A single-molecule dissection of ligand binding to a protein with intrinsic dynamics

Abstract

Protein dynamics have been suggested to have a crucial role in biomolecular recognition, but the precise molecular mechanisms remain unclear. Herein, we performed single-molecule fluorescence resonance energy transfer measurements for wild-type maltose-binding protein (MBP) and its variants to demonstrate the interplay of conformational dynamics and molecular recognition. Kinetic analysis provided direct evidence that MBP recognizes a ligand through an 'induced-fit' mechanism, not through the generally proposed selection mechanism for proteins with conformational dynamics such as MBP. Our results indicated that the mere presence of intrinsic dynamics is insufficient for a 'selection' mechanism. An energetic analysis of ligand binding implicated the critical role of conformational dynamics in facilitating a structural change that occurs upon ligand binding.

Protein conformational dynamics dictate the binding affinity for a ligand

“…The transitions between the two distinct conformational states were clearly detected. As previously reported 12 , the low-and high-FRET states were interpreted as open and partially closed forms, respectively. The intrinsic dynamics of the other MBP mutants could also be observed with a 100-ms time resolution in smFRET measurements ( Supplementary Fig.…”

“…The receptor-ligand interaction process comprises two fundamental steps, namely, the binding and dissociation of a ligand. Our recent single-molecule studies revealed that conformational dynamics plays a negligible role in the ligandbinding step of an MBP 12 . A series of MBP mutants constructed through hinge mutations were shown to have a wide range of intrinsic opening rates and dissociation constants for maltose, enabling us to investigate how protein dynamics determine the ligand dissociation, and thus the binding affinity.…”

“…The role of conformational dynamics in a receptor-ligand interaction has been the subject of intensive studies 3,9,10,12,[16][17][18] . The receptor-ligand interaction process comprises two fundamental steps, namely, the binding and dissociation of a ligand.…”

“…1a). The dye-labelling positions were shown to be sensitive enough such that significant changes in the FRET efficiency (E FRET ) arose during the conformational transitions between the apo-and holo-forms 12 . An amylose-binding assay and a circular dichroism (CD) analysis confirmed that the cysteine introduction and dye labelling have a negligible effect on the biological activity of MBP mutants ( Supplementary Fig.…”

“…In a popular view, termed the conformational selection model, ligand binding was proposed to be strongly coupled to protein dynamics; a ligand selectively binds to a subset of a conformational ensemble, similar to the ligand-bound form, and shifts the equilibrium 9 . However, our recent single-molecule fluorescence resonance energy transfer (smFRET) study on maltose-binding proteins (MBPs) revealed that the presence of conformational dynamics does not dictate the ligand-binding step 12 , leaving the role of protein dynamics in the ligand dissociation an open question.…”