Protein stability as a function of denaturant concentration: the thermal stability of barnase in the presence of urea.


Abstract

The conventional procedure for analyzing urea denaturation curves assumes that the free energy of unfolding (delta GU-F) is linearly related to [urea] that is, delta GU-F = delta GH2O(U-F)--m[urea], where m is a constant, specific for each protein, and delta GH2O(U-F) is the free energy of unfolding in water. This relationship can be measured directly, however, over only a small concentration range of approximately +/- 0.8 M urea around the midpoint of the unfolding transition. A nagging discrepancy (1.6 kcal mol-1) between delta GH2O(U-F) at 298 K of barnase extrapolated from such an equation and the equivalent value obtained from thermal unfolding measurements has stimulated a re-evaluation of the equation. Differential scanning calorimetric measurements have been made of the thermal unfolding of barnase in the presence of concentrations of urea between 0 and 4.5 M, the midpoint of the unfolding transition at 298 K, to test the denaturation equation over a wide range of [urea]. Values for delta GU-F at 298 K (delta G298U-F) for each concentration of urea were extrapolated from the calorimetrically measured enthalpies and the denaturational heat capacity change (delta Cdp) measured for that concentration of urea. A plot of delta G298U-F against [urea] deviates systematically from linearity and fits better the equation: delta G298U-F = 10.5 +/- 0.08 - ((2.65 +/- 0.05) x [urea]) + ((0.08 +/- 0.01) x [urea]2) kcal mol-1. The curvature in the plot leads to apparent values of m that increase when measurements are made at lower concentrations of urea. This could account for increases in m at low values of pH or in destabilized mutants since the protein denatures at lower concentrations of urea. It has been shown previously that small curvature in the free energy of unfolding versus [urea] leads to negligible errors in measurements of delta delta GU-F, the change in free energy of unfolding on mutation, providing that the curvature is similar for all mutants. The calorimetrically measured enthalpies of unfolding are decreased in the presence of urea while delta Cdp is increased. Both of these observations are consistent with an overall exothermic interaction between urea and protein with a net increase on unfolding. Study holds ProTherm entries: 5380 Extra Details: denaturant concentration; heat capacity change;,exothermic interaction; free energies of unfolding

Submission Details

ID: KeDJ3iot3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:29 p.m.

Version: 1

Publication Details
Johnson CM;Fersht AR,Biochemistry (1995) Protein stability as a function of denaturant concentration: the thermal stability of barnase in the presence of urea. PMID:7756311
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
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
1BNR 1995-07-31 BARNASE
2KF5 2009-12-08 Barnase bound to d(CGAC), low pressure
2KF6 2009-12-08 Barnase bound to d(CGAC) high 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
1X1Y 2005-04-26 1.9 Water-mediate interaction at aprotein-protein interface
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
1BNF 1995-07-10 2.0 BARNASE T70C/S92C DISULFIDE MUTANT
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
1BSE 1994-01-31 2.0 CRYSTAL STRUCTURAL ANALYSIS OF MUTATIONS IN THE HYDROPHOBIC CORES OF BARNASE
1BRS 1994-06-22 2.0 PROTEIN-PROTEIN RECOGNITION: CRYSTAL STRUCTURAL ANALYSIS OF A BARNASE-BARSTAR COMPLEX AT 2.0-A RESOLUTION
1BRH 1995-07-10 2.0 BARNASE MUTANT WITH LEU 14 REPLACED BY ALA
1BSA 1994-01-31 2.0 CRYSTAL STRUCTURAL ANALYSIS OF MUTATIONS IN THE HYDROPHOBIC CORES OF BARNASE
2F5W 2006-04-25 2.0 Cross-linked barnase soaked in 3 M thiourea
1BRK 1995-07-10 2.0 BARNASE MUTANT WITH ILE 96 REPLACED BY ALA
1BSB 1994-01-31 2.0 CRYSTAL STRUCTURAL ANALYSIS OF MUTATIONS IN THE HYDROPHOBIC CORES OF BARNASE
1BRJ 1995-07-10 2.0 BARNASE MUTANT WITH ILE 88 REPLACED BY ALA
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
1X1W 2005-04-26 2.1 Water-mediate interaction at aprotein-protein interface
1BNE 1995-07-10 2.1 BARNASE A43C/S80C DISULFIDE MUTANT
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
1BNG 1995-07-10 2.1 BARNASE S85C/H102C 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
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
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
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
3DA7 2009-04-14 2.25 A conformationally strained, circular permutant of barnase
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
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