Role of residual structure in the unfolded state of a thermophilic protein.

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 (DeltaCp degrees ). The lower DeltaCp degrees of the thermophilic protein directly contributes to its higher thermal denaturation temperature (Tm). We propose that this DeltaCp degrees 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 Tm 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.

Energetic evidence for formation of a pH-dependent hydrophobic cluster in the denatured state of Thermus thermophilus ribonuclease H.

NMR studies on the denatured states of proteins indicate that residual structure often resides predominantly in hydrophobic clusters. Such hydrophobic cluster formation implies burial of apolar surface and, consequently, is expected to cause a decrease in heat capacity. We report here that, in the case of ribonuclease H from the thermophile Thermus thermophilus, a sharp decrease in denatured-state heat capacity occurs at about pH 3.8; this result points to the formation of hydrophobic clusters triggered by the protonation of several (about four) carboxylic acid groups, and indicates that the burial of apolar surface is favored by the less hydrophilic character of the uncharged forms of Asp and Glu side-chains. The process is not accompanied by large changes in optically active structure, but appears to be highly cooperative, as indicated by the sharpness of the pH-induced transition in the heat capacity. This acid-induced hydrophobic burial in denatured T.thermophilus ribonuclease H is clearly reflected in the pH dependence of the denaturation temperature (i.e. an abrupt change of slope at about pH 3.8 is seen in the plot of denaturation temperature versus pH), supporting a role for such denatured-state hydrophobic clusters in protein stability. The finding of cooperative protonation of several groups coupled to surface burial in denatured T.thermophilus ribonuclease H emphasizes the potential complexity of denatured-state electrostatics and advises caution when attempting to predict denatured-state properties on the basis of simple electrostatic models. Finally, our results suggest a higher propensity for hydrophobic cluster formation in the denatured state of T.thermophilus ribonuclease H as compared with that of its mesophilic counterpart from Escherichia coli.

Conformational and thermodynamic properties of peptide binding to the human S100P protein.

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 Ca2+ 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 Ca2+-dependent and -independent manner. Ca2+-independent binding occurs with low affinity (Kd approximately 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, Ca2+-dependent binding of melittin to S100P occurs with high affinity (Kd approximately 5 microM) has a stoichiometry of two molecules of melittin per S100P dimer, appears to have positive cooperativity, and is driven by hydrophobic interactions. Furthermore, Ca2+-dependent S100P-melittin complex formation is accompanied by significant conformational changes: Melittin, otherwise unstructured in solution, adopts a helical conformation upon interaction with Ca2+-S100P. These results support a model for the Ca2+-dependent conformational switch in S100P for functional target recognition.

Energetics of heparin binding to human acidic fibroblast growth factor.

The binding of low-molecular-weight heparin to an amino-terminal-truncated, 132-amino-acid, human acidic fibroblast growth factor form has been studied by isothermal titration calorimetry. This technique yields values for the enthalpy change and equilibrium constant, from which the Gibbs energy and entropy change are also calculated. Experiments in different buffers and pH values show that the protonic balance during the reaction is negligible. Experiments made at pH 7.0 with NaCl concentrations ranging from 0.20 to 0.60 M revealed changes in enthalpy and Gibbs energy in the range of -30- -17 and -27- -24 kJ x mol(-1), respectively. Isothermal titration calorimetry was also performed at different temperatures to obtain a value for the heat-capacity change at pH 7.0 and 0.4 M NaCl concentration of -96 J K- x mol(-1). A change in the length of heparin brought about no change in the thermodynamic parameters at 25 degrees C under the same experimental conditions. Changes upon ligand binding in the range of -50- -200 A2 in both polar and non-polar solvent-accessible surface areas were calculated from thermodynamic data by using different parametric equations taken from the literature. These values suggest a negligible overall conformational change in the protein when it binds to heparin and no formation of any protein-protein interface.