Conformational constraint in protein ligand design and the inconsistency of binding entropy

• Published in: Biopolymers 2008-08, Vol.89 (8), p.653-667
• Main Authors: Udugamasooriya, D. Gomika, Spaller, Mark R.
• Format: Article
• Language: English
• Subjects: Benzoates - chemistry; Calorimetry; Entropy; Glutarates - chemistry; Ligands; Membrane Proteins - chemistry; Membrane Proteins - metabolism; Osmotic Pressure; peptides; Peptides, Cyclic - chemistry; Protein Binding; Protein Conformation; Protein Structure, Tertiary; proteins; Protons; Succinates - chemistry; thermodynamics; Water
• ISSN: 0006-3525; 1097-0282
• DOI: 10.1002/bip.20983

It is an accepted practice in ligand design to introduce conformational constraint with the expectation of improving affinity, justified by the theoretical possibility that an unfavorable change in binding entropy will be reduced. This rationale of minimizing the entropic penalty through imposing structural constraints upon a ligand, however, has been voiced more often than verified. Here we examine three modified cyclic peptides, along with multiple versions of their linear control analogs, and determine their thermodynamic parameters when binding the same host, the third PDZ domain (PDZ3) of the mammalian postsynaptic density‐95 (PSD‐95) protein. To begin a two‐stage investigation, the initial evaluation involved solution binding studies with isothermal titration calorimetry (ITC), which provided the changes in Gibbs free energy (ΔG), enthalpy (ΔH), and entropy (TΔS) upon formation of the protein‐ligand complex. In the second stage, a selected macrocycle along with two matched linear controls were subjected to more rigorous analysis by ITC, which included (1) change in heat of buffer ionization (ΔHion) titrations, to examine the role of proton transfer events; (2) change in heat capacity (ΔCp) determinations, to indirectly probe the nature of the binding surface; and (3) osmotic stress experiments, to evaluate desolvation effects and quantitate water release. Together, these demonstrate that the entropic relationship between a macrocyclic ligand and a linear counterpart can be a complex one that is difficult to rationalize. Further, the addition of constraint can, counterintuitively, lead to a less favorable change in binding entropy. This underscores the need to use matched linear control ligands to assure that comparisons are made in a meaningful manner. © 2008 Wiley Periodicals, Inc. Biopolymers 89: 653–667, 2008. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at [e-mail]