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
1STF 1993-04-21T00:00:00+0000 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
2OCT 2006-12-21T00:00:00+0000 1.4 Stefin B (Cystatin B) tetramer
4N6V 2013-10-14T00:00:00+0000 1.8 Partial rotational order disorder structure of human stefin B
1CYU 1995-08-24T00:00:00+0000 0 SOLUTION NMR STRUCTURE OF RECOMBINANT HUMAN CYSTATIN A UNDER THE CONDITION OF PH 3.8 AND 310K
1CYV 1995-08-24T00:00:00+0000 0 SOLUTION NMR STRUCTURE OF RECOMBINANT HUMAN CYSTATIN A UNDER THE CONDITION OF PH 3.8 AND 310K
1DVC 1996-02-26T00:00:00+0000 0 SOLUTION NMR STRUCTURE OF HUMAN STEFIN A AT PH 5.5 AND 308K, NMR, MINIMIZED AVERAGE STRUCTURE
1DVD 1996-02-26T00:00:00+0000 0 SOLUTION NMR STRUCTURE OF HUMAN STEFIN A AT PH 5.5 AND 308K, NMR, 17 STRUCTURES
1GD3 2000-09-08T00:00:00+0000 0 refined solution structure of human cystatin A
1GD4 2000-09-08T00:00:00+0000 0 SOLUTION STRUCTURE OF P25S CYSTATIN A
1N9J 2002-11-25T00:00:00+0000 0 Solution Structure of the 3D domain swapped dimer of Stefin A

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