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Aqueous ionic liquids redistribute local enzyme stability via long-range perturbation pathways.

ABSTRACT: Ionic liquids (IL) and aqueous ionic liquids (aIL) are attractive (co-)solvents for biocatalysis due to their unique properties. On the other hand, the incubation of enzymes in IL or aIL often reduces enzyme activity. Recent studies proposed various aIL-induced effects to explain the reduction, classified as direct effects, e.g., local dehydration or competitive inhibition, and indirect effects, e.g., structural perturbations or disturbed catalytic site integrity. However, the molecular origin of indirect effects has largely remained elusive. Here we show by multi-μs long molecular dynamics simulations, free energy computations, and rigidity analyses that aIL favorably interact with specific residues of Bacillus subtilis Lipase A (BsLipA) and modify the local structural stability of this model enzyme by inducing long-range perturbations of noncovalent interactions. The perturbations percolate over neighboring residues and eventually affect the catalytic site and the buried protein core. Validation against a complete experimental site saturation mutagenesis library of BsLipA (3620 variants) reveals that the residues of the perturbation pathways are distinguished sequence positions where substitutions highly likely yield significantly improved residual activity. Our results demonstrate that identifying these perturbation pathways and specific IL ion-residue interactions there effectively predicts focused variant libraries with improved aIL tolerance.

SUBMITTER: El Harrar T

PROVIDER: S-EPMC8355836 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Deoxyribonucleic acid (DNA) carries the genetic information essential for the growth and functioning of living organisms, playing a significant role in life sciences research. However, the long-term storage and preservation of DNA, while ensuring its bioactivity, are still current challenges to overcome. In this work, aqueous solutions of ionic liquids (ILs) were investigated as potential preservation media for double stranded (dsDNA). A screening of several ILs, by combining the cholinium, tetrabutylammonium, tetrabutylphosphonium, and 1-ethyl-3-methylimidazolium, cations with the anions bromide, chloride, dihydrogen phosphate, acetate, and glycolate, was carried out in order to gather fundamental knowledge on the molecular features of ILs that improve the dsDNA stability. Different IL concentrations and the pH effect were also addressed. Circular dichroism (CD) spectroscopy was used to evaluate the conformational structure and stability of dsDNA. IL-DNA interactions were appraised by UV-Vis absorption spectrophotometry and 31P nuclear magnetic resonance (NMR) spectroscopy. The results obtained demonstrate that pH has a significant effect towards the dsDNA stability. Amongst the ILs investigated, cholinium-based ILs are the most promising class of ILs to preserve the dsDNA structure, in which electrostatic interactions between the cholinium cation and the DNA phosphate groups play a significant role as demonstrated by the 31P NMR data, being more relevant at higher IL concentrations. On the other hand, the denaturation of dsDNA mainly occurs with ILs composed of more hydrophobic cations and able to establish dispersive interactions with the nucleobases environment. Furthermore, the IL anion has a weaker impact when compared to the IL cation effect to interact with DNA molecules. The experimental data of this work provide relevant fundamental knowledge for the application of ILs in the preservation of nucleic acids, being of high relevance in the biotechnology field.

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Aqueous ionic liquids redistribute local enzyme stability via long-range perturbation pathways.

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