Harnessing Pressure Modulation for Exploring Ligand Binding Reactions in Cosolvent Solutions

Abstract

A comprehensive understanding of ligand−protein interactions requires information about all thermodynamic parameters that describe the complexation reaction, and they should be able to provide the necessary information to understand the molecular forces that drive complex formation. Usually, binding studies are performed at ambient pressure conditions. However, in addition to using temperature variation to reveal enthalpic and entropic contributions to ligand binding, complementary pressure-dependent studies providing volumetric properties of the reaction can be beneficial. Changes in partial molar volume can inform about changes in packing and hydration upon ligand binding. Here, after a general discussion of pressure effects on ligand binding reactions, we present a comprehensive study of the effect of pressure and a widely used organic cosolvent, dimethyl sulfoxide (DMSO), on the binding of a small aromatic ligand, proflavine, to the enzyme α-chymotrypsin. We found that DMSO, which acts as a competitive inhibitor for proflavine, has a strong impact on the interaction process, resulting in a decrease of the binding constant. While the reaction performed in neat buffer is basically pressure insensitive, the partial molar volume of the complex in the presence of DMSO is larger compared with the uncomplexed state, rendering the binding constant markedly smaller upon pressurization. We also show that the magnitude and sign of the binding volume provide valuable information about the interaction mechanism and hydration changes, which is of particular interest when cosolvents are present.

Deep sea osmolytes in action: their effect on protein–ligand binding under high pressure stress

“…However, besides using temperature variation to reveal enthalpic and entropic contributions to ligand binding (van't Hoff analysis), also complementary pressure dependent studies may be advantageous. [5][6][7][8] Pressure application enables modulation of intra-and intermolecular interactions (e.g., pressure stabilizes H-bonds and weakens internal salt bridges), and it allows to determine volume changes upon binding with high accuracy (even within fractions of the size of a water molecule), which provides additional information about packing and hydration changes upon ligand binding. [8][9][10][11] According to the Braun-Le Cha ˆtelier principle, pressure favors the state that occupies the smallest possible overall volume.…”

Binding Properties of RNA Quadruplex of SARS-CoV-2 to Berberine Compared to Telomeric DNA Quadruplex

“…The application of high hydrostatic pressure (HPP) is a powerful tool to study the volumetric properties of reactions including complex formation [ 35 ]. However, before being able to explore the effect of HHP on the strength of interaction as described by K b between berberine and the G4Qs, it is mandatory to verify that the structure of 22AG and RG-1 and in complex with berberine is not changing.…”

“…Measurement of the pressure dependence of K b allows to determine the volume change accompanying of the binding reaction, Δ V r , according to [ 35 ]: …”

Ions in the Deep Subsurface of Earth, Mars, and Icy Moons: Their Effects in Combination with Temperature and Pressure on tRNA–Ligand Binding

“…To shed more light on the intimate nature of the binding process, we also employed pressure dependent fluorescence spectroscopy, which provides information about the volumetric changes accompanying the binding event [ 50 ]. To this end, the values of the binding constants were determined in the pressure range between 1 and 2000 bar in the absence and in the presence of the Mg salts at 1 mM concentration.…”

“…From the pressure dependence of K b values, the binding volume, Δ V b , can be determined through the relation [ 50 ]: Here, R is the universal gas constant and T is the temperature (in K units). The binding volume, Δ V b , is defined as the difference in the partial molar volume of the tRNA–ThT complex formed, V complex , and the sum of the partial molar volumes of ThT, V tRNA , and tRNA, V ThT , i.e., Δ V b = V complex − ( V tRNA + V ThT ).…”