Thermodynamic study of the acid denaturation of barnase and its dependence on ionic strength: evidence for residual electrostatic interactions in the acid/thermally denatured state.


Abstract

We have investigated the acid denaturation of barnase and its dependence on ionic strength. From the pH dependence of the protein stability, we have obtained information about the titration properties of the native and denatured protein at temperatures ranging from 15 to 60 degrees C in the absence of chemical denaturant. It appears that both the native and the denatured state of barnase titrates at higher pH values in the presence of salt. The observation suggests that charge interactions are present, not only within the native fold but also within the denatured state, and that these interactions contribute to shift the pKa values from those of isolated model compounds. Upon addition of salt these repulsive interactions are shielded, and the electrostatic free energy of the native state, as well as the denatured state, is reduced. Accordingly, we suggest that the thermally denatured state of barnase is not an extended random coil without residue-residue interactions but is sufficiently compact to contain intramolecular charge-charge repulsions. The results further reveal that the native state of barnase contains at least one residue with a highly anomalous pKa value: At pH 0.3, the difference in degree of protonation between the native and the denatured state is still about 1 mol H+/mol protein. Study holds ProTherm entries: 4498 Extra Details: electrostatic interactions; random coil; protonation;,residue-residue interactions; charge-charge repulsions

Submission Details

ID: rH3frYkY3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:25 p.m.

