Exploring the energy surface of protein folding by structure-reactivity relationships and engineered proteins: observation of Hammond behavior for the gross structure of the transition state and anti-Hammond behavior for structural elements for unfolding/folding of barnase.


Abstract

The structure of alpha-helix 1 (residues 6-18) in the transition state for the unfolding of barnase has been previously characterized by comparing the kinetics and thermodynamics of folding of wild-type protein with those of mutants whose side chains have been cut back, in the main, to that of alanine. The structure of the transition state has now been explored further by comparing the kinetics and thermodynamics of folding of glycine mutants with those of the alanine mutants at solvent-exposed positions in the alpha-helices of barnase. Such "Ala-->Gly scanning" provides a general procedure for examining the structure of solvent-exposed regions in the transition state. A gradual change of structure of the transition state was detected as helix 1 becomes increasingly destabilized on mutation. The extent of change of structure of helix 1 in the transition state for the mutant proteins was probed by a further round of Ala-->Gly scanning of those mutants. Destabilization of the helix 1 was found to cause the overall transition state for unfolding to become closer in structure to that of the folded protein. This is analogous to the conventional Hammond effect in physical-organic chemistry whereby the transition state moves parallel to the reaction coordinate with change in structure. But, paradoxically, the structure of helix 1 itself becomes less folded in the transition state as helix 1 becomes destabilized. This is analogous, however, to the rarer anti-Hammond effect in which there is movement perpendicular to the reaction coordinate. These observations are rationalized by plotting correlation diagrams of degree of formation of individual elements of structure against the degree of formation of overall structure in the transition state. There is a relatively smooth movement of the degree of compactness in the transition state against changes in activation energy on mutation that suggests a smooth movement of the transition state along the energy surface on mutation rather than a switch between two different parallel pathways. The results are consistent with the transition state having closely spaced energy levels. Helix 1, which appears to be an initiation point and forms early in the folding of wild-type protein, may be radically destabilized to the extent that it forms late in the folding of mutants. The order of events in folding may thus not be crucial. Study holds ProTherm entries: 5083, 5084, 5085, 5086, 5087, 5088 Extra Details: alpha-helix; transition state; alanine; Ala-->Gly scanning;,activation energy

Submission Details

ID: EerfcXuL4

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:28 p.m.

Version: 1

Publication Details
Matthews JM;Fersht AR,Biochemistry (1995) Exploring the energy surface of protein folding by structure-reactivity relationships and engineered proteins: observation of Hammond behavior for the gross structure of the transition state and anti-Hammond behavior for structural elements for unfolding/folding of barnase. PMID:7756312
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