Calorimetric measurements of thermal denaturation of stefins A and B

Abstract

Thermal denaturation of two homologous proteins, low-M, 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°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 "C and 90.8 "C for stefins B and A, respectively. The calorimetric enthalpies, which equal the van? Hoff enthalpies to within lo%, 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, 28591 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.Human stefins A and B are small globular proteins of M , 11 000, without disulphide bonds. In their sequence [l-31, there are more than 50% identical amino acid residues. Human stefins A and B are representatives of the stefin family of the cystatin superfamily of the cysteine-proteinases inhibitors [4,5]. The three-dimensional (3D) structure of a closely related cysteine-proteinase inhibitor, the chicken egg-white cystatin, has been determined by Bode and coworkers [6]. The same group determined the 3D structure of the complex between papain and stefin B [7].Spectroscopic study of Gdn/HCl, heat and pH denaturation of the recombinant human stefin B [8] has shown that this protein is much less stable than stefin A. Equilibrium intermediates of molten globular type could be detected in the stefin-B denaturation [8], whereas stefin A seems to denature in an all-or-nothing manner [9].Difference scanning calorimetry yields thermodynamic data on the stability of proteins [lo-121. The temperature dependence of the enthalpy and the entropy of unfolding can be calculated if the change in heat capacity on unfolding is determined. It is a long-standing goal to be able to predict the thermodynamic functions of unfolding as this could contribute towards a rational design of new proteins. A semiempirical method of Ooi and Oobatake [13-151 permits such a calculation from the difference in the accessible surface areas between denatured and native states of a protein, respectively.Correspondence to E. ierovnik, Department of Biochemistry, Fax: f 3 8 61 273 594. Abbreviation. 3D, three-dimensional.Joief Stefkn Institute, Jamova 39, Ljubljana, Slovenia 61 000Our aim was to study the difference in thermal denaturation of the two homologous proteins, of which stefin A shows quite a respective thermal stability and stefin B ...

Design and Validation of a Neutral Protein Scaffold for the Presentation of Peptide Aptamers

“…11,12 SteA shows remarkable thermal stability with a reversible transition observed at 90.8 8C and folding enthalpy of 490 kJ/mol, 16 all suggesting that a SteA-based scaffold would meet criteria (2) and (3). Thus, our preliminary study and the published literature suggested that SteA was a strong candidate for the development of a new scaffold.…”

Characterization of the Equilibrium Intermediates in Acid Denaturation of Human Stefin B

“…Heat capacity differences (&C,) were not determined directly from the thermograms owing to baseline uncertainties. The heat capacity difference on unfolding, obtained from the slope of the calorimetric enthalpy against temperature, was 7.95 kJ K ' mol-' (not shown) which is significantly higher than 6.28 kJ K-' mol-' measured previously [35], or the calculated value of 5.69 kJ K-' mol-' 1451. The slope could be corrected to 5.86 if the fractions of the native and native-like states from CD data were taken into account.…”

“…It was shown, nevertheless, that the active component of the inhibitor was monomeric [34]. Microcalorimetric studies of the two proteins have confirmed that stefin A is much more stable and does not deviate from two-state behavior [35]. Regarding physiological processes, it has been proposed that lower expression of stefin A plays a key role in tumour invasion [36].…”

On the mechanism of human stefin B folding: II. Folding from GuHCl unfolded, TFE denatured, acid denatured, and acid intermediate states

“…23 Thermal denaturation of both proteins by differential scanning calorimetry (DSC) reveals a big difference in melting temperatures, 64.9 o C vs. 94.5 o C for stefins B and A at pH 6.5, respectively. 24 Using structural thermodynamics, underlying structural reasons for different thermodynamic behavior were predicted. 25 It was suggested that the difference in accesible hydrophobic surfaces of the two native proteins, in addition to a difference in the number of h-bonds in the ␤ sheet structure, may account for the differences in thermodynamic behavior.…”