Urea denaturation of barnase: pH dependence and characterization of the unfolded state.


Abstract

To investigate the pH dependence of the conformational stability of barnase, urea denaturation curves were determined over the pH range 2-10. The maximum conformational stability of barnase is 9 kcal mol-1 and occurs between pH 5 and 6. The dependence of delta G on urea concentration increases from 1850 cal mol-1 M-1 at high pH to about 3000 cal mol-1 M-1 near pH 3. This suggests that the unfolded conformations of barnase become more accessible to urea as the net charge on the molecule increases. Previous studies suggested that in 8 M urea barnase unfolds more completely than ribonuclease T1, even with the disulfide bonds broken [Pace, C.N., Laurents, D. V., & Thomson, J.A. (1990) Biochemistry 29, 2564-2572]. In support of this, solvent perturbation difference spectroscopy showed that in 8 M urea the Trp and Tyr residues in barnase are more accessible to perturbation by dimethyl sulfoxide than in ribonuclease T1 with the disulfide bonds broken. Study holds ProTherm entries: 4015, 4016, 4017, 4018, 4019, 4020, 4021 Extra Details: pH dependence; accessible; disulfide bonds;,solvent perturbation

Submission Details

ID: 9uMrzDGS

Submitter: Connie Wang

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

Version: 1

Publication Details
Pace CN;Laurents DV;Erickson RE,Biochemistry (1992) Urea denaturation of barnase: pH dependence and characterization of the unfolded state. PMID:1547213
Additional Information

Study Summary

Number of data points 21
Proteins Ribonuclease ; Ribonuclease
Unique complexes 1
Assays/Quantities/Protocols Experimental Assay: Cm temp:20.0 C, pH:2.5, buffers:diglycine: 100 mM ; Experimental Assay: m temp:20.0 C, pH:2.5, buffers:diglycine: 100 mM ; Experimental Assay: dG_H2O temp:20.0 C, pH:2.5, buffers:diglycine: 100 mM ; Experimental Assay: Cm pH:2.98, temp:20.0 C, buffers:diglycine: 100 mM ; Experimental Assay: m pH:2.98, temp:20.0 C, buffers:diglycine: 100 mM ; Experimental Assay: dG_H2O pH:2.98, temp:20.0 C, buffers:diglycine: 100 mM ; Experimental Assay: Cm pH:2.76, buffers:diglycine: 30 mM, temp:20.0 C, ionic:NaCl: 0.5 M ; Experimental Assay: m pH:2.76, temp:20.0 C, buffers:diglycine: 30 mM, ionic:NaCl: 0.5 M ; Experimental Assay: dG_H2O pH:2.76, buffers:diglycine: 30 mM, temp:20.0 C, ionic:NaCl: 0.5 M ; Experimental Assay: Cm buffers:diglycine: 30 mM, ionic:NaCl: 0.1 M, pH:2.82, temp:20.0 C ; Experimental Assay: m temp:20.0 C, ionic:NaCl: 0.1 M, pH:2.82, buffers:diglycine: 30 mM ; Experimental Assay: dG_H2O buffers:diglycine: 30 mM, ionic:NaCl: 0.1 M, pH:2.82, temp:20.0 C ; Experimental Assay: Cm buffers:diglycine: 30 mM, pH:2.87, temp:20.0 C ; Experimental Assay: m temp:20.0 C, pH:2.87, buffers:diglycine: 30 mM ; Experimental Assay: dG_H2O buffers:diglycine: 30 mM, pH:2.87, temp:20.0 C ; Experimental Assay: Cm pH:3.17, buffers:diglycine: 30 mM, temp:20.0 C ; Experimental Assay: m pH:3.17, temp:20.0 C, buffers:diglycine: 30 mM ; Experimental Assay: dG_H2O pH:3.17, buffers:diglycine: 30 mM, temp:20.0 C ; Experimental Assay: Cm buffers:MOPS: 30 mM, temp:25.0 C, pH:7.0 ; Experimental Assay: m buffers:MOPS: 30 mM, temp:25.0 C, pH:7.0 ; Experimental Assay: dG_H2O buffers:MOPS: 30 mM, temp:25.0 C, pH:7.0
Libraries Mutations for sequence AQVINTFDGVADYLQTYHKLPDNYITKSEAQALGWVASKGNLADVAPGKSIGGDIFSNREGKLPGKSGRTWREADINYTSGFRNSDRILYSSDWLIYKTTDHYQTFTKIR

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
2KF6 2009-12-08 Barnase bound to d(CGAC) high pressure
2KF4 2009-12-08 Barnase high pressure structure
1BNR 1995-07-31 BARNASE
2KF3 2009-12-08 Barnase, low pressure reference NMR structure
1FW7 2003-06-10 NMR STRUCTURE OF 15N-LABELED BARNASE
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
1X1Y 2005-04-26 1.9 Water-mediate interaction at aprotein-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
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
1BSB 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
1B21 1998-12-09 2.0 DELETION OF A BURIED SALT BRIDGE IN BARNASE
1BSC 1994-01-31 2.0 CRYSTAL STRUCTURAL ANALYSIS OF MUTATIONS IN THE HYDROPHOBIC CORES OF BARNASE
1BRH 1995-07-10 2.0 BARNASE MUTANT WITH LEU 14 REPLACED BY ALA
2F5W 2006-04-25 2.0 Cross-linked barnase soaked in 3 M thiourea
1BSA 1994-01-31 2.0 CRYSTAL STRUCTURAL ANALYSIS OF MUTATIONS IN THE HYDROPHOBIC CORES OF BARNASE
1BNF 1995-07-10 2.0 BARNASE T70C/S92C DISULFIDE MUTANT
1BRJ 1995-07-10 2.0 BARNASE MUTANT WITH ILE 88 REPLACED BY ALA
1BSE 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
1BNE 1995-07-10 2.1 BARNASE A43C/S80C DISULFIDE MUTANT
1BNI 1995-09-15 2.1 BARNASE WILDTYPE STRUCTURE AT PH 6.0
1B2U 1998-12-09 2.1 STRUCTURAL RESPONSE TO MUTATION AT A PROTEIN-PROTEIN INTERFACE
1BNG 1995-07-10 2.1 BARNASE S85C/H102C DISULFIDE MUTANT
1BNJ 1995-09-15 2.1 BARNASE WILDTYPE STRUCTURE AT PH 9.0
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
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
1BAN 1993-10-31 2.2 THE CONTRIBUTION OF BURIED HYDROGEN BONDS TO PROTEIN STABILITY: THE CRYSTAL STRUCTURES OF TWO BARNASE MUTANTS
1BAO 1993-10-31 2.2 THE CONTRIBUTION OF BURIED HYDROGEN BONDS TO PROTEIN STABILITY: THE CRYSTAL STRUCTURES OF TWO BARNASE MUTANTS
1YVS 1999-02-02 2.2 Trimeric domain swapped barnase
1BRG 1994-06-22 2.2 CRYSTALLOGRAPHIC ANALYSIS OF PHE->LEU SUBSTITUTION IN THE HYDROPHOBIC CORE OF BARNASE
3DA7 2009-04-14 2.25 A conformationally strained, circular permutant of barnase
1X1X 2005-04-26 2.3 Water-mediate interaction at aprotein-protein interface
1X1U 2005-04-26 2.3 Water-mediate interaction at aprotein-protein interface
1BSD 1994-01-31 2.3 CRYSTAL STRUCTURAL ANALYSIS OF MUTATIONS IN THE HYDROPHOBIC CORES OF BARNASE
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