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
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:34 p.m.
|Number of data points||20|
|Proteins||Cystatin-A ; Cystatin-A ; Papain ; Cystatin-B|
|Assays/Quantities/Protocols||Experimental Assay: dG temp:50.0 C ; Experimental Assay: dG temp:45.0 C ; Experimental Assay: dG temp:25.0 C ; Experimental Assay: dG temp:10.0 C ; Experimental Assay: dHcal pH:8.1, buffers:HEPES: 0.01 M ; Experimental Assay: Tm pH:8.1, buffers:HEPES: 0.01 M ; Experimental Assay: dHvH pH:8.1, buffers:HEPES: 0.01 M ; Experimental Assay: dHcal buffers:Sodium phosphate: 0.01 M, pH:6.5 ; Experimental Assay: Tm buffers:Sodium phosphate: 0.01 M, pH:6.5 ; Experimental Assay: dHvH buffers:Sodium phosphate: 0.01 M, pH:6.5 ; Experimental Assay: dHcal pH:5.0, buffers:Sodium acetate: 0.02 M ; Experimental Assay: Tm pH:5.0, buffers:Sodium acetate: 0.02 M ; Experimental Assay: dHvH pH:5.0, buffers:Sodium acetate: 0.02 M|
|Libraries||Mutations for sequence E:IPEYVDWRQKGAVTPVKNQGSCGSCWAFSAVVTIEGIIKIRTGNLNQYSEQELLDCDRRSYGCNGGYPWSALQLVAQYGIHYRNTYPYEGVQRYCRSREKGPYAAKTDGVRQVQPYNQGALLYSIANQPVSVVLQAAGKDFQLYRGGIFVGPCGNKVDHAVAAVGYGPNYILIKNSWGTGWGENGYIRIKRGTGNSYGVCGLYTSSFYPVKN/I:MMSGAPSATQPATAETQHIADQVRSQLEEKYNKKFPVFKAVSFKSQVVAGTNYFIKVHVGDEDFVHLRVFQSLPHENKPLTLSNYQTNKAKHDELTYF ; Mutations for sequence MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTNETYGKLEAVQYKTQVVAGTNYYIKVRAGDNKYMHLKVFKSLPGQNEDLVLTGYQVDKNKDDELTGF|