03-08 2.3 X-RAY CRYSTAL STRUCTURE THE TWO SITE-SPECIFIC MUTANTS ILE7SER AND PHE110SER OF AZURIN FROM PSEUDOMONAS AERUGINOSA
2XV3 2010-12-29 2.3 Pseudomonas aeruginosa Azurin with mutated metal-binding loop sequence (CAAAAHAAAAM), chemically reduced, pH5.3
3FSV 2009-03-10 2.3 Pseudomonas aeruginosa Azurin with mutated metal-binding loop sequence (CAAAHAAAM)
1BEX 1998-10-14 2.3 STRUCTURE OF RUTHENIUM-MODIFIED PSEUDOMONAS AERUGINOSA AZURIN
3IN0 2009-10-27 2.35 Crystal structure of the F114P/M121Q variant of Pseudomonas aeruginosa azurin in the Cu(II) state
5SYD 2017-08-16 2.4 Circularly permutated azurin (cpAz) based on P. aeruginosa azurin sequence
1AZR 1993-07-15 2.4 CRYSTAL STRUCTURE OF PSEUDOMONAS AERUGINOSA ZINC AZURIN MUTANT ASP47ASP AT 2.4 ANGSTROMS RESOLUTION
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