Extrapolation to water of kinetic and equilibrium data for the unfolding of barnase in urea solutions.


Abstract

Assumptions about the dependence of protein unfolding on the concentration of urea have been examined by an extensive survey of the equilibrium unfolding of barnase and many of its mutants measured by urea denaturation and differential scanning calorimetry. The free energy of equilibrium unfolding and the activation energy for the kinetics of unfolding of proteins are generally assumed to change linearly with [urea]. A slight downward curvature is detected, however, in plots of highly precise measurements of logku versus [urea] (where ku is the observed rate constant for the unfolding of barnase). The data fit the equation logku = logkuH2O* + mku*.[urea] - 0.014[urea]2, where mku* is a variable which depends on the mutation. The constant 0.014 was measured directly on four destabilized mutants and wild-type, and was also determined from a global analysis of data from > 60 mutants of barnase. Any equivalent deviations from linearity in the equilibrium unfolding are small and in the same region, as determined from measurements on 166 mutants. The free energy of unfolding of barnase, delta GU-F, appears significantly larger by 1.6 kcal mol-1 when measured by calorimetry than when determined by urea denaturation. However, the changes in delta GU-F on mutation, delta delta GU-F, determined by calorimetry and by urea denaturation are identical. We show analytically how, in general, the curvature in plots of activation or equilibrium energies against [denaturant] should not affect the changes of these values on mutation provided measurements are made over the same concentration ranges of denaturant and the curvature is independent of mutation. Study holds ProTherm entries: 1709, 1710, 1711, 1712, 1713, 1714, 1715, 1716, 1717, 1718, 1719, 1720, 1721, 1722, 1723, 1724, 1725, 1726, 11793, 11794, 11795, 11796, 11797, 11798, 11799, 11800, 11801, 11802, 11803, 11804, 11805, 11806, 11807, 11808, 11809, 11810 Extra Details: measurements were made in the presence of 20mM acetate calorimetry; protein folding; urea

Submission Details

ID: Yi2PUzBM3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:17 p.m.

Version: 1

Publication Details
Matouschek A;Matthews JM;Johnson CM;Fersht AR,Protein Eng. (1994) Extrapolation to water of kinetic and equilibrium data for the unfolding of barnase in urea solutions. PMID:7831279
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
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