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© 2022 The Authors. Published by American Chemical Society, Paramyxoviruses are enveloped viruses harboring a negative-sense RNA genome that must enter the host’s cells to replicate. In the case of the parainfluenza virus, the cell entry process starts with the recognition and attachment to target receptors, followed by proteolytic cleavage of the fusion glycoprotein (F) protein, exposing the fusion peptide (FP) region. The FP is responsible for binding to the target membrane, and it is believed to play a crucial role in the fusion process, but the mechanism by which the parainfluenza FP (PIFP) promotes membrane fusion is still unclear. To elucidate this matter, we performed biophysical experimentation of the PIFP in membranes, together with coarse grain (CG) and atomistic (AA) molecular dynamics (MD) simulations. The simulation results led to the pinpointing of the most important PIFP amino acid residues for membrane fusion and show that, at high concentrations, the peptide induces the formation of a water-permeable porelike structure. This structure promotes lipid head intrusion and lipid tail protrusion, which facilitates membrane fusion. Biophysical experimental results validate these findings, showing that, depending on the peptide/lipid ratio, the PIFP can promote fusion and/or membrane leakage. Our work furthers the understanding of the PIFP-induced membrane fusion process, which might help foster development in the field of viral entry inhibition., This work was financially supported by FCT-Fundação para a Ciência e a Tecnologia, Portugal, through project PTDC/CCI-BIO/28200/2017 and by the European Union (H2020-FETOPEN-2018-2019-2020-01, grant no. 828774). This work was also financially supported by Project LISBOA-01-0145-FEDER-007660 (Microbiologia Molecular, Estrutural e Celular) funded by FEDER funds through COMPETE2020_Programa Operacional Competitividade e Internacionalização (POCI). M.V. and D.A.M. thank FCT for their PhD fellowships (SFRH/BD/148542/2019 and PD/BD/136752/2018, respectively). M.N.M. thanks FCT for the Post-Doc fellowship CEECIND/04124/2017. M.N.M. and D.L. thank the MACC for the computing hours in their HPC center (CPCA/A0/7329/2020 and CPCA/A0/7305/2020).

A key step in infections by enveloped viruses, such as influenza, is the fusion between the viral envelope and the host cell membrane, which allows the virus to insert its genetic material into the host cell and replicate. The influenza virus fusion process is promoted by hemagglutinin (HA), a glycoprotein that contains three identical monomers composed of two polypeptide chains (HA1 and HA2). Early studies on this protein revealed that HA‐mediated fusion involves the insertion of the HA2 N‐terminal segment into the host membrane and that this segment, known as the fusion peptide, is a key player in the fusion process. This mini‐review highlights the main findings that have been obtained by experimental and computational studies on the HA fusion peptide, which give us a glimpse of its mode of action., A key step in influenza infection is the fusion of the viral and host membranes, promoted by the protein hemagglutinin, which contains a fusion peptide that inserts and destabilizes the host membrane. This review highlights the major findings of experimental and computational studies, which analyzed the properties of this peptide, including the role of key residues for its fusogenic activity.

