Two fragments of pancreatic ribonuclease A, a truncated version of S-peptide (residues 1-15) and S-protein (residues 21-124), combine to give a catalytically active complex designated ribonuclease S. Residue 13 in the peptide is methionine. According to the X-ray structure of the complex of S-protein and S-peptide (1-20), this residue is almost fully buried. We have substituted Met-13 with seven other hydrophobic residues ranging in size from glycine to phenylalanine and have determined the thermodynamic parameters associated with the binding of these analogues to S-protein by titration calorimetry at 25 degrees C. These data should provide useful quantitative information for evaluating the contribution of hydrophobic interactions in the stabilization of protein structures.

Two fragments of pancreatic ribonuclease A, a truncated version of S-peptide (residues 1-15) and S-protein (residues 21-124), combine to give a catalytically active complex designated ribonuclease S. We have substituted the wild-type residue Met-13 with six other hydrophobic residues ranging in size from alanine to phenylalanine and have determined the thermodynamic parameters associated with binding of these analogues to S-protein by titration calorimetry in the temperature range 5-25 degrees C. The heat capacity change (delta Cp) associated with binding was obtained from a global analysis of the temperature dependences of the free energies and enthalpies of binding. The delta Cp's were not correlated in any simple fashion with the nonpolar surface area (delta Anp) buried upon binding.

Parallel measurements of the thermodynamics (free-energy, enthalpy, entropy and heat-capac change) of ligand binding to FK506 binding protein First suggested >70 years ago (1, 2) and particularly emphasized by Pauling et al. (3,4), the hydrogen bond is now recognized as an interaction of fundamental importance in determining the structures of proteins and their complexes with ligands (5). Nevertheless, conflicting views are still held on the relative energetic contributions ofhydrogen bonds and interactions involving nonpolar groups to the thermodynamics of protein folding and ligand binding (6-8). Models have been presented arguing that the overall enthalpic contribution of interactions involving nonpolar moieties (at 250C) to the process of protein folding is highly unfavorable (7, 9), highly favorable (8), or essentially zero (10, 11). Arguments have also been advanced stating that the overall contribution of amide hydrogen-bond formation to the enthalpy of protein folding (or ligand-protein binding) at 250C is favorable (7), negligible (12) and possibly unfavorable (8,13). Clarification of the present controversy requires estimates of the enthalpies of formation of specific hydrogen bonds in particular regions of known protein structures. Such measurements are essential for understanding the energetic basis of protein folding and for obtaining structure-based predictions of the energies of ligand binding for the purpose of drug design.Part of the difficulty in dissecting the relative energetic contribution of hydrogen bonds to folding or binding reactions of proteins is that the formation of hydrogen bonds between atoms in reaction products is often accompanied by the release ofwater molecules that were hydrogen-bonded to these atoms prior to reaction. Tacrolimus (also known as FK506) and rapamycin are large macrocyclic compounds each of which binds with high affinity to a common cytosolic protein of 12 kDa known as FK506 binding protein . Recently, the structures of FKBP-12 in solution and in the crystalline state have been determined (14, 15), as have the crystal structures of the tacrolimus-FKBP-12 and rapamyci-FKBP-12 complexes (16-18). One of the structural features common to both protein-ligand complexes is a hydrogen bond between the Tyr-82 hydroxyl hydrogen (donor) and the amide carbonyl oxygen (acceptor) of either rapamycin or tacrolimus, buried in the hydrophobic interface (Fig. 1). As determined by x-ray crystallography, an important feature of the unliganded protein structure is the welldefined hydration of this tyrosine hydroxyl group. Furthermore, the NMR structure of tacrolimus in chloroform has been reported by Karuso et al. (19); the low solubility of tacrolimus prevents the determination of its structure in water. However, the interaction of unbound tacrolimus with water has been investigated through molecular dynamics simulation (20). The simulation reveals that the amide carbonyl oxygen of tacrolimus is exposed to solvent and makes an average of 1.6 hydrogen bonds with water mol...

High-sensitivity differential scanning calorimetry has been applied to the study of the reversible thermal unfolding of the lysozyme of T4 bacteriophage in which the threonine residue at position 157 has been replaced by seven different residues. High-resolution structures derived from X-ray crystallography have been reported for these and six other mutants by Alber et al. [Alber, T., Dao-Pin, S., Wilson, K., Wozniak, J. A., Cook, S. P., & Matthews, B. W. (1987) Nature 330, 41-46]. At pH 2.5 the changes relative to the wild-type protein in the standard free energy of unfolding produced by these mutations indicate apparent destabilizations of 0.6 kcal mol-1 (T157R) to 1.9 kcal mol-1 (T157I), whereas the changes in enthalpy of unfolding range from -5.8 kcal mol-1 (T157N) to 11.9 kcal mol-1 (T157E). Since the denaturations are in all cases accompanied by large changes in heat capacity amounting to 2.5 kcal K-1 mol-1, both the free energies and enthalpies are functions of temperature. An intriguing feature of the present results is the relatively large enthalpy changes and the corresponding compensating entropy changes. Our present understanding of the intramolecular energetics of proteins is insufficient to account for these changes.

Hydrophobic interactions, believed to be important determinants of the stability of many protein structures, multisubunit protein complexes, and protein/ligand complexes, may be considered to be the result of two component interaction processes: the removal of nonpolar groups from water (hydration) and the packing of these groups within proteins or protein complexes. Early studies by Kauzmann (8) and Tanford (9) emphasized the similarities of protein unfolding and the transfer of nonpolar substances from water to nonpolar phases. More recently, striking correlations between the thermodynamics of protein folding and the aqueous dissolution of hydrophobic substances have been noted by Sturtevant (10), Baldwin (11), and Murphy et al. (12). Among the most intriguing of the observations are the linear relationships observed between (i) enthalpy and heat capacity changes and (ii) entropy and heat capacity changes, for solutes within a given class (gaseous, solid, or liquid hydrophobic compound or protein). Consideration of these relationships has led to differing theories regarding the contribution of hydrophobic interactions to the stabilization of protein structures (11, 13, 14). A key common feature of the theories is the dissection of the free energy of unfolding into its various contributions, with the heat capacity playing a central role in determining the free energy of hydrophobicinteraction.An early analysis by Sturtevant (10) indicated that heat capacity changes for protein unfolding are determined largely by the exposure of nonpolar groups to water but that one must also consider contributions due to the changes in the numbers of easily excitable vibrational modes upon unfolding. The concept of solvent-accessible surface area introduced by Lee and Richards (15) provided an important structural parameter with which to describe the interaction of water with proteins. Recently, several reports have focused on the relationship between heat capacity changes and changes in solvent-accessible surface areas accompanying protein unfolding. Spolar et al. (16) and Livingstone et al. (17) analyzed the heat capacity changes for the unfolding of proteins for which high-resolution x-ray crystallographic information was available. Their analyses indicated that the heat capacity change for unfolding proteins is proportional to the difference in solvent-accessible nonpolar surface area between native and denatured states. Another investigation on the binding of various S peptides to S protein casts doubt on the generalization of the relationship between nonpolar surface and heat capacity (18,19).To clarify the structural determinants for heat capacities and their relationship to the free energy of hydrophobic interaction, it is important to obtain high-precision measurements of heat capacity changes, in addition to other thermotTo whom reprint requests should be addressed. 4781The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertis...