The effect of pressure and guanidine hydrochloride on azurins mutated in the hydrophobic core.


Abstract

The unfolding of the blue-copper protein azurin from Pseudomonas aeruginosa by guanidine hydrochloride, under nonreducing conditions, has been studied by fluorescence techniques and circular dichroism. The denaturation transition may be fitted by a simple two-state model. The total free energy change from the native to the unfolded state was 9.4 +/- 0.4 kcal.mol-1, while a lower value (6.4 +/- 0.4 kcal.mol-1) was obtained for the metal depleted enzyme (apo-azurin) suggesting that the copper atom plays an important stabilization role. Azurin and apo-azurin were practically unaffected by hydrostatic pressure up to 3000 bar. Site-directed mutagenesis has been used to destabilize the hydrophobic core of azurin. In particular either hydrophobic residue Ile7 or Phe110 has been substituted with a serine. The free energy change of unfolding by guanidinium hydrochloride, resulted to be 5.8 +/- 0.3 kcal.mol-1 and 4.8 +/- 0.3 kcal.mol-1 for Ile7Ser and Phe110Ser, respectively, showing that both mutants are much less stable than the wild-type protein. The mutated apoproteins could be reversible denatured even by high pressure, as demonstrated by steady-state fluorescence measurements. The change in volume associated to the pressure-induced unfolding was estimated to be -24 mL.mol-1 for Ile7Ser and -55 mL.mol-1 for Phe110Ser. These results show that the tight packing of the hydrophobic residues that characterize the inner structure of azurin is fundamental for the protein stability. This suggests that the proper assembly of the hydrophobic core is one of the earliest and most crucial event in the folding process, bearing important implication for de novo design of proteins. Study holds ProTherm entries: 12978, 12979, 12980, 12981, 12982, 12983 Extra Details: azurin; protein folding; hydrophobic interaction;,dynamic fluorescence; high pressure

Submission Details

ID: ySNAheyj

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:44 p.m.

Version: 1

Publication Details
Mei G;Di Venere A;Campeggi FM;Gilardi G;Rosato N;De Matteis F;Finazzi-Agrò A,Eur. J. Biochem. (1999) The effect of pressure and guanidine hydrochloride on azurins mutated in the hydrophobic core. PMID:10504393
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
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)
2FNW 2006-10-03 1.4 Pseudomonas aeruginosa E2Q/H83Q/M109H-azurin RE(PHEN)(CO)3
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)
2I7O 2007-08-14 1.5 Structure of Re(4,7-dimethyl-phen)(Thr124His)(Lys122Trp)(His83Gln)AzCu(II), a Rhenium modified Azurin mutant
1XB3 2004-10-19 1.5 The D62C/K74C double mutant of Pseudomonas Aeruginosa Azurin
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'
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
2GHZ 2006-07-04 1.6 Crystal structure of Azurin Phe114Pro mutant
1JZE 2001-10-17 1.6 Pseudomonas aeruginosa Azurin Ru(bpy)2(im)(His83)
6GYI 2019-01-30 1.6 Azurin fom Pseudomonas aeruginosa treated with hydrosulfide
2XV2 2010-12-29 1.6 Pseudomonas aeruginosa Azurin with mutated metal-binding loop sequence (CAAHAAM), chemically reduced, pH4.2
2XV0 2010-12-29 1.6 Pseudomonas aeruginosa Azurin with mutated metal-binding loop sequence (CAAHAAM), chemically reduced, pH4.8
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
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
3UGE 2012-11-07 1.7 Silver Metallated Pseudomonas aeruginosa Azurin at 1.70 A
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)
2HX9 2007-01-30 1.7 Crystal structure of Cu(I) Azurin with the metal-binding loop sequence 'CTFPGHSALM' replaced with 'CSPHQGAGM', at pH4
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
1JZJ 2001-10-24 1.8 Pseudomonas aeruginosa Azurin Os(bpy)2(im)(His83)
1I53 2001-10-17 1.8 RE(I)-TRICARBONYL DIIMINE (Q107H)) AZURIN
3JTB 2009-11-17 1.8 Cu(II) N47S/F114N variant of Pseudomonas Aeruginosa Azurin
3FT0 2009-03-10 1.8 Pseudomonas aeruginosa Azurin with mutated metal-binding loop sequence (CAAAAHAAAAM), chemically reduced
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
3FQY 2009-11-10 1.9 Azurin C112D
1R1C 2003-09-30 1.9 PSEUDOMONAS AERUGINOSA W48F/Y72F/H83Q/Y108W-AZURIN RE(PHEN)(CO)3(HIS107)
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
3FQ1 2009-11-10 1.9 Azurin C112D/M121I
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
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
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
1E5Z 2000-08-19 2.0 Azurin from Pseudomonas aeruginosa, reduced form, pH 9.0
4QLW 2014-08-13 2.0 Azurin mutant M121E with iron
1XB8 2004-10-19 2.0 Zn substituted form of D62C/K74C double mutant of Pseudomonas Aeruginosa Azurin
4HHW 2012-11-21 2.0 Crystal structure of the Pseudomonas aeruginosa azurin, H124NO YOH122
6IAV 2019-01-30 2.0 CO-AZURIN FROM PSEUDOMONAS AERUGINOSA TREATED WITH HYDROSULFIDE
3FSZ 2009-03-10 2.0 Pseudomonas aeruginosa Azurin with mutated metal-binding loop sequence (CAAAAHAAAAM)
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)
1E5Y 2000-08-19 2.0 Azurin from Pseudomonas aeruginosa, reduced form, pH 5.5
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?
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)
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
3FPY 2009-11-10 2.1 Azurin C112D/M121L
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
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
2OJ1 2007-11-27 2.3 Disulfide-linked dimer of azurin N42C/M64E double mutant
2TSB 1996-11-08 2.3 AZURIN MUTANT M121A-AZIDE
2XV3 2010-12-29 2.3 Pseudomonas aeruginosa Azurin with mutated metal-binding loop sequence (CAAAAHAAAAM), chemically reduced, pH5.3
1ETJ 1997-04-21 2.3 AZURIN MUTANT WITH MET 121 REPLACED BY GLU
1ILU 1996-03-08 2.3 X-RAY CRYSTAL STRUCTURE THE TWO SITE-SPECIFIC MUTANTS ILE7SER AND PHE110SER OF AZURIN FROM PSEUDOMONAS AERUGINOSA
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
3OQR 2010-10-13 2.4 C112D/M121E Azurin, pH 10.0
1AG0 1997-10-29 2.4 STRUCTURE OF CYS 112 ASP AZURIN FROM PSEUDOMONAS AERUGINOSA
1AZR 1993-07-15 2.4 CRYSTAL STRUCTURE OF PSEUDOMONAS AERUGINOSA ZINC AZURIN MUTANT ASP47ASP AT 2.4 ANGSTROMS RESOLUTION
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
100.0 Azurin P00282 AZUR_PSEAE
99.2 Azurin B3EWN9 AZUR_PSEAI