Effects of a novel disulfide bond and engineered electrostatic interactions on the thermostability of azurin.


Abstract

Identification and evaluation of factors important for thermostability in proteins is a growing research field with many industrial applications. This study investigates the effects of introducing a novel disulfide bond and engineered electrostatic interactions with respect to the thermostability of holo azurin from Pseudomonas aeruginosa. Four mutants were selected on the basis of rational design and novel temperature-dependent atomic displacement factors from crystal data collected at elevated temperatures. The atomic displacement parameters describe the molecular movement at higher temperatures. The thermostability was evaluated by optical spectroscopy as well as by differential scanning calorimetry. Although azurin has a high inherent stability, the introduction of a novel disulfide bond connecting a flexible loop with small alpha-helix (D62C/K74C copper-containing mutant), increased the T(m) by 3.7 degrees C compared with the holo protein. Furthermore, three mutants were designed to introduce electrostatic interactions, K24R, D23E/K128R, and D23E/K128R/K24R. Mutant K24R stabilizes loops between two separate beta-strands and D23E/K128R was selected to stabilize the C-terminus of azurin. Furthermore, D23E/K128R/K24R was selected to reflect the combination of the electrostatic interactions in D23E/K128R and K24R. The mutants involving electrostatic interactions had a minor effect on the thermostability. The crystal structures of the copper-containing mutants D62C/K74C and K24R have been determined to 1.5 and 1.8 A resolution. In addition the crystal structure of the zinc-loaded mutant D62C/K74C has also been completed to 1.8 A resolution. These structures support the selected design and provide valuable information for evaluating effects of the modifications on the thermostability of holo azurin. Study holds ProTherm entries: 18348, 18349, 18350, 18351, 18352, 18353, 18354, 18355, 18356, 18357, 18358, 18359, 18360, 18362, 18363, 18364, 18365, 18366, 18367, 18368, 18369, 18370, 18371, 18372, 18373, 18374, 18375, 18376, 18377 Extra Details: Experiment was done at the scan rate of 20 degrees C/hour disulfide bond; electrostatic interactions; atomic displacement parameters; copper-containing mutants

Submission Details

ID: mfNDjX43

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:50 p.m.

Version: 1

Publication Details
Tigerström A;Schwarz F;Karlsson G;Okvist M;Alvarez-Rúa C;Maeder D;Robb FT;Sjölin L,Biochemistry (2004) Effects of a novel disulfide bond and engineered electrostatic interactions on the thermostability of azurin. PMID:15449946
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
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'
2XV0 2010-12-29 1.6 Pseudomonas aeruginosa Azurin with mutated metal-binding loop sequence (CAAHAAM), chemically reduced, pH4.8
2XV2 2010-12-29 1.6 Pseudomonas aeruginosa Azurin with mutated metal-binding loop sequence (CAAHAAM), chemically reduced, pH4.2
2HX8 2007-01-30 1.6 Crystal structure of Cu(I) Azurin with the metal-binding loop sequence 'CTFPGHSALM' replaced with 'CSPHQGAGM', at pH5
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
2GHZ 2006-07-04 1.6 Crystal structure of Azurin Phe114Pro mutant
4HHG 2012-11-21 1.6 Crystal structure of the Pseudomonas aeruginosa azurin, RuH107NO YOH109
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
1JZH 2001-10-17 1.7 Pseudomonas aeruginosa Azurin Ru(tpy)(bpy)(His83)
3UGE 2012-11-07 1.7 Silver Metallated Pseudomonas aeruginosa Azurin at 1.70 A
2HX9 2007-01-30 1.7 Crystal structure of Cu(I) Azurin with the metal-binding loop sequence 'CTFPGHSALM' replaced with 'CSPHQGAGM', at pH4
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)
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)
1I53 2001-10-17 1.8 RE(I)-TRICARBONLY DIIMINE (Q107H)) AZURIN
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
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
1VLX 1997-03-12 1.9 STRUCTURE OF ELECTRON TRANSFER (COBALT-PROTEIN)
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
3FQ1 2009-11-10 1.9 Azurin C112D/M121I
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
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
4HIP 2012-11-21 1.9 Crystal structure of the Pseudomonas aeruginosa azurin, H126NO YOH109
1R1C 2003-09-30 1.9 PSEUDOMONAS AERUGINOSA W48F/Y72F/H83Q/Y108W-AZURIN RE(PHEN)(CO)3(HIS107)
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
4QLW 2014-08-13 2.0 Azurin mutant M121E with iron
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
3FSZ 2009-03-10 2.0 Pseudomonas aeruginosa Azurin with mutated metal-binding loop sequence (CAAAAHAAAAM)
1E5Y 2000-08-19 2.0 Azurin from Pseudomonas aeruginosa, reduced form, pH 5.5
3FSW 2009-03-10 2.0 Pseudomonas aeruginosa Azurin with mutated metal-binding loop sequence (CAAAAHAAAM)
1JVL 2002-01-04 2.0 Azurin dimer, covalently crosslinked through bis-maleimidomethylether
1E5Z 2000-08-19 2.0 Azurin from Pseudomonas aeruginosa, reduced form, pH 9.0
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
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
3NP3 2010-10-13 2.1 C112D/M121E 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
3FPY 2009-11-10 2.1 Azurin C112D/M121L
1E67 2000-08-16 2.14 Zn-Azurin from Pseudomonas aeruginosa
4HZ1 2012-12-12 2.2 Crystal Structure of Pseudomonas aeruginosa azurin with iron(II) at the copper-binding site.
1ILS 1996-03-08 2.2 X-RAY CRYSTAL STRUCTURE THE TWO SITE-SPECIFIC MUTANTS ILE7SER AND PHE110SER OF 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
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
3NP4 2010-10-13 2.25 C112D/M121E Pseudomonas aeruginosa Azurin
2IDF 2008-03-18 2.25 P. aeruginosa azurin N42C/M64E double mutant, BMME-linked dimer
1ETJ 1997-04-21 2.3 AZURIN MUTANT WITH MET 121 REPLACED BY GLU
2OJ1 2007-11-27 2.3 Disulfide-linked dimer of azurin N42C/M64E double mutant
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)
2TSB 1996-11-08 2.3 AZURIN MUTANT M121A-AZIDE
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
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
1AZN 1994-10-15 2.6 CRYSTAL STRUCTURE OF THE AZURIN MUTANT PHE114ALA FROM PSEUDOMONAS AERUGINOSA AT 2.6 ANGSTROMS RESOLUTION
3IN2 2009-11-17 2.6 Crystal structure of the N47S/M121L variant of Pseudomonas aeruginosa azurin in the Cu(II) state
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