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
1B2U 1998-12-01T00:00:00+0000 2.1 STRUCTURAL RESPONSE TO MUTATION AT A PROTEIN-PROTEIN INTERFACE
1B2S 1998-11-30T00:00:00+0000 1.82 STRUCTURAL RESPONSE TO MUTATION AT A PROTEIN-PROTEIN INTERFACE
1B2Z 1998-12-03T00:00:00+0000 2.03 DELETION OF A BURIED SALT BRIDGE IN BARNASE
1B2X 1998-12-03T00:00:00+0000 1.8 BARNASE WILDTYPE STRUCTURE AT PH 7.5 FROM A CRYO_COOLED CRYSTAL AT 100K
3KCH 2009-10-21T00:00:00+0000 1.94 Baranase crosslinked by glutaraldehyde
1B21 1998-12-03T00:00:00+0000 2.0 DELETION OF A BURIED SALT BRIDGE IN BARNASE
1B20 1998-12-03T00:00:00+0000 1.7 DELETION OF A BURIED SALT-BRIDGE IN BARNASE
1A2P 1998-01-07T00:00:00+0000 1.5 BARNASE WILDTYPE STRUCTURE AT 1.5 ANGSTROMS RESOLUTION
1FW7 2000-09-22T00:00:00+0000 0 NMR STRUCTURE OF 15N-LABELED BARNASE
1BGS 1993-11-02T00:00:00+0000 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