Protein folding in the absence of chemical denaturants. Reversible pressure denaturation of the noncovalent complex formed by the association of two protein fragments.


Abstract

Small monomeric proteins are the best models for studying protein folding, but they are often too stable for denaturation using pressure as the sole perturbant. In the present work we subject [CI-2(1-40).(41-64)], a noncovalent complex formed by the association of two complementary fragments of the chymotrypsin inhibitor-2, to high pressure to investigate the folding mechanism of a model protein. Pressures up to 3.5 kilobar do not affect the intact protein, but it can be unfolded reversibly by pressure in the presence of subdenaturing concentrations of guanidine chloride, with free energy and molar volume changes of 2.5 kcal mol-1 and 42.5 ml mol-1, respectively. In contrast, the complex can be reversibly denatured by high pressure without the addition of chemical denaturants. However, the process is clearly independent of the protein concentration, indicating lack of dissociation. We determined a change in the free energy of 1.4 kcal mol-1 and a molar volume change of 35 ml mol-1 for the pressure denaturation of the complex. A persistent quenching of the tryptophan adds further evidence for the presence of residual structure in the high pressure-denatured state. This state also appears to be compact as the small volume change indicates, compared with pressure denaturation of naturally occurring dimers. Based on observations of a number of pressure-denatured states and on characteristics of large CI-2 fragments with a solvent accessible core but maintaining tertiary interactions, the structure of the pressure-denatured state of the CI-2 complex could be explained by an ordered molten globule-like conformation. Study holds ProTherm entries: 6415 Extra Details: molar volume; free energy; pressure denaturation;,solvent accessible core; tertiary interaction; molten globule

Submission Details

ID: oK9bZvCK

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:32 p.m.

Version: 1

Publication Details
Mohana-Borges R;Lima Silva J;de Prat-Gay G,J. Biol. Chem. (1999) Protein folding in the absence of chemical denaturants. Reversible pressure denaturation of the noncovalent complex formed by the association of two protein fragments. PMID:10075663
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
3CI2 1993-10-31 REFINEMENT OF THE THREE-DIMENSIONAL SOLUTION STRUCTURE OF BARLEY SERINE PROTEINASE INHIBITOR 2 AND COMPARISON WITH THE STRUCTURES IN CRYSTALS
1CIS 1993-10-31 CONTEXT DEPENDENCE OF PROTEIN SECONDARY STRUCTURE FORMATION. THE THREE-DIMENSIONAL STRUCTURE AND STABILITY OF A HYBRID BETWEEN CHYMOTRYPSIN INHIBITOR 2 AND HELIX E FROM SUBTILISIN CARLSBERG
1CIR 1996-01-29 COMPLEX OF TWO FRAGMENTS OF CI2 [(1-40)(DOT)(41-64)]
1LW6 2002-08-21 1.5 Crystal Structure of the Complex of Subtilisin BPN' with Chymotrypsin Inhibitor 2 at 1.5 Angstrom Resolution
1YPC 1994-01-31 1.7 DIRECT OBSERVATION OF BETTER HYDRATION AT THE N-TERMINUS OF AN ALPHA-HELIX WITH GLYCINE RATHER THAN ALANINE AS N-CAP
1CQ4 1998-11-25 1.8 CI2 MUTANT WITH TETRAGLUTAMINE (MGQQQQGM) REPLACING MET59
5FFN 2016-05-18 1.8 Complex of subtilase SubTY from Bacillus sp. TY145 with chymotrypsin inhibitor CI2A
5FBZ 2016-05-18 1.9 Structure of subtilase SubHal from Bacillus halmapalus - complex with chymotrypsin inhibitor CI2A
1YPA 1994-01-31 2.0 DIRECT OBSERVATION OF BETTER HYDRATION AT THE N-TERMINUS OF AN ALPHA-HELIX WITH GLYCINE RATHER THAN ALANINE AS N-CAP
1YPB 1994-01-31 2.0 DIRECT OBSERVATION OF BETTER HYDRATION AT THE N-TERMINUS OF AN ALPHA-HELIX WITH GLYCINE RATHER THAN ALANINE AS N-CAP
2CI2 1988-09-07 2.0 CRYSTAL AND MOLECULAR STRUCTURE OF THE SERINE PROTEINASE INHIBITOR CI-2 FROM BARLEY SEEDS
2SNI 1988-09-07 2.1 STRUCTURAL COMPARISON OF TWO SERINE PROTEINASE-PROTEIN INHIBITOR COMPLEXES. EGLIN-C-SUBTILISIN CARLSBERG AND CI-2-SUBTILISIN NOVO
1COA 1994-01-31 2.2 THE EFFECT OF CAVITY CREATING MUTATIONS IN THE HYDROPHOBIC CORE OF CHYMOTRYPSIN INHIBITOR 2
1CIQ 1996-03-08 2.2 COMPLEX OF TWO FRAGMENTS OF CI2, RESIDUES 1-40 AND 41-64

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
92.6 Subtilisin-chymotrypsin inhibitor-2A P08626 ICI3_HORVU
100.0 Subtilisin-chymotrypsin inhibitor-2A P01053 ICI2_HORVU