Differential Scanning Calorimetry Study of the Thermodynamic Stability of Some Mutants of Sso7d from Sulfolobus solfataricus ² Francesca Catanzano, ‡ Giuseppe Graziano, § Paola Fusi, | Paolo Tortora, | and Guido Barone* ,‡ Dipartimento di Chimica, UniVersita ` di Napoli “Federico II”, Via Mezzocannone, 4, 80134 Napoli, Italy, Facolta ` di Scienze, UniVersita ` del Sannio, Via Marmorale, 82020 Paduli (BN), Italy, and Dipartimento di Fisiologia e Biochimica Generali, UniVersita ` di Milano, Via Celoria, 26, 20133 Milano, Italy ReceiVed December 4, 1997; ReVised Manuscript ReceiVed May 4, 1998 ABSTRACT: Sso7d from the thermoacidophilic archaebacterium Sulfolobus solfataricus is a small globular protein with a known three-dimensional structure. Inspection of the structure reveals that Phe31 is a member of the aromatic cluster forming the protein hydrophobic core, whereas Trp23 is located on the protein surface and its side chain exposed to the solvent. The thermodynamic consequences of the substitution of these two residues in Sso7d have been investigated by comparing the temperature-induced denaturation of Sso7d with that of three mutants: F31A-Sso7d, F31Y-Sso7d, and W23A-Sso7d. The denaturation processes proved to be reversible for all proteins, and represented well by the two-state N S D transition model in a wide range of pH. All three mutants are less thermally stable than the parent protein; in particular, in the pH range of 5.0-7.0, the F31A substitution leads to a decrease of 24 °C in the denaturation temperature, the F31Y substitution to a decrease of 10 °C, and the W23A substitution to a decrease of 6 °C. A careful thermodynamic analysis of such experimental data is carried out. The physicochemical rationalization of the relationship between a given tertiary structure and its thermodynamic stability has attracted much interest in recent years. Such research is closely related to the classic problem of protein folding, since Anfinsen (1) demonstrated that this funda- mental process is ruled by the “thermodynamic hypothesis”. All the experimental investigations performed so far have pointed out that the native structure of globular proteins from mesophilic organisms possesses only marginal stability (2). However, thermophilic microorganisms are able to live under extreme temperature conditions; the upper temperature limit of viability seems to be approximately 150 °C(3). As microorganisms are isothermal in their habitat, they cannot avoid the thermal stress and must adapt their biological functions to the extreme temperature. In fact, proteins extracted from thermophilic microorganisms are still fully active at temperatures near or above the boiling point of water. Thermophilic proteins are made up of the same 20 natural amino acids, and the stability of the native conforma- tion is ensured by the same noncovalent interactions that are operating in mesophilic proteins. It is hard to try to understand how the balance among stabilizing and destabi- lizing factors has been optimized during evolution to cope with such high temperatures (4). A thermodynamic study of the extrastability of thermo- philic proteins requires a quantitative determination of the changes in the state functions associated with the denaturation process, ∆ d G, ∆ d H, ∆ d S, and ∆ d C p . The best approach for performing such a task is provided by differential scanning calorimetry (DSC) 1 which allows the direct evaluation of the denaturation enthalpy and heat capacity changes, and, provided that the process is a reversible two-state transition, allows the calculation of the Gibbs energy and entropy changes. Sso7d has been recently isolated from Sulfolobus solfa- taricus (5, 6), a thermoacidophilic archaebacterium that lives at 87 °C and acidic pH in volcanic hot springs (7). Formerly, we referred to this molecule as P2 (6); however, on the basis of amino acid sequence, P2 and Sso7d were proven to be the same molecule. We have subsequently cloned the protein-encoding gene and expressed it in Escherichia coli (8). The recombinant protein was identical to the natural form, the only difference being the absence of monomethy- lation at Lys4 and Lys6. Sso7d is a small basic (pI ) 10.2), 63-residue protein that contains no histidine or asparagine, and no cysteine. The physiological role of the protein is not clear yet; it is able to bind DNA nonspecifically, thus protecting DNA from thermal denaturation (9, 10), but also has a ribonuclease activity (6). Its tertiary structure in solution, determined by NMR (11, 12), shows that the protein is folded into a compact globular unit composed of a three- stranded antiparallel -sheet which is orthogonal to a two- ² Work supported by a PRIN grant from the Italian Ministry for University and Scientific and Technological Research (MURST, Rome) and funds from the Italian National Research Council (CNR, Rome). * To whom correspondence should be addressed. Fax: +39/81/ 5527771. E-mail: barone@chemna.dichi.unina.it. ‡ Universita ` di Napoli “Federico II”. § Universita ` del Sannio. | Universita ` di Milano. 1 Abbreviations: Sso7d, 7 kDa DNA-binding protein from Sulfolobus solfataricus; F31A-Sso7d and F31Y-Sso7d, mutants of Sso7d where phenylalanine 31 is replaced by alanine and tyrosine, respectively; W23A-Sso7d, mutant of Sso7d where tryptophan 23 is replaced by alanine; CD, circular dichroism; DSC, differential scanning calorimetry; ASA, accessible surface area; NMR, nuclear magnetic resonance; FTIR, Fourier transform infrared spectroscopy; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 10493 Biochemistry 1998, 37, 10493-10498 S0006-2960(97)02994-2 CCC: $15.00 © 1998 American Chemical Society Published on Web 06/27/1998