Thermochemistry of Binary and Ternary Protein Interactions Measured by Titration Calorimetry: Complex Formation of CD4, HIV gp120, and Anti-gp120

This chapter describes the use of solution biophysical technologies for quantitative analysis of protein-protein interactions. Biophysical technologies have the potential for revealing detailed, molecular information about the binding mechanism of protein interactions, such as stoichiometry, binding thermodynamics, coupled protonation events, and accurate determination of affinity. However, binding interactions between proteins can be more complex than often assumed. An accurate interpretation of biophysical data in terms of molecular binding mechanism normally requires the use of multiple biophysical methods to evaluate the roles of various processes that may be coupled to binding such as oligomerization, protein folding, protonation, and changes in hydration. This chapter presents two applications. The first is the analysis of the binary interaction between the human receptor soluble CD4 and the human immunodeficiency vius envelop protein gp 120. A strategy is outlined for characterizing binary protein interactions, and evaluating the roles of various commonly occuring side reactions such as oligomerization, protonation, protein folding, and conformational change. The sCD4-gp 120 reaction has one of the largest binding enthalpy energies of any protein interaction. The origin of this highly unusual feature is assignes, after considering several possible molecular origins, to a large conformational change within gp 120 that is coupled to binding of sCD4. The second application builds on the first, and includes the quantitative analysis of ternary protein complex formation of sCD4, gp 120, and the anti-gp 120 monoclonal antibody 48d. The importance of characterizing ternary interactions within the energetic constrainst set by a thermodynamic cycle analysis for equilibrium binding reactions is described.