Version: 1

Publication Details
Oliveberg M;Vuilleumier S;Fersht AR,Biochemistry (1994) Thermodynamic study of the acid denaturation of barnase and its dependence on ionic strength: evidence for residual electrostatic interactions in the acid/thermally denatured state. PMID:8038174
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
1BNR 1995-07-31 BARNASE
2KF3 2009-12-08 Barnase, low pressure reference NMR structure
2KF4 2009-12-08 Barnase high pressure structure
1FW7 2003-06-10 NMR STRUCTURE OF 15N-LABELED BARNASE
2KF6 2009-12-08 Barnase bound to d(CGAC) high pressure
2KF5 2009-12-08 Barnase bound to d(CGAC), low pressure
2C4B 2005-11-21 1.3 Inhibitor cystine knot protein McoEeTI fused to the catalytically inactive barnase mutant H102A
1A2P 1998-04-29 1.5 BARNASE WILDTYPE STRUCTURE AT 1.5 ANGSTROMS RESOLUTION
2ZA4 2008-05-20 1.58 Crystal Structural Analysis of Barnase-barstar Complex
1B20 1998-12-09 1.7 DELETION OF A BURIED SALT-BRIDGE IN BARNASE
1BRN 1994-01-31 1.76 SUBSITE BINDING IN AN RNASE: STRUCTURE OF A BARNASE-TETRANUCLEOTIDE COMPLEX AT 1.76 ANGSTROMS RESOLUTION
1B2X 1998-12-09 1.8 BARNASE WILDTYPE STRUCTURE AT PH 7.5 FROM A CRYO_COOLED CRYSTAL AT 100K
1B2S 1998-12-08 1.82 STRUCTURAL RESPONSE TO MUTATION AT A PROTEIN-PROTEIN INTERFACE
1RNB 1992-07-15 1.9 CRYSTAL STRUCTURE OF A BARNASE-D(*GP*C) COMPLEX AT 1.9 ANGSTROMS RESOLUTION
1BRI 1995-07-10 1.9 BARNASE MUTANT WITH ILE 76 REPLACED BY ALA
1X1Y 2005-04-26 1.9 Water-mediate interaction at aprotein-protein interface
3KCH 2010-03-09 1.94 Baranase crosslinked by glutaraldehyde
2F5M 2006-04-25 1.95 Cross-linked barnase soaked in bromo-ethanol
2F56 2006-04-25 1.96 Barnase cross-linked with glutaraldehyde soaked in 6M urea
1BRS 1994-06-22 2.0 PROTEIN-PROTEIN RECOGNITION: CRYSTAL STRUCTURAL ANALYSIS OF A BARNASE-BARSTAR COMPLEX AT 2.0-A RESOLUTION
2F5W 2006-04-25 2.0 Cross-linked barnase soaked in 3 M thiourea
1BNF 1995-07-10 2.0 BARNASE T70C/S92C DISULFIDE MUTANT
1BSA 1994-01-31 2.0 CRYSTAL STRUCTURAL ANALYSIS OF MUTATIONS IN THE HYDROPHOBIC CORES OF BARNASE
1BRK 1995-07-10 2.0 BARNASE MUTANT WITH ILE 96 REPLACED BY ALA
1BRJ 1995-07-10 2.0 BARNASE MUTANT WITH ILE 88 REPLACED BY ALA
1BSC 1994-01-31 2.0 CRYSTAL STRUCTURAL ANALYSIS OF MUTATIONS IN THE HYDROPHOBIC CORES OF BARNASE
1B21 1998-12-09 2.0 DELETION OF A BURIED SALT BRIDGE IN BARNASE
1BRH 1995-07-10 2.0 BARNASE MUTANT WITH LEU 14 REPLACED BY ALA
1BSE 1994-01-31 2.0 CRYSTAL STRUCTURAL ANALYSIS OF MUTATIONS IN THE HYDROPHOBIC CORES OF BARNASE
1BSB 1994-01-31 2.0 CRYSTAL STRUCTURAL ANALYSIS OF MUTATIONS IN THE HYDROPHOBIC CORES OF BARNASE
1B2Z 1998-12-09 2.03 DELETION OF A BURIED SALT BRIDGE IN BARNASE
1BNS 1994-06-22 2.05 STRUCTURAL STUDIES OF BARNASE MUTANTS
1BNG 1995-07-10 2.1 BARNASE S85C/H102C DISULFIDE MUTANT
1X1W 2005-04-26 2.1 Water-mediate interaction at aprotein-protein interface
1B27 1998-12-09 2.1 STRUCTURAL RESPONSE TO MUTATION AT A PROTEIN-PROTEIN INTERFACE
1BNJ 1995-09-15 2.1 BARNASE WILDTYPE STRUCTURE AT PH 9.0
1B2U 1998-12-09 2.1 STRUCTURAL RESPONSE TO MUTATION AT A PROTEIN-PROTEIN INTERFACE
1BNI 1995-09-15 2.1 BARNASE WILDTYPE STRUCTURE AT PH 6.0
1BNE 1995-07-10 2.1 BARNASE A43C/S80C DISULFIDE MUTANT
2F4Y 2006-04-25 2.15 Barnase cross-linked with glutaraldehyde
3Q3F 2012-01-25 2.17 Engineering Domain-Swapped Binding Interfaces by Mutually Exclusive Folding: Insertion of Ubiquitin into position 103 of Barnase
1YVS 1999-02-02 2.2 Trimeric domain swapped barnase
1BAO 1993-10-31 2.2 THE CONTRIBUTION OF BURIED HYDROGEN BONDS TO PROTEIN STABILITY: THE CRYSTAL STRUCTURES OF TWO BARNASE MUTANTS
1BRG 1994-06-22 2.2 CRYSTALLOGRAPHIC ANALYSIS OF PHE->LEU SUBSTITUTION IN THE HYDROPHOBIC CORE OF BARNASE
1BAN 1993-10-31 2.2 THE CONTRIBUTION OF BURIED HYDROGEN BONDS TO PROTEIN STABILITY: THE CRYSTAL STRUCTURES OF TWO BARNASE MUTANTS
3DA7 2009-04-14 2.25 A conformationally strained, circular permutant of barnase
1BSD 1994-01-31 2.3 CRYSTAL STRUCTURAL ANALYSIS OF MUTATIONS IN THE HYDROPHOBIC CORES OF BARNASE
1X1U 2005-04-26 2.3 Water-mediate interaction at aprotein-protein interface
1X1X 2005-04-26 2.3 Water-mediate interaction at aprotein-protein interface
1B3S 1998-12-09 2.39 STRUCTURAL RESPONSE TO MUTATION AT A PROTEIN-PROTEIN INTERFACE
1BGS 1994-04-30 2.6 RECOGNITION BETWEEN A BACTERIAL RIBONUCLEASE, BARNASE, AND ITS NATURAL INHIBITOR, BARSTAR

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
97.3 Ribonuclease P35078 RN_BACCI
100.0 Ribonuclease P00648 RNBR_BACAM