Thermodynamics of transient conformations in the folding pathway of barnase: reorganization of the folding intermediate at low pH.


Abstract

New classes of small proteins have recently been found that refold rapidly with two-state kinetics from a substantially unfolded conformation ("U") and without the accumulation of a folding intermediate. Barnase, on the other hand, is representative of a class of proteins that display multistate kinetics and refold from a partly structured conformation, a folding intermediate (I). The accumulation of I on the folding pathway of barnase is highly dependent on the experimental conditions: a transition from multistate to two-state folding behavior can be induced simply by changing the reaction conditions away from physiological, i.e., elevated temperatures, high concentration of denaturant, or low pH. We argue that the change in folding behavior results from the denatured state changing under different conditions. The denatured state seems compact and partly structured at conditions that favor folding but is disorganized at denaturing conditions. At physiological pH and temperature, the denatured state (Dphys) is the folding intermediate because it is the most stable of the denatured conformation, i.e., Dphys is identical to I. At high temperature or [urea], however, Dphys becomes destabilized relative to less structured denatured states ("U"). Kinetics under these extreme conditions is two-state because the refolding reaction is from "U" to the native state with no significant accumulation of Dphys (identical to I) which is here a high-energy intermediate. The two-state behavior at low pH results from a different cause. The acid-denatured state of barnase (Dacid) is not as unfolded as "U" but energetically similar to Dphys (identical to I). It appears that protonation of Dphys has only marginal effects on its stability, so that the protonated form of Dphys constitutes the acid-denatured state at equilibrium. The energetic similarity between Dphys and Dacid gives rise to two-state kinetics at low pH, although the refolding is from a compact denatured state throughout the pH range. Protonation of Dphys to give Dacid causes the structure to become more disorganized and hydrated. The heat capacity of Dphys (identical to I) at pH 6.3 is in between that of "U" and the native protein. We suggest that protonation of folding intermediates disrupts their structural integrity and allows isoenergetic reorganizations that increase the solvation of charged residues. Such protonated and reorganized folding intermediates may then constitute the molten globules, which are compact denatured states that are sometimes observed at equilibrium at low pH and high ionic strength. Under all experimental conditions, the heat capacity of the major transition state is close to that of the native protein. This, together with its titration properties, shows that the transition state is an expanded form of the native state with a weakened but poorly hydrated hydrophobic core, and with disrupted surface regions. Study holds ProTherm entries: 4991 Extra Details:

Submission Details

ID: citVK6cq3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:27 p.m.

Version: 1

Publication Details
Oliveberg M;Fersht AR,Biochemistry (1996) Thermodynamics of transient conformations in the folding pathway of barnase: reorganization of the folding intermediate at low pH. PMID:8611580
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
2KF5 2009-12-08 Barnase bound to d(CGAC), low pressure
2KF3 2009-12-08 Barnase, low pressure reference NMR structure
1BNR 1995-07-31 BARNASE
1FW7 2003-06-10 NMR STRUCTURE OF 15N-LABELED BARNASE
2KF6 2009-12-08 Barnase bound to d(CGAC) high pressure
2KF4 2009-12-08 Barnase high pressure structure
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
1BRJ 1995-07-10 2.0 BARNASE MUTANT WITH ILE 88 REPLACED BY ALA
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
1BSB 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
1BRK 1995-07-10 2.0 BARNASE MUTANT WITH ILE 96 REPLACED BY ALA
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
2F5W 2006-04-25 2.0 Cross-linked barnase soaked in 3 M thiourea
1BRS 1994-06-22 2.0 PROTEIN-PROTEIN RECOGNITION: CRYSTAL STRUCTURAL ANALYSIS OF A BARNASE-BARSTAR COMPLEX AT 2.0-A RESOLUTION
1BSC 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
1B2U 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
1B27 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
1X1W 2005-04-26 2.1 Water-mediate interaction at aprotein-protein interface
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
1BAN 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
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
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