Thermodynamics of Folding and Binding in an Affibody:Affibody Complex

Affibody binding proteins are selected from phage-displayed libraries of variants of the 58 residue Z domain. ZTaq is an affibody originally selected as a binder to Taq DNA polymerase. The anti-ZTaq affibody was selected as a binder to ZTaq and the ZTaq:anti-ZTaq complex is formed with a dissociation constant Kd = 0.1 μM. We have determined the structure of the ZTaq:anti-ZTaq complex as well as the free state structures of ZTaq and anti-ZTaq using NMR. Here we complement the structural data with thermodynamic studies of ZTaq and anti-ZTaq folding and complex formation. Both affibody proteins show cooperative two-state thermal denaturation at melting temperatures TM ∼ 56 °C. ZTaq:anti-ZTaq complex formation at 25 °C in 50 mM NaCl and 20 mM phosphate buffer (pH 6.4) is enthalpy driven with ΔH°bind = –9.0 (±0.1) kcal mol−1. The heat capacity change ΔCP°,bind = –0.43 (±0.01) kcal mol−1 K−1 is in accordance with the predominantly non-polar character of the binding surface, as judged from calculations based on changes in accessible surface areas. A further dissection of the small binding entropy at 25 °C (–TΔS°bind = –0.6 (±0.1) kcal mol−1) suggests that a favourable desolvation of non-polar surface is almost completely balanced by unfavourable conformational entropy changes and loss of rotational and translational entropy. Such effects can therefore be limiting for strong binding also when interacting protein components are stable and homogeneously folded. The combined structure and thermodynamics data suggest that protein properties are not likely to be a serious limitation for the development of engineered binding proteins based on the Z domain.