Dominant Alcohol-Protein Interaction via Hydration-Enabled Enthalpy-Driven Binding Mechanism

Abstract

Water plays an important role in weak associations of small drug molecules with proteins. Intense focus has been on binding-induced structural changes in the water network surrounding protein binding sites, especially their contributions to binding thermodynamics. However, water is also tightly coupled to protein conformations and dynamics, and so far little is known about the influence of water-protein interactions on ligand binding. Alcohols are a type of low-affinity drugs, and it remains unclear how water affects alcohol-protein interactions. Here, we present alcohol adsorption isotherms under controlled protein hydration using in situ NMR detection. As functions of hydration level, Gibbs free energy, enthalpy, and entropy of binding were determined from the temperature dependence of isotherms. Two types of alcohol binding were found. The dominant type is low-affinity nonspecific binding, which is strongly dependent on temperature and the level of hydration. At low hydration levels, this nonspecific binding only occurs above a threshold of alcohol vapor pressure. An increased hydration level reduces this threshold, with it finally disappearing at a hydration level of h ≈ 0.2 (g water/g protein), gradually shifting alcohol binding from an entropy-driven to an enthalpy-driven process. Water at charged and polar groups on the protein surface was found to be particularly important in enabling this binding. Although further increase in hydration has smaller effects on the changes of binding enthalpy and entropy, it results in a significant negative change in Gibbs free energy due to unmatched enthalpy-entropy compensation. These results show the crucial role of water-protein interplay in alcohol binding.

Figure 1

Isotherms of (A) EtOH and (B) TFE in dry BSA at 6°C, 15°C, and 25°C. The insets show isotherms below P/P₀~0.7. Thresholds of relative vapor pressure in the isotherms are recognized. The threshold of relative vapor pressure is P/P₀~0.15 for EtOH (Inset of A) and P/P₀~0.3 for TFE (Inset of B). The sorption of both alcohols shows little temperature dependence below this threshold and is marked with shade in yellow. Above the threshold, alcohol sorption is enhanced by temperature. Sharp alcohol uptake above the relative vapor pressure of P/P₀~0.7 occurs for both alcohols, associated with protein denaturation. At each alcohol vapor pressure, NMR signal was measured five times when the interaction reached equilibrium, and then the standard deviations of NMR peak areas were used to calibrate the error bars in the isotherms.

Figure 2

(A) Isotherms of TFE in hydrated BSA at 15°C below P/P₀=0.3 at various hydration levels. Dotted straight lines associated with the isotherms of h=0.11, 0.16 and 0.18 illustrate how the threshold (the intercept of the dotted line with the horizontal line of y=0) is determined for a given isotherm associated with nonspecific alcohol binding. (B) The determined alcohol relative vapor pressure threshold is plotted versus h at 15°C. The threshold decreases linearly with h and reaches zero at h~0.2. Inset: the number of bound TFE versus h at 15°C at P/P₀=0.01 and P/P₀=0.03. The value of thresholds and the number of bound TFE were determined from Figure 2A. (C) Isotherms of TFE in hydrated BSA at h=0.11 and 15°C and 25°C, and at h=0.32 and 15°C and 25°C. The isotherms at h=0.11 show a relative pressure threshold at P/P₀~0.25, while no threshold is seen in isotherms at h=0.32. At each alcohol vapor pressure, NMR signal was measured five times when the interaction reached equilibrium, and then the standard deviations of NMR peak areas were used to calibrate the error bars in the isotherms.

Figure 3

Water sorption isotherms of BSA at 6°C, 15°C, and 25°C. The isotherms show temperature dependence above h~0.2. The inset shows a typical ¹H NMR spectrum at 25°C and h~0.2. The peak associated with water component and the peak associated with protein component are obtained by Lorentz line fitting. At each water vapor pressure, NMR signal was measured five times when the interaction reached equilibrium, and then the standard deviations of NMR peak areas were used to calibrate the error bars in the isotherms.

Figure 4

ΔG, ΔH, and TΔS of TFE binding in hydrated BSA versus hydration level h at 20°C. Isotherms at the same hydration levels in Supporting Information Figure S2 were used to calculate ΔG, ΔH, and TΔS at 20°C (see Methods). Relative vapor pressures in the region where nonspecific binding dominates the isotherms (after completion of binding to high-affinity sites and before denaturation taking place) were used in the calculation (dry, P/P₀~0.4; h=0.11, P/P₀~0.4; h=0.21, P/P₀~0.2; h=0.32, P/P₀~0.1). ΔG, ΔH, and TΔS were calculated at such relative vapor pressures and plotted in Figure 4. The error bars of isotherms in Supporting Information Figure S2 were propagated to calculate the error bars in Figure 4.