Calorimetric measurements of thermal denaturation of stefins A and B. Comparison to predicted thermodynamics of stefin-B unfolding.


Abstract

Thermal denaturation of two homologous proteins, low-M(r) cysteine-proteinase inhibitors stefins A and B, has been investigated by microcalorimetry. Calorimetric enthalpies, as well as the temperatures at maximum heat capacity, were determined as a function of pH for each protein. Transitions were found reversible at all pH values examined (5.0, 6.5, 8.1) for the thermally more stable stefin A, in contrast to stefin B. Stefin B shows a sharp irreversible transition around 65 degrees C at pH 6.5 and 8.1, probably due to unfolding of a dimeric state followed by oligomerisation. At pH 5.0, both proteins exhibit a reversible transition with temperatures of half-denaturation at 50.2 degrees C and 90.8 degrees C for stefins B and A, respectively. The calorimetric enthalpies, which equal the van't Hoff enthalpies to within 10%, are 293 kJ/mol and 490 kJ/mol for stefins B and A, respectively. Using the predictive method of Ooi and Oobatake (1991) [Proc. Natl Acad. Sci. USA 88, 2859] the thermodynamic functions of unfolding were calculated for stefin B, whose three-dimensional structure has been determined. The calculated enthalpy, heat-capacity change on unfolding and the temperature of half denaturation compare well to the microcalorimetric data. Study holds ProTherm entries: 7597, 7598, 7599, 7600, 7601, 7602, 7603, 7604, 7605, 7606 Extra Details: low-M(r) cysteine-proteinase inhibitors stefins A and B;,dimeric state; oligomerisation; heat-capacity change

Submission Details

ID: AxBw8mza

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:34 p.m.