Funding Information: Funding: This study was financially supported by the Portuguese Fundação para a Ciência e Tec-nologia (FCT), grants PTDC/BIA-BQM/27959/2017 and PTDC/BIA-BQM/0562/2020, and Project MOSTMICRO-ITQB with references UIDB/04612/2020 and UIDP/04612/2020. This project has also received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement 810856. MCM is the recipient of FCT grant SFRH/BD/143651/2019. BAS is the recipient of FCT grant DFA/BD/8066/2020. Funding Information: This study was financially supported by the Portuguese Funda??o para a Ci?ncia e Tecnologia (FCT), grants PTDC/BIA-BQM/27959/2017 and PTDC/BIA-BQM/0562/2020, and Project MOSTMICRO-ITQB with references UIDB/04612/2020 and UIDP/04612/2020. This project has also received funding from the European Union?s Horizon 2020 research and innovation program under grant agreement 810856. MCM is the recipient of FCT grant SFRH/BD/143651/2019. BAS is the recipient of FCT grant DFA/BD/8066/2020. Publisher Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. Flavodiiron proteins (FDPs) are a family of modular and soluble enzymes endowed with nitric oxide and/or oxygen reductase activities, producing N2O or H2O, respectively. The FDP from Escherichia coli, which, apart from the two core domains, possesses a rubredoxin-like domain at the C-terminus (therefore named flavorubredoxin (FlRd)), is a bona fide NO reductase, exhibiting O2 reducing activity that is approximately ten times lower than that for NO. Among the flavorubredoxins, there is a strictly conserved amino acids motif,-G[S,T]SYN-, close to the catalytic diiron center. To assess its role in FlRd’s activity, we designed several site-directed mutants, replacing the conserved residues with hydrophobic or anionic ones. The mutants, which maintained the general characteristics of the wild type enzyme, including cofactor content and integrity of the diiron center, revealed a decrease of their oxygen reductase activity, while the NO reductase activity—specifically, its physiological function—was almost completely abolished in some of the mutants. Molecular modeling of the mutant proteins pointed to subtle changes in the predicted structures that resulted in the reduction of the hydration of the regions around the conserved residues, as well as in the elimination of hydrogen bonds, which may affect proton transfer and/or product release. published

Understanding SARS-CoV-2 evolution and host immunity is critical to control COVID-19 pandemics. At the core is an arms-race between SARS-CoV-2 antibody and angiotensin-converting enzyme 2 (ACE2) recognition, a function of the viral protein spike. Mutations in spike impacting antibody and/or ACE2 binding are appearing worldwide, with the effect of mutation synergy still incompletely understood. We engineered 25 spike-pseudotyped lentiviruses containing individual and combined mutations, and confirmed that E484K evades antibody neutralization elicited by infection or vaccination, a capacity augmented when complemented by K417N and N501Y mutations. In silico analysis provided an explanation for E484K immune evasion. E484 frequently engages in interactions with antibodies but not with ACE2. Importantly, we identified a novel amino acid of concern, S494, which shares a similar pattern. Using the already circulating mutation S494P, we found that it reduces antibody neutralization of convalescent and post-immunization sera, particularly when combined with E484K and N501Y. Our analysis of synergic mutations provides a landscape for hotspots for immune evasion and for targets for therapies, vaccines and diagnostics. One-Sentence SummaryAmino acids in SARS-CoV-2 spike protein implicated in immune evasion are biased for binding to neutralizing antibodies but dispensable for binding the host receptor angiotensin-converting enzyme

A challenging problem in Bioinformatics is to predict protein structure, properties, activities or interactions from their aminoacid sequences. Sequence-derived physicochemical features of proteins have been used to support the development of Machine Learning (ML) models. However, tools and platforms to calculate features from protein sequences and train ML models are scarce and have limitations in terms of performance, user-friendliness and domains of application., This study was supported by FCT through project PTDC/CCI-BIO/28200/2017 and the strategic funding of UID/BIO/04469/2020, and also by the European Regional Development Fund under the scope of Norte2020, through the projects DeepBio (ref. NORTE-01-0247-FEDER-039831). This work was also financially supported by Project LISBOA-01-0145-FEDER-007660 (Microbiologia Molecular, Estrutural e Celular) funded by FEDER funds through COMPETE2020 - Programa Operacional Competitividade e Internacionalização (POCI) and by national funds through FCT - Fundação para a Ciência e a Tecnologia., info:eu-repo/semantics/publishedVersion

