Affibody Complex Formation: An In-Depth Thermodynamic Analysis Using Isothermal Titration Calorimetry

This study investigates the thermodynamics of binding between the affibody proteins ZTaq and anti-ZTaq across a broad temperature range, aiming to deepen the understanding of the underlying mechanisms governing their interaction. Affibodies are small, engineered proteins of notable stability and practical utility, serving as robust models for molecular recognition processes. Here, the anti-idiotypic binders ZTaq and anti-ZTaq were expressed and purified, and their interaction was characterized using isothermal titration calorimetry (ITC). The analysis revealed that the formation of the ZTaq:anti-ZTaq complex is marked by a large negative free energy of binding ΔG_b that is virtually unaffected by changes in salt concentration, in contrast to typical protein-polyelectrolyte systems where ionic strength plays a major role. Furthermore, the thermodynamic data indicated a large, negative heat capacity change ΔC_p, which is primarily attributed to conformational transitions, especially the disruption of the molten-globule-like (MG) state of anti-ZTaq above 303 K. By comparing thermodynamic and structural properties with related affibody systems, the study aims to clarify how specific sequence features contribute to the exceptional binding properties of these proteins, providing new insights into protein engineering for high-affinity molecular recognition.