A method is described for the inclusion of the effects of hydration in empirical conformational energy computations on polypeptides. The free energy of hydration is composed of additive contributions of various functional groups. The hydration of each group is assumed to be proportional to the accessible surface area of the group. The constants of proportionality, representing the free energy of hydration per unit area of accessible surface, have been evaluated for seven classes of groups (occurring in peptides) by least-squares fitting to experimental free energies of solution of small monofunctional aliphatic and aromatic molecules. The same method has also been applied to the modeling of the enthalpy and heat capacity ofhydration, each of which is computed from the accessible surface area.The free energy of folding of a protein consists of the sum of contributions from the energy of its intramolecular interactions (1, 2) and from the free energy of interaction of the molecule with the surrounding solvent water. Exact computation of the latter contribution still poses problems (3). As a practical approach, hydration-shell models have been used. In these models, the free energy of interaction of water molecules with the solute is expressed in the form of an averaged effective potential of interaction of atoms (and functional groups) of a solute molecule with a layer of solvent around each atom (4-10)-i.e., in terms ofa potential ofmean force (3). An empirical free energy of hydration is assigned to every atom and group. When the conformation of the protein changes, some water is eliminated from the hydration shell whenever groups on the protein approach each other. The free energy change accompanying this process depends on the total free energy of hydration of the groups and on the amount of water being eliminated from the hydration shells. This amount, in turn, depends on the size and distance of separation of the groups that approach each other, and it can be computed by geometrical methods from the volumes of overlapping spheres (4-6, 10, 11).The hydration-shell model contains several approximations, which may be sources of error and also reduce the speed of computer-based numerical computations (8), such as the thickness of the shell, the apportioning of the free energy between overlapping hydration shells of covalently connected atoms, and the calculation of the volume of overlap of three or more hydration spheres that belong to nearby atoms. The latter problem can be overcome, however, by modifying the computing procedures (10, 11).We have initiated an alternative approach, in order to avoid these problems. We assume that the extent of interaction of any functional group i of a solute with the solvent is proportional to the solvent-accessible surface area Ai of group i (12-14) because the group can interact directly only with the water molecules that are in contact with the group at this surface. Thus, the total free energy of hydration of a solute molecule is given by Eq. 1: AGh = E Ai [1] where...

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Although bovine ␤-lactoglobulin assumes a monomeric native structure at pH 3 in the absence of salt, the addition of salts stabilizes the dimer. Thermodynamics of the monomer-dimer equilibrium dependent on the salt concentration were studied by sedimentation equilibrium. The addition of NaCl, KCl, or guanidine hydrochloride below 1 M stabilized the dimer in a similar manner. On the other hand, NaClO 4 was more effective than other salts by about 20-fold, suggesting that anion binding is responsible for the salt-induced dimer formation, as observed for acid-unfolded proteins. The addition of guanidine hydrochloride at 5 M dissociated the dimer into monomers because of the denaturation of protein structure. In the presence of either NaCl or NaClO 4 , the dimerization constant decreased with an increase in temperature, indicating that the enthalpy change (⌬H D ) of dimer formation is negative. The heat effect of the dimer formation was directly measured with an isothermal titration calorimeter by titrating the monomeric ␤-lactoglobulin at pH 3.0 with NaClO 4 . The net heat effects after subtraction of the heat of salt dilution, corresponding to ⌬H D , were negative, and were consistent with those obtained by the sedimentation equilibrium. From the dependence of dimerization constant on temperature measured by sedimentation equilibrium, we estimated the ⌬H D value at 20°C and the heat capacity change (⌬C p ) of dimer formation. In both NaCl and NaClO 4 , the obtained ⌬C p value was negative, indicating the dominant role of burial of the hydrophobic surfaces upon dimer formation. The observed ⌬C p values were consistent with the calculated value from the X-ray dimeric structure using a method of accessible surface area. These results indicated that monomer-dimer equilibrium of ␤-lactoglobulin at pH 3 is determined by a subtle balance of hydrophobic and electrostatic effects, which are modulated by the addition of salts or by changes in temperature.