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
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
100.0 Azurin P00282 AZUR_PSEAE
99.2 Azurin B3EWN9 AZUR_PSEAI