Cosolvent Dimethyl Sulfoxide Influences Protein–Ligand Binding Kinetics via Solvent Viscosity Effects: Revealing the Success Rate of Complex Formation Following Diffusive Protein–Ligand Encounter

Abstract

Protein−ligand-exchange kinetics determines the duration of biochemical signals and consequently plays an important role in drug design. Binding studies commonly require solubilization of designed ligands in solvents such as dimethyl sulfoxide (DMSO), resulting in residual amounts of DMSO following titration of solubilized ligands into aqueous protein samples. Therefore, it is critical to establish whether DMSO influences protein−ligand binding. Here, we address the general and indirect effect of DMSO on protein−ligand binding caused by solvent viscosity, which is strongly dependent on the relative concentrations of DMSO and water. As a model system, we studied the binding of a drug-like ligand to the carbohydrate recognition domain of galectin-3 in the presence of variable amounts of DMSO. We used isothermal titration calorimetry to characterize binding thermodynamics and 15 N NMR relaxation to monitor kinetics. The binding enthalpy is not affected, but we observe a subtle trend of increasingly unfavorable entropy of binding, and consequently decreased affinity, with increasing DMSO concentration. The increasing concentration of DMSO results in a reduced association rate of binding, while the dissociation rate is less affected. The observed association rate is inversely proportional to the viscosity of the DMSO−water mixture, as expected from theory, but significantly reduced from the diffusion-controlled limit. By comparing the viscosity dependence of the observed association rate with that of the theoretical diffusion-controlled association rate, we estimate the success rate of productive complex formation following an initial encounter of proteins and ligands, showing that only one out of several hundred binding "attempts" are successful.

Ligand-induced protein transition state stabilization switches the binding pathway from conformational selection to induced fit

“…Thus, the experimental value of k on is significantly lower than k onD , which is expected because the former includes not only the diffusion-limited rate of encounter but also the diffusive “search” by the ligand across the protein surface from the initial point of contact to the immediate neighborhood of the binding site. This result is also in keeping with our recent work, showing that several hundred ligand–galectin-3C encounters occur for each successful binding event ( 53 ).…”

DMSO‐Related Effects on Ligand‐Binding Properties of Lysine Methyltransferases G9a and SETD8

“…Another possible explanation of this effect on binding kinetics could be the higher solvent viscosity at high percentage of DMSO, causing a decrease in diffusion coefficient of the analyte. [23]…”

Conformational energies of biomolecules in solution: Extending the MPCONF196 benchmark with explicit water molecules

“…Solute‐solvent interactions play a crucial role for the properties of molecules in solution such as the reactivity towards certain reaction pathways, the association of molecules, and conformational energies 1‐4 . Besides industrial and laboratory processes like stereoselective reactions in water 5 or various catalytic reactions, 6,7 the solvent effect for biomolecules is of special interest as it can influence, for example, the folding and dynamics of proteins 8,9 and their binding to ligands 10,11 . Moreover, the conformations of a protein in solution can deviate from the gas‐phase ones and can have different properties 12,13 .…”