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
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