Ribonucleases H from the thermophilic bacterium Thermus thermophilus and the mesophile Escherichia coli demonstrate a dramatic and surprising difference in their change in heat capacity upon unfolding (⌬Cp°). The lower ⌬Cp°of the thermophilic protein directly contributes to its higher thermal denaturation temperature (T m). We propose that this ⌬Cp°difference originates from residual structure in the unfolded state of the thermophilic protein; we verify this hypothesis by using a mutagenic approach. Residual structure in the unfolded state may provide a mechanism for balancing a high T m with the optimal thermodynamic stability for a protein's function. Structure in the unfolded state is shown to differentially affect the thermodynamic profiles of thermophilic and mesophilic proteins.T hermophilic organisms thrive at temperatures where proteins from mesophilic organisms are often completely unfolded and nonfunctional. Understanding the mechanisms by which proteins function at such high temperatures will help to optimize and design thermostable functional proteins for a variety of biotechnological applications. To learn how proteins from thermophilic organisms (thermophilic proteins) function at such elevated temperatures, we need to understand what makes these proteins different from their mesophilic homologs. This difference clearly does not reside in the overall structure of the native conformation; structures of numerous pairs of homologous proteins show that the thermophilic and mesophilic proteins invariably adopt the same fold (1). Examining the differences between individual amino acids and their specific interactions has led to the conclusion that the rules are extremely complex (2, 3).Thermodynamic comparisons of thermophilic and mesophilic pairs of proteins can provide a rational framework for understanding the functional differences between thermophilic and mesophilic proteins. A protein's thermodynamic stability is defined as the difference between the free energies of the native and the unfolded states (⌬G unf ϭ G U Ϫ G N ). The manner in which protein stability depends on temperature is illustrated by the so-called ''protein stability curve,'' which is defined by the Gibbs-Helmholtz equation (4, 5) (for an example, see Fig. 4). The temperature at which the ⌬G unf°e quals zero is the thermal denaturation midpoint (T m ), and the curvature of the stability curve, determined under standard conditions, is given by the heat capacity change upon unfolding (⌬Cp°). There are several ways in which a protein stability curve can be altered to result in a larger T m . An increase in the number of enthalpic interactions will raise the curve and make the protein more stable at every temperature. Alternatively, a lowering of the ⌬Cp°produces a ''flatter'' curve, which results in a higher T m for the same stability maximum. The question then becomes, how do thermophilic proteins alter these protein stability curves and how are they encoded in the structure and sequence?Thermus thermophilus and Escherichia coli RNa...

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S100P is a member of the S100 subfamily of calcium-binding proteins that are believed to be associated with various diseases, and in particular deregulation of S100P expression has been documented for prostate and breast cancer. Previously, we characterized the effects of metal binding on the conformational properties of S100P and proposed that S100P could function as a Ca 2+ conformational switch. In this study we used fluorescence and CD spectroscopies and isothermal titration calorimetry to characterize the target-recognition properties of S100P using a model peptide, melittin. Based on these experimental data we show that S100P and melittin can interact in a Ca 2+ -dependent and -independent manner. Ca 2+ -independent binding occurs with low affinity (K d ≈ 0.2 mM), has a stoichiometry of four melittin molecules per S100P dimer and is presumably driven by favorable electrostatic interactions between the acidic protein and the basic peptide. In contrast, Ca 2+ -dependent binding of melittin to S100P occurs with high affinity (K d ≈ 5 M) has a stoichiometry of two molecules of melittin per S100P dimer, appears to have positive cooperativity, and is driven by hydrophobic interactions. Furthermore, Ca 2+ -dependent S100P-melittin complex formation is accompanied by significant conformational changes: Melittin, otherwise unstructured in solution, adopts a helical conformation upon interaction with Ca 2+ -S100P. These results support a model for the Ca 2+ -dependent conformational switch in S100P for functional target recognition.Keywords: Isothermal titration calorimetry; circular dichroism spectroscopy; fluorescence spectroscopy; structural thermodynamics; conformational transitionThe human protein S100P belongs to the S100 subfamily of calcium-binding proteins that share a common Ca 2+ -binding structural motif, the EF-hand (Kretsinger 1976;Chazin 1995). Twenty members of the subfamily have been identified to date (Donato 1999;Gribenko et al. 2001). These proteins have two EF-hand motifs in their primary structure, various metal-binding properties, and are expressed in a tissue-and cell-type-specific fashion (Hilt and Kligman 1991;Zimmer et al. 1995;Schafer and Heizmann 1996). Increased or altered expression of S100 proteins has been documented for many human diseases and tumors (Donato 1999). S100P was shown to be differentially expressed in androgen-dependent and androgen-independent prostate cancer cell lines (Averboukh et al. 1996). Recently it has also been shown that S100P overexpression is associated with immortalization of human breast epithelial cells in vitro and early stages of breast cancer development in vivo (Guerreiro Da Silva et al. 2000).In our previous report we characterized the oligomerization and divalent cation-binding properties of S100P and proposed a Ca 2+ /Mg 2+ switch model for S100P function

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The binding of myo-inositol hexasulfate to an N-terminal truncated 132-amino-acid human acidic fibroblast growth factor form was studied by isothermal titration calorimetry. The technique yields values for the enthalpy change and equilibrium constant, from which the Gibbs energy and entropy change can also be calculated. Experiments in different buffers and pH values show that the proton balance in the reaction is negligible. Experiments at pH 7.0 in the presence of 0.2±0.6 m NaCl showed that the enthalpy and Gibbs energy changes parallel behaviour with ionic strength change, with values in the 221 to 211 kJ´mol 21 range in the first case and in the 231 to 222 kJ´mol 21 range in the second. No dependence of entropy on ionic strength was found, with a constant value of < 35 J´K 21´m ol 21 at all ionic strengths studied. The results can be interpreted in molecular terms by a model in which competitive binding of 3±4 chloride ions to the myo-inositol-binding site is assumed. Isothermal titration calorimetry was also performed at different temperatures and yielded a value of 2142^13 J´K 21´m ol 21 for the heat-capacity change at pH 7.0 and 0.4 m NaCl. Using different parametric equations in the literature, changes on ligand binding in the range 2100 to 2200 A Ê 2 in solvent-accessible surface areas, both polar and apolar, were calculated from thermodynamic data. These values suggest a negligible overall conformational change in the protein when the ligand binds and agree closely with calculations performed with NMR structural data, in which it is shown that the most important negative change in total solvent-accessible surface area occurs in the amino acids Ile56, Gln57, Leu58 and Leu149, in the high-affinity receptor-binding region of the protein.

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