The most common strategy in the development of HIV-1 protease inhibitors has been the design of high affinity transition state analogs that effectively compete with natural substrates for the active site. A second approach has been the development of compounds that inactivate the protease by destabilizing its quaternary or tertiary structure. A successful optimization of these strategies requires an accurate knowledge of the energetics of structural stabilization and binding, and the identification of those regions in the protease molecule that are critical to stability and function. Here the energetics of stabilization of the HIV-1 protease has been measured for the first time by high sensitivity differential scanning calorimetry. These studies have permitted the evaluation of the different components of the Gibbs energy of stabilization (the enthalpy, entropy and heat capacity changes). The stability of the protease is pH-dependent and due to its dimeric nature is also concentration-dependent. At pH 3.4 the Gibbs energy of stabilization is close to 10 kcal/mol at 25 degreesC, consistent with a dissociation constant of 5x10(-8) M. The stability of the protease increases at higher pH values. At pH 5, the Gibbs energy of stabilization is 14.5 kcal/mol at 25 degreesC, consistent with a dissociation constant of 2.3x10(-11) M. The pH dependence of the Gibbs energy of stabilization indicates that between pH 3.4 and pH 5 an average of 3-4 ionizable groups per dimer become protonated upon unfolding. A structure-based thermodynamic analysis of the protease molecule indicates that most of the Gibbs energy of stabilization is provided by the dimerization interface and that the isolated subunits are intrinsically unstable. The Gibbs energy, however, is not uniformly distributed along the dimerization interface. The dimer interface is characterized by the presence of clusters of residues (hot spots) that contribute significantly and other regions that contribute very little to subunit association. At the dimerization interface, residues located at the carboxy and amino termini contribute close to 75% of the total Gibbs energy (Cys95, Thr96, Leu97, Asn98 and Phe99 and Pro1, Ile3, Leu5). Residues Thr26, Gly27 and Asp29 located at the base of the active site are also important, and to a lesser extent Gly49, Ile50, Gly51 located at the tip of the flap region. The structure-based thermodynamic analysis also predicts the existence of regions of the protease with only marginal stability and a high propensity to undergo independent local unfolding. In particular, the flap region occupies a very shallow energy minimum and its conformation can easily be affected by relatively small perturbations. This property of the protease can be related to the ability of some mutations to elicit resistance towards certain inhibitors.