Pyruvate dehydrogenase complex (PDC) catalyzes the oxidative decarboxylation of pyruvate to acetyl-coenzyme A, hinging glycolysis and the tricarboxylic acid cycle. PDC deficiency, an inborn error of metabolism, has a broad phenotypic spectrum. Symptoms range from fatal lactic acidosis or progressive neuromuscular impairment in the neonatal period, to chronic neurodegeneration. Most disease-causing mutations in PDC deficiency affect the PDHA1 gene, encoding the α subunit of the PDC-E1 component. Detailed biophysical analysis of pathogenic protein variants is a challenging approach to support the design of therapies based on improving and correcting protein structure and function. Herein, we report the characterization of clinically relevant PDC-E1α variants identified in Portuguese PDC deficient patients. These variants bear amino acid substitutions in different structural regions of PDC-E1α. The structural and functional analyses of recombinant heterotetrameric (αα’ββ’) PDC-E1 variants, combined with molecular dynamics (MD) simulations, show a limited impact of the amino acid changes on the conformational stability, apart from the increased propensity for aggregation of the p.R253G variant as compared to wild-type PDC-E1. However, all variants presented a functional impairment in terms of lower residual PDC-E1 enzymatic activity and ≈3–100 × lower affinity for the thiamine pyrophosphate (TPP) cofactor, in comparison with wild-type PDC-E1. MD simulations neatly showed generally decreased stability (increased flexibility) of all variants with respect to the WT heterotetramer, particularly in the TPP binding region. These results are discussed in light of disease severity of the patients bearing such mutations and highlight the difficulty of developing chaperone-based therapies for PDC deficiency.

The bioenergetics of anaerobic metabolism frequently relies on redox loops performed by membrane complexes with substrate- and quinone-binding sites on opposite sides of the membrane. However, in sulfate respiration (a key process in the biogeochemical sulfur cycle), the substrate- and quinone-binding sites of the QrcABCD complex are periplasmic, and their role in energy conservation has not been elucidated. Here we show that the QrcABCD complex of Desulfovibrio vulgaris is electrogenic, as protons and electrons required for quinone reduction are extracted from opposite sides of the membrane, with a H+/e− ratio of 1. Although the complex does not act as a H+-pump, QrcD may include a conserved proton channel leading from the N-side to the P-side menaquinone pocket. Our work provides evidence of how energy is conserved during dissimilatory sulfate reduction, and suggests mechanisms behind the functions of related bacterial respiratory complexes in other bioenergetic contexts., The bacterial complex QrcABCD plays a key role in the bioenergetics of sulfate respiration. Here, Duarte et al. show that this complex is electrogenic, with protons and electrons required for quinone reduction being extracted from opposite sides of the membrane.

© The Author(s) 2020. Open Access. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/., The influenza virus fusion process, whereby the virus fuses its envelope with the host endosome membrane to release the genetic material, takes place in the acidic late endosome environment. Acidification triggers a large conformational change in the fusion protein, hemagglutinin (HA), which enables the insertion of the N-terminal region of the HA2 subunit, known as the fusion peptide, into the membrane of the host endosome. However, the mechanism by which pH modulates the molecular properties of the fusion peptide remains unclear. To answer this question, we performed the first constant-pH molecular dynamics simulations of the influenza fusion peptide in a membrane, extending for 40 µs of aggregated time. The simulations were combined with spectroscopic data, which showed that the peptide is twofold more active in promoting lipid mixing of model membranes at pH 5 than at pH 7.4. The realistic treatment of protonation introduced by the constant-pH molecular dynamics simulations revealed that low pH stabilizes a vertical membrane-spanning conformation and leads to more frequent contacts between the fusion peptide and the lipid headgroups, which may explain the increase in activity. The study also revealed that the N-terminal region is determinant for the peptide's effect on the membrane., This work was financially supported by FCT—Fundação para a Ciência e a Tecnologia, Portugal, through projects PTDC/QUI-BIQ/114774/2009, PTDC/CCI-BIO/28200/2017 and Pest-OE/EQB/LA0004/2011. This work was also financially supported by Project LISBOA-01-0145-FEDER-007660 (Microbiologia Molecular, Estrutural e Celular) funded by FEDER funds through COMPETE2020—Programa Operacional Competitividade e Internacionalização (POCI) and by national funds through FCT—Fundação para a Ciência e a Tecnologia. DL was supported by FCT post-doc fellowship SFRH/BPD/92537/2013.

