Thermodynamics of Folding, Stabilization, and Binding in an Engineered Protein−Protein Complex

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Abstract
We analyzed the thermodynamics of a complex protein−protein binding interaction using the (engineered) ZSPA-1 affibody and it's Z domain binding partner as a model. Free ZSPA-1 exists in an equilibrium between a molten-globule-like (MG) state and a completely unfolded state, wheras a well-ordered structure is observed in the Z:ZSPA-1 complex. The thermodynamics of the MG state unfolding equilibrium can be separated from the thermodynamics of binding and stabilization by combined analysis of isothermal titration calorimetry data and a separate van't Hoff analysis of thermal unfolding. We find that (i) the unfolding equilibrium of free ZSPA-1 has only a small influence on effective binding affinity, that (ii) the Z:ZSPA-1 interface is inconspicuous and structure-based energetics calculations suggest that it should be capable of supporting strong binding, but that (iii) the conformational stabilization of the MG state to a well-ordered structure in the Z:ZSPA-1 complex is associated with a large change in conformational entropy that opposes binding.