High pressure spectrofluorimetry – a tool to determine protein-ligand binding volume

Skvarnavičius, Gediminas; Toleikis, Zigmantas; Grigaliunas, Martynas; Smirnovienė, Joana; Norvaišas, Povilas; Cimmperman, P.; Matulis, Daumantas; Petrauskas, Vytautas

doi:10.1088/1742-6596/950/4/042001

Cited by 5 publications

(13 citation statements)

References 24 publications

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“…There are several techniques that are capable of determining the volume of protein–ligand binding: the fluorescent pressure shift assay (PressureFluor, FPSA) (Toleikis et al ., 2011, 2016; Skvarnavičius et al ., 2017), vibrating tube densitometry(Barbosa et al ., 2003; Son et al ., 2012; Toleikis et al ., 2016), and the high-pressure NMR (Wilton et al ., 2009; Roche et al ., 2012; Toleikis et al ., 2016). They can determine the change of system volume and compressibility caused by the bound ligand.…”

Section: Assays To Determine Small Molecule Inhibitor Interaction Witmentioning

confidence: 99%

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Thermodynamic, kinetic, and structural parameterization of human carbonic anhydrase interactions toward enhanced inhibitor design

Linkuvienė

Zubrienė

Manakova

et al. 2018

Quart. Rev. Biophys.

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Section: Assays To Determine Small Molecule Inhibitor Interaction Witmentioning

confidence: 99%

“…(2015); Skvarnavičius et al . (2017). However, there is relatively little data available in the literature on any protein–ligand system, not mentioning the CA, and it is difficult to judge yet if these approaches will be useful in the design of ligands with desired binding properties.…”

Section: Assays To Determine Small Molecule Inhibitor Interaction Witmentioning

confidence: 99%

Thermodynamic, kinetic, and structural parameterization of human carbonic anhydrase interactions toward enhanced inhibitor design

Linkuvienė

Zubrienė

Manakova

et al. 2018

Quart. Rev. Biophys.

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“…The obtained Δ V b values of the CA I and CA II interaction with the ligands were within the range of −22 to −28 mL/mol. The binding volume of −32 mL/mol of the CA I interaction with the other compound bearing a primary sulfonamide group in its structureacetazolamidewas determined previously by a fluorescent pressure shift assay . Acetazolamide has a higher affinity toward CA I and more negative Δ V b than structurally similar compounds 1 and 2 .…”

Section: Discussionmentioning

confidence: 89%

“…The binding volume of −32 mL/mol of the CA I interaction with the other compound bearing a primary sulfonamide group in its structure—acetazolamide—was determined previously by a fluorescent pressure shift assay. 61 Acetazolamide has a higher affinity toward CA I 40 and more negative Δ V b than structurally similar compounds 1 and 2 . These results were compatible with previous findings showing correlations between protein–ligand binding volumes and affinities.…”

Section: Discussionmentioning

confidence: 97%

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

et al. 2021

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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) 1 H– 15 N 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.

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“…The deep oceans on earth are populated by many organisms that are constantly exposed to high hydrostatic pressures (HHP), reaching values even beyond 1000 bar (100 MPa) at the deepest ocean trenches and subseafloor region. , Hence, knowledge about high hydrostatic pressure effects on biological systems is also fundamental for our understanding of life being exposed to such harsh conditions and of the physical limits of life in general. The molecular effects of pressure on binding equilibria are still largely unknown, however, and the literature on pressure effects on ligand-binding reactions is still scarce. − …”

Section: Introductionmentioning

confidence: 99%

Harnessing Pressure Modulation for Exploring Ligand Binding Reactions in Cosolvent Solutions

et al. 2021

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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.

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High pressure spectrofluorimetry – a tool to determine protein-ligand binding volume

Cited by 5 publications

References 24 publications

Thermodynamic, kinetic, and structural parameterization of human carbonic anhydrase interactions toward enhanced inhibitor design

Thermodynamic, kinetic, and structural parameterization of human carbonic anhydrase interactions toward enhanced inhibitor design

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

Harnessing Pressure Modulation for Exploring Ligand Binding Reactions in Cosolvent Solutions

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