NMR Relaxation Analysis of Ligand-Dependent Backbone Dynamics and Conformational Entropy in Galectin-3C

Abstract

Understanding the role of conformational entropy in protein-ligand interactions remains a major challenge in rational drug design. In this work Galectin-3C (Gal-3C) complexes with glycomimetic macrocyclic ligands FS435 and FS471 were investigated using NMR spectroscopy.
The ligands exhibit similar binding free energies, and calorimetric studies revealed significant enthalpy-entropy compensation. Surprisingly, the structurally pre-organized ligand FS435 displayed a less favorable entropy contribution upon binding than the more flexible ligand FS471. To investigate whether this behavior arises from changes in protein conformational entropy, 15N relaxation measurements (R1, R2, and heteronuclear NOE) were acquired at 14.1 T and 18.8 T and analyzed using isotropic and anisotropic model-free approaches.
Chemical shift perturbation analysis demonstrated largely conserved binding modes for the two ligands, although ligand-specific perturbations were observed near the binding interface. The fitted global rotational correlation times were highly similar for the two complexes (τm ≈ 7.3 – 7.4 ns), indicating comparable overall tumbling behavior and solvent conditions. Model-free analysis revealed subtle but spatially organized differences in backbone dynamics. The FS435-bound complex displayed increased rigidity in regions proximal to the binding site, whereas neighboring and distal regions displayed compensatory increases in flexibility. This suggests redistribution of backbone dynamics throughout the protein rather than uniform rigidification upon ligand binding.
Conformational entropy analysis indicated a general trend towards greater backbone conformational entropy in the FS471-bound complex relative to the FS435-bound complex. These findings support the hypothesis that modulation of Gal-3C backbone dynamics contributes to the thermodynamic differences observed between the two complexes.

Popular Abstract

When researchers design new drug molecules, it is easy to imagine binding as a lock-and-key problem: the drug molecule should fit as well as possible into the binding site of the protein. However, reality is much more complex. Proteins are not rigid structures, they are in constant motion, and these motions can influence how favorably a molecule can bind.

In this thesis, galectin-3C was studied. Galectin-3C is part of the protein galectin-3, which has been proven to be involved in cancer and cardiovascular diseases. The protein has been investigated as a possible target for future drug development. The work focused on two small “keys”, also known as ligands, denoted as FS435 and FS471. These ligands are structurally similar but differ in how they interact with galectin-3C.

One of the ligands (FS435) is more preorganized. This means that it already resembles the shape it adopts when bound to the protein and therefore does not need to rearrange as much during binding. The other ligand, FS471, is more open and flexible and can adopt several conformations. A common idea in ligand design is that a preorganized ligand should in fact be more favorable to the protein. Favorable means that the ligand does not lose as much conformational freedom upon binding than a flexible ligand. However, previous data suggests the opposite trend for these two ligands. One possible explanation is that the preorganized ligand causes the protein to reorganize in a more rigid manner, which shifts the entropic cost from the ligand to the protein.

To investigate this paradox, nuclear magnetic resonance (NMR) was used. NMR utilizes strong magnetic fields which can help to examine the movement of the backbone of the protein. In this thesis, NMR relaxation experiments were used to examine the motions of the backbone of galectin-3C when bound to FS435 and FS471.

The results showed that both ligands in complex with galectin-3C did not affect the overall rotation of the protein in any different way. The observable differences between the two ligands were observed in how the two ligands affected the local amino acid residues around the protein. These differences suggest that Galectin-3C retains more backbone motional freedom when bound to the flexible FS471 than when bound to the preorganized ligand FS435.

This means that a more preorganized ligand is not necessarily more favorable for the entire binding process. Although FS435 may lose less flexibility itself, it appears to restrict the protein backbone more than FS471.

In conclusion, protein motions can help to explain why two similar structural molecules exhibit different binding properties. For drug design, this means that researchers should not only ask how well a molecule fits into a protein structure, but also how the molecule affects the movements of the protein.

• author: Carlander, Jonatan
• supervisor: Mikael Akke; Johan Wallerstein
• organization: Biophysical Chemistry
• course: KFKM05 20261
• year: 2026
• type: H3 - Professional qualifications (4 Years - )
• subject: Technology and Engineering; Biology and Life Sciences; Chemistry
• keywords: biophysical chemistry, galectin-3C, NMR relaxation, conformational entropy, protein-ligand interactions, model-free analysis
• language: English
• id: 9233559
• date added to LUP: 2026-06-10 08:59:04
• date last changed: 2026-06-10 08:59:04