Critical assessment of structure-based approaches to improve protein resistance in aqueous ionic liquids by enzyme-wide saturation mutagenesis

Harrar, Till El; Davari, Mehdi D.; Jaeger, Karl‐Erich; Schwaneberg, Ulrich; Gohlke, Holger

doi:10.1016/j.csbj.2021.12.018

Cited by 8 publications

(8 citation statements)

References 105 publications

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“…Moreover, considering the solvent variation can thermodynamically and kinetically affect the process of unfolding and aggregation events, the fine-tuning of cation/anion and hydration solvation by introducing surface substitution might offer intriguing prospects for improving native enzymatic function by preventing the irreversible protein aggregation. Besides the employed halogen-anion (i.e., Cl – ) in the current study, more and more favorable ILs are being applied in the biocatalysis field, ,,− such as the IL containing the hydrophobic anion (e.g., bis(trifluoromethylsulfonyl)amide ([TFSA] − ) and bis(fluorosulfonyl)amide ([FSA] − )). In addition, the studied observables (e.g., structural change, hydration, IL layer) in MD simulation can be easily transferred to other protein–IL systems, which offer the researchers the key to open the protein–IL interaction door.…”

Section: Resultsmentioning

confidence: 99%

“…Protein engineering emerges as a powerful tool to optimize enzymes to achieve the desired properties. − Charge engineering/modification on enzymes has been shown to be a promising approach to tailoring the enzyme resistance in ILs. ,− In this regard, Nordwald et al fine-tuned the positive-to-negative charged amino acid ratio of chymotrypsin (from bovine pancreas) and observed an increased half-life (1.6- and 4.3-fold) relative to wild-type chymotrypsin in 1-butyl-3-methylimidazolium chloride ([BMIM]Cl, 20 wt %) and 1-ethyl-3-methylimidazolium ethylsulfate ([EMIM][EtSO 4 ], 20 wt %), respectively . Moreover, half-lives of Candida rugosa lipase and Carica papaya papain in [BMIM]Cl were also enhanced by applying the similarly charged amino acid ratio strategy …”

Section: Introductionmentioning

confidence: 99%

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How Does Surface Charge Engineering of Bacillus subtilis Lipase A Improve Ionic Liquid Resistance? Lessons Learned from Molecular Dynamics Simulations

Pramanik

Cui

Dhoke

et al. 2022

ACS Sustainable Chem. Eng.

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Biocatalysis in ionic liquids (ILs) gained substantial interest due to solvent properties of the ILs, such as near-zero vapor pressure, high thermal stability, and wide tunability. Enzymes are often not catalytically active in ILs; therefore, understanding and improving enzyme resistance in ILs are essential to enable efficient biocatalysis in ionic liquids. Surface charge engineering has repeatedly been reported to enhance enzyme resistance toward ILs. However, the molecular knowledge about how substitutions to charged amino acids improve enzyme activity in an IL is far from being understood. Here, we report a comprehensive study to provide some principles of how surface charged amino acid substitutions (negatively and positively) strengthen the IL resistance of the Bacillus subtilis lipase A (BSLA) in [BMIM]Cl. Twenty typical BSLA substitutions (ten beneficial and ten nonbeneficial, obtained from the BSLA-SSM library) were studied by molecular dynamics (MD) simulations in the [BMIM]Cl system. The BSLA-IL interaction patterns were printed by analyzing several structural-and solvation-based observables. Lessons learned by analyzing the SSM library of BSLA comprise the following: (i) A general trend was found where both negatively and positively charged substitutions increased the essential water molecules locally at the substitution site, thereby contributing to the overall protein hydration shell. (ii) Electrostatic repulsion of both IL ions and the refined hydration shell are ultimately the two main drivers to enhanced IL resistance. The analysis of 20 BSLA substitutions and the identified common interactions reveals that surface charge engineering is very likely to be a general protein engineering strategy to enhance lipase/enzyme activity in ILs. Moreover, this study also suggests that MD is a valuable technique to screen for beneficial substitutions that repel/recruit surface solvation.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

How Does Surface Charge Engineering of Bacillus subtilis Lipase A Improve Ionic Liquid Resistance? Lessons Learned from Molecular Dynamics Simulations

Pramanik

Cui

Dhoke

et al. 2022

ACS Sustainable Chem. Eng.

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“…Moreover, the anion type has also been validated as a contributor to the reduction of enzyme activity. Interestingly, structural weak spots identified by the rigidity theory showed better gain-in-precision (GiP) values in enhancing enzyme resistance in ILs . Up to now, it is well recognized that five factors affect the characterization of enzymes in ILs: (a) changing the tertiary structure elements, , (b) disrupting the hydrogen-bond/salt bridges; (c) forming a cation–anion network; (d) reshaping electronic interaction and π–π interaction; ,, and (e) competing for the binding site with the substrate .…”

Section: Introductionmentioning

confidence: 99%

“…Interestingly, structural weak spots identified by the rigidity theory showed better gain-in-precision (GiP) values in enhancing enzyme resistance in ILs. 16 Up to now, it is well recognized that five factors affect the characterization of enzymes in ILs: (a) changing the tertiary structure elements, 11,17 (b) disrupting the hydrogen-bond/salt bridges; 18 (c) forming a cation−anion network; (d) reshaping electronic interaction and π−π interaction; 11,19,20 and (e) competing for the binding site with the substrate. 11 The latter correlates closely with the physicochemical properties of ILs, 21 for example, hydrophilicity/hydrophobicity, constituent cations and anions, and cation alkyl chain length.…”

Section: Introductionmentioning

confidence: 99%

Enzyme Hydration: How to Retain Resistance in Ionic Liquids

Cui

Zhang

Yildiz

et al. 2022

ACS Sustainable Chem. Eng.

