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

Molecules, vol. 31, pp. 2500

Abstract

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 ΔGb 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 ΔCp, 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.

Authors 2

1. Jacek Janusz Walkowiak corresponding Aachen DWI-Leibniz-Institute for Interactive Materials e.V Institute of Technical and Macromolecular Chemistry

   RWTH Aachen University · DWI – Leibniz Institute for Interactive Materials · Maastricht University · University of Bayreuth

   Affiliation as printed

   Aachen-Maastricht Institute for Biobased Materials (AMIBM), Maastricht University, Urmonderbaan 22, 6167 RD Geleen, The Netherlands

   DWI—Leibniz-Institute for Interactive Materials e.V., Forckenbeckstraße 50, 52074 Aachen, Germany

   Department of Chemistry, Inorganic Chemistry III, Northern Bavarian NMR Centre, University of Bayreuth, Universitätsstrasse 30, 95440 Bayreuth, Germany

   Institute of Technical and Macromolecular Chemistry, RWTH Aachen University, Worringerweg 2, 52074 Aachen, Germany

2. Julian Karl Aachen DWI-Leibniz-Institute for Interactive Materials e.V

   DWI – Leibniz Institute for Interactive Materials

   Affiliation as printed

   DWI—Leibniz-Institute for Interactive Materials e.V., Forckenbeckstraße 50, 52074 Aachen, Germany