Protein-Ligand Binding Volume Determined from a Single 2D NMR Spectrum with Increasing Pressure.

Gediminas Skvarnavičius,Zigmantas Toleikis,Daumantas Matulis,Vytautas Petrauskas,Christian Roumestand,Vilma Michailovienė

doi:10.1021/acs.jpcb.1c02917

Abstract

Proteins undergo changes in their partial volumes in numerous biological processes such as enzymatic catalysis, unfolding–refolding, and ligand binding. The change in the protein volume upon ligand binding—a parameter termed the protein–ligand binding volume—can be extensively studied by high-pressure NMR spectroscopy. In this study, we developed a method to determine the protein–ligand binding volume from a single two-dimensional (2D) 1H–15N heteronuclear single quantum coherence (HSQC) spectrum at different pressures, if the exchange between ligand-free and ligand-bound states of a protein is slow in the NMR time-scale. This approach required a significantly lower amount of protein and NMR time to determine the protein–ligand binding volume of two carbonic anhydrase isozymes upon binding their ligands. The proposed method can be used in other protein–ligand systems and expand the knowledge about protein volume changes upon small-molecule binding.

Highlights

Most protein−ligand binding studies are performed at ambient pressure and provide the standard Gibbs energy of binding
High pressure provides an additional approach to study the protein−ligand binding by revealing the volume changes occurring upon protein interaction with a ligand
We aimed to show that ΔVb can be obtained from 1H−15N heteronuclear single quantum coherence (HSQC) spectra at various pressures using a single concentration of protein and ligand

Summary

Introduction

Most protein−ligand binding studies are performed at ambient pressure and provide the standard Gibbs energy of binding. High pressure provides an additional approach to study the protein−ligand binding by revealing the volume changes occurring upon protein interaction with a ligand. This approach provides insight into the changes in the protein partial volume while performing native functions, unfolding, and binding small molecules. These reactions cause the rearrangement of volume-related protein cavities, clefts, and the hydration shell.[1−5]. This parameter may have alternative names, such as the reaction or interaction volume

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