Hydrogenases are efficient biocatalysts for H2 production and oxidation with various potential biotechnological applications.[NiFe]-class hydrogenases are highly active in both production and oxidation processes—albeit primarily biased to the latter—but suffer from being sensitive to O2.[NiFeSe] hydrogenases are a subclass of [NiFe] hydrogenases with, usually, an increased insensitivity to aerobic environments. In this study we aim to understand the structural causes of the low sensitivity of a [NiFeSe]-hydrogenase, when compared with a [NiFe] class enzyme, by studying the diffusion of O2. To unravel the differences between the two enzymes, we used computational methods comprising Molecular Dynamics simulations with explicit O2 and Implicit Ligand Sampling methodologies. With the latter, we were able to map the free energy landscapes for O2 permeation in both enzymes. We derived pathways from these energy landscapes and selected the kinetically more relevant ones with reactive flux analysis using transition path theory. These studies evidence the existence of quite different pathways in both enzymes and predict a lower permeation efficiency for O2 in the case of the [NiFeSe]-hydrogenase when compared with the [NiFe] enzyme. These differences can explain the experimentally observed lower inhibition by O2 on [NiFeSe]-hydrogenases, when compared with [NiFe]-hydrogenases. A comprehensive map of the residues lining the most important O2 pathways in both enzymes is also presented.

Histone deacetylases are involved in chromatin remodelling and thus play a vital role in the epigenetic regulation of gene expression. HDAC inhibitors alter the acetylation status of histone and non-histone proteins to regulate various cellular events such as transcription. Novel HDAC inhibitors were designed and synthesised to promote higher levels of recombinant protein production in tobacco cell cultures. The effect of these chemical enhancers on the epigenetic profiles in plant cells has been evaluated by molecular docking, in vitro and in vivo studies. The addition of these novel enhancers led to an increase in histone H3 acetylation levels that promoted an increase in the accumulation levels of the recombinant protein in cell culture. These results can pave the way for the application of these enhancers to improve the production of high value products in plant cell based systems.

At a late stage in spore development in Bacillus subtilis, the mother cell directs synthesis of a layer of peptidoglycan known as the cortex between the two forespore membranes, as well as the assembly of a protective protein coat at the surface of the forespore outer membrane. SafA, the key determinant of inner coat assembly, is first recruited to the surface of the developing spore and then encases the spore under the control of the morphogenetic protein SpoVID. SafA has a LysM peptidoglycan-binding domain, SafALysM, and localizes to the cortex-coat interface in mature spores. SafALysM is followed by a region, A, required for an interaction with SpoVID and encasement. We now show that residues D10 and N30 in SafALysM, while involved in the interaction with peptidoglycan, are also required for the interaction with SpoVID and encasement. We further show that single alanine substitutions on residues S11, L12, and I39 of SafALysM that strongly impair binding to purified cortex peptidoglycan affect a later stage in the localization of SafA that is also dependent on the activity of SpoVE, a transglycosylase required for cortex formation. The assembly of SafA thus involves sequential protein-protein and protein-peptidoglycan interactions, mediated by the LysM domain, which are required first for encasement then for the final localization of the protein in mature spores. IMPORTANCEBacillus subtilis spores are encased in a multiprotein coat that surrounds an underlying peptidoglycan layer, the cortex. How the connection between the two layers is enforced is not well established. Here, we elucidate the role of the peptidoglycan-binding LysM domain, present in two proteins, SafA and SpoVID, that govern the localization of additional proteins to the coat. We found that SafALysM is a protein-protein interaction module during the early stages of coat assembly and a cortex-binding module at late stages in morphogenesis, with the cortex-binding function promoting a tight connection between the cortex and the coat. In contrast, SpoVIDLysM functions only as a protein-protein interaction domain that targets SpoVID to the spore surface at the onset of coat assembly.