Version: 1

Publication Details
Zerovnik E;Lohner K;Jerala R;Laggner P;Turk V,Eur. J. Biochem. (1992) Calorimetric measurements of thermal denaturation of stefins A and B. Comparison to predicted thermodynamics of stefin-B unfolding. PMID:1446674
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
1GD4 2001-09-08 SOLUTION STRUCTURE OF P25S CYSTATIN A
1N9J 2003-02-25 Solution Structure of the 3D domain swapped dimer of Stefin A
1GD3 2001-09-08 refined solution structure of human cystatin A
1CYV 1995-12-07 SOLUTION NMR STRUCTURE OF RECOMBINANT HUMAN CYSTATIN A UNDER THE CONDITION OF PH 3.8 AND 310K
1CYU 1995-12-07 SOLUTION NMR STRUCTURE OF RECOMBINANT HUMAN CYSTATIN A UNDER THE CONDITION OF PH 3.8 AND 310K
1DVC 1996-08-01 SOLUTION NMR STRUCTURE OF HUMAN STEFIN A AT PH 5.5 AND 308K, NMR, MINIMIZED AVERAGE STRUCTURE
1DVD 1996-08-01 SOLUTION NMR STRUCTURE OF HUMAN STEFIN A AT PH 5.5 AND 308K, NMR, 17 STRUCTURES
2OCT 2007-04-03 1.4 Stefin B (Cystatin B) tetramer
2CIO 2006-05-18 1.5 The high resolution x-ray structure of papain complexed with fragments of the Trypanosoma brucei cysteine protease inhibitor ICP.
1PPN 1994-01-31 1.6 STRUCTURE OF MONOCLINIC PAPAIN AT 1.60 ANGSTROMS RESOLUTION
1KHQ 2003-09-09 1.6 ORTHORHOMBIC FORM OF PAPAIN/ZLFG-DAM COVALENT COMPLEX
9PAP 1986-10-24 1.65 STRUCTURE OF PAPAIN REFINED AT 1.65 ANGSTROMS RESOLUTION
1PIP 1993-10-31 1.7 CRYSTAL STRUCTURE OF PAPAIN-SUCCINYL-GLN-VAL-VAL-ALA-ALA-P-NITROANILIDE COMPLEX AT 1.7 ANGSTROMS RESOLUTION: NONCOVALENT BINDING MODE OF A COMMON SEQUENCE OF ENDOGENOUS THIOL PROTEASE INHIBITORS
1CVZ 2000-08-30 1.7 CRYSTAL STRUCTURE ANALYSIS OF PAPAIN WITH CLIK148(CATHEPSIN L SPECIFIC INHIBITOR)
3KSE 2010-12-01 1.71 Unreduced cathepsin L in complex with stefin A
4N6V 2014-04-09 1.8 Partial rotational order disorder structure of human stefin B
3E1Z 2009-01-27 1.86 Crystal structure of the parasite protesase inhibitor chagasin in complex with papain
1PPP 1994-01-31 1.9 CRYSTAL STRUCTURE OF PAPAIN-E64-C COMPLEX. BINDING DIVERSITY OF E64-C TO PAPAIN S2 AND S3 SUBSITES
4QRV 2015-08-12 1.98 Crystal structure of I86F mutant of papain
3KFQ 2010-11-03 1.99 Unreduced cathepsin V in complex with stefin A
1KHP 2003-09-09 2.0 Monoclinic form of papain/ZLFG-DAM covalent complex
1PPD 1985-01-02 2.0 RESTRAINED LEAST-SQUARES REFINEMENT OF THE SULFHYDRYL PROTEASE PAPAIN TO 2.0 ANGSTROMS
3IMA 2010-02-16 2.03 Complex strcuture of tarocystatin and papain
1POP 1993-10-31 2.1 X-RAY CRYSTALLOGRAPHIC STRUCTURE OF A PAPAIN-LEUPEPTIN COMPLEX
6H8T 2018-09-12 2.1 Crystal structure of Papain modify by achiral Ru(II)complex
1PE6 1993-04-15 2.1 REFINED X-RAY STRUCTURE OF PAPAIN(DOT)E-64-C COMPLEX AT 2.1-ANGSTROMS RESOLUTION
1BP4 1999-08-12 2.2 USE OF PAPAIN AS A MODEL FOR THE STRUCTURE-BASED DESIGN OF CATHEPSIN K INHIBITORS. CRYSTAL STRUCTURES OF TWO PAPAIN INHIBITOR COMPLEXES DEMONSTRATE BINDING TO S'-SUBSITES.
1STF 1994-01-31 2.37 THE REFINED 2.4 ANGSTROMS X-RAY CRYSTAL STRUCTURE OF RECOMBINANT HUMAN STEFIN B IN COMPLEX WITH THE CYSTEINE PROTEINASE PAPAIN: A NOVEL TYPE OF PROTEINASE INHIBITOR INTERACTION
1NB5 2003-02-18 2.4 Crystal structure of stefin A in complex with cathepsin H
1BQI 1999-08-16 2.5 USE OF PAPAIN AS A MODEL FOR THE STRUCTURE-BASED DESIGN OF CATHEPSIN K INHIBITORS. CRYSTAL STRUCTURES OF TWO PAPAIN INHIBITOR COMPLEXES DEMONSTRATE BINDING TO S'-SUBSITES.
4QRG 2015-08-05 2.5 Crystal structure of I86L mutant of papain
3LFY 2010-07-21 2.6 CTD of Tarocystatin in complex with papain
3K9M 2009-11-03 2.61 Cathepsin B in complex with stefin A
3TNX 2012-09-12 2.62 Structure of the precursor of a thermostable variant of papain at 2.6 Angstroem resolution
1NB3 2003-02-18 2.8 Crystal structure of stefin A in complex with cathepsin H: N-terminal residues of inhibitors can adapt to the active sites of endo-and exopeptidases
5PAD 1977-04-12 2.8 BINDING OF CHLOROMETHYL KETONE SUBSTRATE ANALOGUES TO CRYSTALLINE PAPAIN
2PAD 1977-04-12 2.8 BINDING OF CHLOROMETHYL KETONE SUBSTRATE ANALOGUES TO CRYSTALLINE PAPAIN
1PAD 1977-04-12 2.8 Binding of chloromethyl ketone substrate analogues to crystalline papain
4PAD 1977-04-12 2.8 Binding of chloromethyl ketone substrate analogues to crystalline papain
6PAD 1977-04-12 2.8 Binding of chloromethyl ketone substrate analogues to crystalline papain
4QRX 2015-08-05 3.14 Crystal structure of pro-papain mutant at pH 4.0
3USV 2012-11-28 3.8 Structure of the precursor of a thermostable variant of papain at 3.8 A resolution from a crystal soaked at pH 4

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Cystatin-A P01040 CYTA_HUMAN
98.6 E Papain P00784 PAPA1_CARPA
94.9 Cystatin-B Q862Z5 CYTB_MACFU
99.0 Cystatin-B Q8I030 CYTB_PANTR
100.0 Cystatin-B P60576 CYTB_PONPY
100.0 Cystatin-B P60575 CYTB_PANPA
100.0 Cystatin-B P04080 CYTB_HUMAN
100.0 Cystatin-B Q76LA0 CYTB_GORGO