The Effect of Dimethyl Sulfoxide on the Lysozyme Unfolding Kinetics, Thermodynamics, and Mechanism.

Timur Magsumov,Timur Mukhametzyanov,Alisa Fatkhutdinova,Igor Sedov

doi:10.3390/biom9100547

Timur Magsumov, Timur Mukhametzyanov + Show 2 more

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https://doi.org/10.3390/biom9100547

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Journal: Biomolecules	Publication Date: Sep 29, 2019
Citations: 10	License type: CC BY 4.0

Affiliation: Kazan Federal University

Abstract

The thermal stability of proteins in the presence of organic solvents and the search for ways to increase this stability are important topics in industrial biocatalysis and protein engineering. The denaturation of hen egg-white lysozyme in mixtures of water with dimethyl sulfoxide (DMSO) with a broad range of compositions was studied using a combination of differential scanning calorimetry (DSC), circular dichroism (CD), and spectrofluorimetry techniques. In this study, for the first time, the kinetics of unfolding of lysozyme in DMSO–water mixtures was characterized. In the presence of DMSO, a sharp decrease in near-UV CD and an increase in the fluorescence signal were observed at lower temperatures than the DSC denaturation peak. It was found that differences in the temperatures of the CD and DSC signal changes increase as the content of DMSO increases. Changes in CD and fluorescence are triggered by a break of the tertiary contacts, leading to an intermediate state, while the DSC peak corresponds to a subsequent complete loss of the native structure. In this way, the commonly used two-state model was proven to be unsuitable to describe the unfolding of lysozyme in the presence of DMSO. In kinetic studies, it was found that even high concentrations of DMSO do not drastically change the activation energy of the initial stage of unfolding associated with a disruption of the tertiary structure, while the enthalpy of denaturation shows a significant dependence on DMSO content. This observation suggests that the structure of the transition state upon unfolding remains similar to the structure of the native state.

Highlights

The use of mixed aqueous–organic solvents allows enzyme-catalyzed reactions to be conducted with substrates that are poorly soluble in water, to change the selectivity of these reactions, and sometimes to make them proceed in the reverse direction [1,2,3]
Besides the lower thermodynamic stability of proteins in aqueous–organic mixtures reflected in lower denaturation temperatures, the kinetic stability of the native state and the thermodynamic functions of activation of the unfolding process are affected by the presence of organic cosolvents
While there are many papers dedicated to the effects of organic cosolvents on the thermodynamic stability of proteins [9,14,15,16,17,18], the kinetics and mechanisms of protein unfolding in binary aqueous–organic solvents remain poorly explored

Summary

Introduction

The use of mixed aqueous–organic solvents allows enzyme-catalyzed reactions to be conducted with substrates that are poorly soluble in water, to change the selectivity of these reactions, and sometimes to make them proceed in the reverse direction [1,2,3]. With few exceptions [4,5], the addition of organic cosolvents leads to a decrease in the stability of the native structure of the enzyme proteins [6], a decrease in the denaturation temperature [7,8,9,10], and a reduction of the catalytic activity [11,12,13]. Besides the lower thermodynamic stability of proteins in aqueous–organic mixtures reflected in lower denaturation temperatures, the kinetic stability of the native state and the thermodynamic functions of activation of the unfolding process are affected by the presence of organic cosolvents. While there are many papers dedicated to the effects of organic cosolvents on the thermodynamic stability of proteins [9,14,15,16,17,18], the kinetics and mechanisms of protein unfolding in binary aqueous–organic solvents remain poorly explored.

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