At a late stage in spore development in Bacillus subtilis, the mother cell directs synthesis of a layer of peptidoglycan known as the cortex between the two forespore membranes, as well as the assembly of a protective protein coat at the surface of the forespore outer membrane. SafA, the key determinant of inner coat assembly, is first recruited to the surface of the developing spore and then encases the spore under the control of the morphogenetic protein SpoVID. SafA has a LysM peptidoglycan-binding domain, SafA(LysM), and localizes to the cortex-coat interface in mature spores. SafA(LysM) is followed by a region, A, required for an interaction with SpoVID and encasement. We now show that residues D10 and N30 in SafA(LysM), while involved in the interaction with peptidoglycan, are also required for the interaction with SpoVID and encasement. We further show that single alanine substitutions on residues S11, L12, and I39 of SafA(LysM) that strongly impair binding to purified cortex peptidoglycan affect a later stage in the localization of SafA that is also dependent on the activity of SpoVE, a transglycosylase required for cortex formation. The assembly of SafA thus involves sequential protein-protein and protein-peptidoglycan interactions, mediated by the LysM domain, which are required first for encasement then for the final localization of the protein in mature spores. IMPORTANCE Bacillus subtilis spores are encased in a multiprotein coat that surrounds an underlying peptidoglycan layer, the cortex. How the connection between the two layers is enforced is not well established. Here, we elucidate the role of the peptidoglycan-binding LysM domain, present in two proteins, SafA and SpoVID, that govern the localization of additional proteins to the coat. We found that SafA(LysM) is a protein-protein interaction module during the early stages of coat assembly and a cortex-binding module at late stages in morphogenesis, with the cortex-binding function promoting a tight connection between the cortex and the coat. In contrast, SpoVID(LysM) functions only as a protein-protein interaction domain that targets SpoVID to the spore surface at the onset of coat assembly.

Propagation of some Olea europaea L. cultivars is strongly limited due to recalcitrant behavior in adventitious root formation by semi-hardwood cuttings. One example is the cultivar ”Galega vulgar”. The formation of adventitious roots is considered a morphological response to stress. Alternative oxidase (AOX) is the terminal oxidase of the alternative pathway of the plant mitochondrial electron transport chain. This enzyme is well known to be induced in response to several biotic and abiotic stress situations. This work aimed to characterize the alternative oxidase 1 (AOX1)-subfamily in olive and to analyze the expression of transcripts during the indole-3-butyric acid (IBA)-induced in vitro adventitious rooting (AR) process. OeAOX1a (acc. no. MF410318) and OeAOX1d (acc. no. MF410319) were identified, as well as different transcript variants for both genes which resulted from alternative polyadenylation events. A correlation between transcript accumulation of both OeAOX1a and OeAOX1d transcripts and the three distinct phases (induction, initiation, and expression) of the AR process in olive was observed. Olive AOX1 genes seem to be associated with the induction and development of adventitious roots in IBA-treated explants. A better understanding of the molecular mechanisms underlying the stimulus needed for the induction of adventitious roots may help to develop more targeted and effective rooting induction protocols in order to improve the rooting ability of difficult-to-root cultivars. published

During the infection process, the influenza fusion peptide (FP) inserts into the host membrane, playing a crucial role in the fusion process between the viral and host membranes. In this work we used a combination of simulation and experimental techniques to analyse the molecular details of this process, which are largely unknown. Although the FP structure has been obtained by NMR in detergent micelles, there is no atomic structure information in membranes. To answer this question, we performed bias-exchange metadynamics (BE-META) simulations, which showed that the lowest energy states of the membrane-inserted FP correspond to helical-hairpin conformations similar to that observed in micelles. BE-META simulations of the G1V, W14A, G12A/G13A and G4A/G8A/G16A/G20A mutants revealed that all the mutations affect the peptide’s free energy landscape. A FRET-based analysis showed that all the mutants had a reduced fusogenic activity relative to the WT, in particular the mutants G12A/G13A and G4A/G8A/G16A/G20A. According to our results, one of the major causes of the lower activity of these mutants is their lower membrane affinity, which results in a lower concentration of peptide in the bilayer. These findings contribute to a better understanding of the influenza fusion process and open new routes for future studies.

A recent X-ray structure has enabled the location of chloride and cesium ions on the surface of subtilisin Carlsberg in acetonitrile soaked crystals. (1) To complement the previous study and analyze the system in solution, molecular dynamics (MD) simulations, in acetonitrile, were performed using this structure. Additionally, Cl(-) and Cs(+) ions were docked on the protein surface and this system was also simulated. Our results indicate that chloride ions tend to stay close to the protein, whereas cesium ions frequently migrate to the solvent. The distribution of the ions around the enzyme surface is not strongly biased by their initial locations. Replacing cesium by sodium ions showed that the distribution of the two cations is similar, indicating that Cs(+) can be used to find the binding sites of cations like Na(+) and K(+), which, unlike Cs(+), have physiological and biotechnological roles. The Na(+)Cl(-) is more stable than the Cs(+)Cl(-) ion pair, decreasing the probability of interaction between Cl(-) and subtilisin. The comparison of water and acetonitrile simulations indicates that the solvent influences the distribution of the ions. This work provides an extensive theoretical analysis of the interaction between ions and the model enzyme subtilisin in a nonaqueous medium.