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Application of ionic liquids (ILs) as media in biocatalysis has enormous potential for synthesizing valuable compounds and bulk products in pharmaceuticals and bioenergy due to their unique solvent properties such as volatility, flammability, and solubility. However, ILs as reaction media are often limited by poor enzymatic activity and stability in ILs. We printed a comprehensive IL−enzyme interaction map by studying 45 molecular observables of 30 lipase A from Bacillus subtilis (BSLA) variants in four ILs and a substitutional landscape with 1504 BSLA variants. The results demonstrated that the enzyme hydration shell is the deciding and independent factor determining the enzyme's IL resistance. A universal positive correlation (up to R 2 = 0.96 in 1-butyl-3-methylimidazolium trifluoromethanesulfonate ([BMIM][TfO]) and R 2 = 0.85 in 1-butyl-3methylimidazolium chloride ([BMIM]Cl)) was verified, and an experimentally derived ranking of amino acid substitutions is summarized in a list to provide benefits for better protein engineering practice. Hydration-guided engineering yielded a supremely tolerant BSLA variant I12R/D34K/A132K with 8.1-fold, 8.6-fold, 6.6-fold, and 4.6-fold improved tolerance toward [BMIM]Cl, [BMIM]Br, [BMIM]I, and [BMIM][TfO], respectively, when compared to the wild-type BSLA. The obtained knowledge provides a lesson learned on forecasting enzyme stability in ILs and simplifies a rational design of the IL-tolerant enzymes.

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“…Surprisingly, we obtained low improvements in the prediction precision of favorable substitutions for FoldX compared to random mutagenesis, whereas predictions based on experimental thermostability data from CNA resulted in markedly higher improvements. 11 One of the reasons for the subpar results may be that the FoldX (free) energy function has been trained on data for the thermodynamic stability of proteins in water. 13 Hence, the function does not consider effects on interactions between protein residues by solvents other than water, e.g., organic solvents or ionic liquids.…”

Section: Introductionmentioning

confidence: 99%

Cumulative Millisecond-Long Sampling for a Comprehensive Energetic Evaluation of Aqueous Ionic Liquid Effects on Amino Acid Interactions

Harrar

Gohlke

2022

J. Chem. Inf. Model.

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The interactions of amino acid side-chains confer diverse energetic contributions and physical properties to a protein’s stability and function. Various computational tools estimate the effect of changing a given amino acid on the protein’s stability based on parametrized (free) energy functions. When parametrized for the prediction of protein stability in water, such energy functions can lead to suboptimal results for other solvents, such as ionic liquids (IL), aqueous ionic liquids (aIL), or salt solutions. However, to our knowledge, no comprehensive data are available describing the energetic effects of aIL on intramolecular protein interactions. Here, we present the most comprehensive set of potential of mean force (PMF) profiles of pairwise protein–residue interactions to date, covering 50 relevant interactions in water, the two biotechnologically relevant aIL [BMIM/Cl] and [BMIM/TfO], and [Na/Cl]. These results are based on a cumulated simulation time of >1 ms. aIL and salt ions can weaken, but also strengthen, specific residue interactions by more than 3 kcal mol–1, depending on the residue pair, residue–residue configuration, participating ions, and concentration, necessitating considering such interactions specifically. These changes originate from a complex interplay of competitive or cooperative noncovalent ion–residue interactions, changes in solvent structural dynamics, or unspecific charge screening effects and occur at the contact distance but also at larger, solvent-separated distances. This data provide explanations at the atomistic and energetic levels for complex IL effects on protein stability and should help improve the prediction accuracies of computational tools that estimate protein stability based on (free) energy functions.

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Critical assessment of structure-based approaches to improve protein resistance in aqueous ionic liquids by enzyme-wide saturation mutagenesis

Cited by 8 publications

References 105 publications

How Does Surface Charge Engineering of Bacillus subtilis Lipase A Improve Ionic Liquid Resistance? Lessons Learned from Molecular Dynamics Simulations

How Does Surface Charge Engineering of Bacillus subtilis Lipase A Improve Ionic Liquid Resistance? Lessons Learned from Molecular Dynamics Simulations

Enzyme Hydration: How to Retain Resistance in Ionic Liquids

Cumulative Millisecond-Long Sampling for a Comprehensive Energetic Evaluation of Aqueous Ionic Liquid Effects on Amino Acid Interactions

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