Abstract
Our understanding of what determines ligand affinity of proteins is poor, even with high-resolution structures available. Both the non-covalent ligand–protein interactions and the relative free energies of available conformations contribute to the affinity of a protein for a ligand. Distant, non-binding site residues can influence the ligand affinity by altering the free energy difference between a ligand-free and ligand-bound conformation. Our hypothesis is that when different ligands induce distinct ligand-bound conformations, it should be possible to tweak their affinities by changing the free energies of the available conformations. We tested this idea for the maltose-binding protein (MBP) from Escherichia coli. We used single-molecule Förster resonance energy transfer (smFRET) to distinguish several unique ligand-bound conformations of MBP. We engineered mutations, distant from the binding site, to affect the stabilities of different ligand-bound conformations. We show that ligand affinity can indeed be altered in a conformation-dependent manner. Our studies provide a framework for the tuning of ligand affinity, apart from modifying binding site residues.
Highlights
Insight in the principles behind ligand affinity of proteins is fundamental to all biological processes
Our hypothesis is that when different ligands induce distinct ligand-bound conformations, it should be possible to tweak their affinities by changing the free energies of the available conformations. We tested this idea for the maltose-binding protein (MBP) from Escherichia coli
We further developed the theoretical framework of closed-conformation dependent ligand affinity tuning by describing how single mutations outside the binding pocket can affect the stability of ligand-induced closed MBP conformations
Summary
Insight in the principles behind ligand affinity of proteins is fundamental to all biological processes. Mutations that affect the direct interactions with structures but can probe the conformational the ligand in and around the binding pocket are dynamics of a protein, for instance by singleexpected to primarily alter DGbind instead of DGconf In such mutants the DDG between the open and ligand-bound MBP molecule FRET. The mutation resulted in a DDG of 0.8 kcal/mol between the open and the closed-liganded conformations of all three ligands (Figure 3(C)) This indicates that the conformational change probably did not significantly alter the free energy of the maltotetraose-induced closed conformation compared to the closed conformation that is induced by either maltose or maltotriose. The new insights help us to understand the sometimes-surprising differences in ligand affinity between homologous proteins and may be used for a more rational optimization of biocatalysts
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