The phenomenon known as "ligand imprinting" or "ligand-induced enzyme memory" was first reported in 1988, when Russell and Klibanov observed that lyophilizing subtilisin in the presence of competitive inhibitors (that were subsequently removed) could significantly enhance its activity in an apolar solvent. (Russell and Klibanov, J Biol Chem 1988;263:11624-11626). They further observed that this enhancement did not occur when similar assays were carried out in water. Herein, we shed light on the molecular determinants of ligand imprinting using a molecular dynamics (MD) approach. To simulate the effect of placing an enzyme in the presence of a ligand before its lyophilization, an inhibitor was docked in the active site of subtilisin and 20 ns MD simulations in water were performed. The ligand was then removed and the resulting structure was used for subsequent MD runs using hexane and water as solvents. As a control, the same simulation setup was applied using the structure of subtilisin in the absence of the inhibitor. We observed that the ligand maintains the active site in an open conformation and that this configuration is retained after the removal of the inhibitor, when the simulations are carried out in hexane. In agreement with experimental findings, the structural configuration induced by the ligand is lost when the simulations take place in water. Our analysis of fluctuations indicates that this behavior is a result of the decreased flexibility displayed by enzymes in an apolar solvent, relatively to the aqueous situation.

The emergence of an influenza pandemic is one of the biggest health threats of our time and there is an urgent need to develop vaccines and drugs against a broad spectrum of influenza viruses (IV). A promising strategy to combat IV is to inactivate the fusion process between the viral and host membranes, which is mediated by the surface protein hemagglutinin (HA). During this process, the N-terminal region of HA, known as fusion peptide (FP), inserts into the host membrane. Although it has been shown that the FP plays a crucial role in the fusion process, the molecular effect of the peptide remains unclear. To analyse the molecular determinants underlying the IV FP, we used state of the art simulation techniques, including metadynamics and constant pH molecular dynamics. The simulation results were combined spectroscopic methods to analyse the peptide's affinity for lipid membranes and its ability to promote lipid-mixing. This allowed us to obtain a detailed molecular characterization of the peptide's conformational properties and its effect on the host membrane. Our work also sheds light into the effect of mutations and external conditions, such as pH, on the FP activity. These results can be useful for the design of novel therapies against this devastating pathogen.

The peptide Ac-(cyclo-2,6)-R[KAAAD]-NH2 (cyc-RKAAAD) is a short cyclic peptide known to adopt a remarkably stable single turn α-helix in water. Due to its simplicity and the availability of thermodynamic and kinetic experimental data, cyc-RKAAAD poses as an ideal model for evaluating the aptness of current molecular dynamics (MD) simulation methodologies to accurately sample conformations that reproduce experimentally observed properties. In this work, we extensively sample the conformational space of cyc-RKAAAD using microsecond-timescale MD simulations. We characterize the peptide conformational preferences in terms of secondary structure propensities and, using Cartesian-coordinate principal component analysis (cPCA), construct its free energy landscape, thus obtaining a detailed weighted discrimination between the helical and nonhelical subensembles. The cPCA state discrimination, together with a Markov model built from it, allowed us to estimate the free energy of unfolding (-0.57 kJ/mol) and the relaxation time (∼0.435 μs) at 298.15 K, which are in excellent agreement with the experimentally reported values (-0.22 kJ/mol and 0.42 μs, Serrano, A. L.; Tucker, M. J.; Gai, F. J. Phys. Chem. B, 2011, 115, 7472-7478.). Additionally, we present simulations conducted using two enhanced sampling methods: replica-exchange molecular dynamics (REMD) and bias-exchange metadynamics (BE-MetaD). We compare the free energy landscape obtained by these two methods with the results from MD simulations and discuss the sampling and computational gains achieved. Overall, the results obtained attest to the suitability of modern simulation methods to explore the conformational behavior of peptide systems with a high level of realism.

