Stability of Ligand-induced Protein Conformation Influences Affinity in Maltose-binding Protein |
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Institution: | Department of Biochemistry, Groningen Biomolecular Science and Biotechnology Institute, University of Groningen, Nijenborgh 4, Groningen 9747 AG, the Netherlands |
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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. |
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Keywords: | ligand-binding conformational dynamics ligand affinity single-molecule FRET thermodynamics of ligand binding |
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