The catalytic activities of cutinase immobilized on zeolite NaY and Candida antarctica lipase B immobilized on an acrylic resin (Novozym 453) were measured in a model transesterification reaction in three imidazolium cation-based ionic liquids (RTILs), supercritical ethane (sc-ethane), sc-CO2 and n-hexane, at a water activity (aW) of 0.2 and 0.7. The transesterification activity of cutinase was highest and similar in 1-n-butyl-3-methylimidazolium hexafluorophosphate ([C4mim][PF6]), sc-ethane and n-hexane, more than one order of magnitude lower in sc-CO2, and increased with an increase in aW. Hydrolysis was not detected in sc-fluids and n-hexane, and was observed in RTILs at aW 0.7 only. Both initial rates of transesterification and of hydrolysis of Novozym decreased with an increase in aW. sc-CO2 did not have a deleterious effect on Novozym activity, which was as high as in sc-ethane and n-hexane. The low reaction rates obtained in this case in RTILs suggested the existence of internal diffusion limitations absent in the cutinase preparation where the enzyme is only adsorbed at the surface of the support. sc-CO2 did not adversely affect the catalytic activity of cutinase suspended in [C4mim][PF6], suggesting a protective effect of the RTIL. In the case of Novozym, a marked increase in the rate of transesterification was obtained in the [C4mim][PF6]/sc-CO2 system, compared to the RTIL alone. This may reflect improved mass transfer of solutes to the pores of the immobilization matrix due to a high concentration of dissolved CO2 and a reduction in viscosity of the RTIL.

The technological value of nonaqueous enzymology has been recognized for more than thirty years. A detailed understanding of the molecular determinants of enzyme behaviour in nonaqueous media is essential to explore their potential. Computer simulations have provided valuable contributions to this field, having elucidated how the solvent affects the structural and dynamic properties of enzymes, as well as their activity and enantioselectivity. They have also helped to shed light on the effect of hydration and the role of counterions. In this perspective, we describe the major challenges and achievements of molecular simulations of enzymes in different types of nonaqueous solvents, including organic solvents, ionic liquids and supercritical fluids.

Influenza virus is responsible for worldwide outbreaks of flu, causing hundreds of thousands of deaths every year, which rise to millions in pandemic years. In order to infect the host cells, influenza virus promotes the fusion of the viral and host membranes. This process is mediated by hemagglutinin (HA), a glycoprotein located on the surface of the viral envelope. Influenza HA has a highly conserved N-terminal domain, comprising ∼20 residues, which inserts into the host membrane. This region is critical for destabilizing target membranes during the fusion process and is known as fusion peptide (FP).To elucidate the molecular determinants that lead to the destabilization of biological membranes by the FP, we used a molecular dynamics (MD) approach. We performed simulations with the wild type and four different FP mutants: G1V, W14A, G4A/G8A/G16A/G20A, and G12A/G13A, which are known or expected to have an impaired fusogenic activity. Our aim is to understand how these mutations affect the conformational distribution of the peptide and its ability to destabilize the membrane.Given that lipid membranes are very viscous, it is difficult to obtain reasonable sampling using standard MD. Therefore, in addition to standard MD simulations, we also performed bias-exchange metadynamics simulations, both in water and in a DMPC membrane. These simulations enable us to compare the energy landscapes of the wild type and mutant peptides and explain how these mutations affect the fusogenic ability of the peptide.

Repair of Iron Centres (RICs) are a widely-spread family of diiron proteins involved in the protection of iron–sulphur-containing enzymes from nitrosative and oxidative stress. Here, homology-based modelling was used to predict putative ligands of the RIC diiron centre in E. coli. Site-directed mutagenesis studies showed that several conserved residues modulate the spectroscopic properties of the diiron centre, and mutations in H129, E133 and E208 abrogated RIC ability to protect aconitase. Taken together, these data led to a structural model of a diiron centre inserted in a four-helix bundle fold and coordinated by H84, H129, H160, H204, E133 and E208. Moreover, two μ-carboxylate bridges involving E133 and E208 were found to be required for assembly of a stable diiron centre.