RESUMO
Hel308 helicases promote genome stability in archaea and are conserved in metazoans, where they are known as HELQ. Their helicase mechanism is well characterised, but it is unclear how they specifically contribute to genome stability in archaea. We show here that a highly conserved motif of Hel308/HELQ helicases (motif IVa, F/YHHAGL) modulates both DNA unwinding and a newly identified strand annealing function of archaeal Hel308. A single amino acid substitution in motif IVa results in hyper-active DNA helicase and annealase activities of purified Hel308 in vitro. All-atom molecular dynamics simulations using Hel308 crystal structures provided a molecular basis for these differences between mutant and wild type Hel308. In archaeal cells, the same mutation results in 160000-fold increased recombination, exclusively as gene conversion (non-crossover) events. However, crossover recombination is unaffected by the motif IVa mutation, as is cell viability or DNA damage sensitivity. By contrast, cells lacking Hel308 show impaired growth, increased sensitivity to DNA cross-linking agents, and only moderately increased recombination. Our data reveal that archaeal Hel308 suppresses recombination and promotes DNA repair, and that motif IVa in the RecA2 domain acts as a catalytic switch to modulate the separable recombination and repair activities of Hel308.
Assuntos
Archaea , DNA Helicases , Humanos , Archaea/genética , DNA Helicases/metabolismo , Reparo do DNA , DNA/química , Recombinação Genética , Instabilidade GenômicaRESUMO
Mitochondrial adenine nucleotide translocase (ANT) exchanges ADP for ATP to maintain energy production in the cell. Its protonophoric function in the presence of long-chain fatty acids (FA) is also recognized. Our previous results imply that proton/FA transport can be best described with the FA cycling model, in which protonated FA transports the proton to the mitochondrial matrix. The mechanism by which ANT1 transports FA anions back to the intermembrane space remains unclear. Using a combined approach involving measurements of the current through the planar lipid bilayers reconstituted with ANT1, site-directed mutagenesis and molecular dynamics simulations, we show that the FA anion is first attracted by positively charged arginines or lysines on the matrix side of ANT1 before moving along the positively charged protein-lipid interface and binding to R79, where it is protonated. We show that R79 is also critical for the competitive binding of ANT1 substrates (ADP and ATP) and inhibitors (carboxyatractyloside and bongkrekic acid). The binding sites are well conserved in mitochondrial SLC25 members, suggesting a general mechanism for transporting FA anions across the inner mitochondrial membrane.
Assuntos
Bicamadas Lipídicas , Prótons , Ácidos Graxos/metabolismo , Translocases Mitocondriais de ADP e ATP/metabolismo , Ânions/metabolismo , Trifosfato de Adenosina/metabolismoRESUMO
Molecular dynamics (MD) simulations of uncoupling proteins (UCP), a class of transmembrane proteins relevant for proton transport across inner mitochondrial membranes, represent a complicated task due to the lack of available structural data. In this work, we use a combination of homology modelling and subsequent microsecond molecular dynamics simulations of UCP2 in the DOPC phospholipid bilayer, starting from the structure of the mitochondrial ATP/ADP carrier (ANT) as a template. We show that this protocol leads to a structure that is impermeable to water, in contrast to MD simulations of UCP2 structures based on the experimental NMR structure. We also show that ATP binding in the UCP2 cavity is tight in the homology modelled structure of UCP2 in agreement with experimental observations. Finally, we corroborate our results with conductance measurements in model membranes, which further suggest that the UCP2 structure modeled from ANT protein possesses additional key functional elements, such as a fatty acid-binding site at the R60 region of the protein, directly related to the proton transport mechanism across inner mitochondrial membranes.
Assuntos
Proteínas Mitocondriais/química , Simulação de Dinâmica Molecular , Conformação Proteica , Proteína Desacopladora 2/química , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Ácidos Graxos/química , Ácidos Graxos/metabolismo , Transporte de Íons , Proteínas de Membrana/química , Camundongos , Proteínas Mitocondriais/metabolismo , Ligação Proteica , Estabilidade Proteica , Relação Estrutura-Atividade , Proteína Desacopladora 2/metabolismoRESUMO
Adenine nucleotide translocase (ANT) is a well-known mitochondrial exchanger of ATP against ADP. In contrast, few studies have shown that ANT also mediates proton transport across the inner mitochondrial membrane. The results of these studies are controversial and lead to different hypotheses about molecular transport mechanisms. We hypothesized that the H+-transport mediated by ANT and uncoupling proteins (UCP) has a similar regulation pattern and can be explained by the fatty acid cycling concept. The reconstitution of purified recombinant ANT1 in the planar lipid bilayers allowed us to measure the membrane current after the direct application of transmembrane potential ΔΨ, which would correspond to the mitochondrial states III and IV. Experimental results reveal that ANT1 does not contribute to a basal proton leak. Instead, it mediates H+ transport only in the presence of long-chain fatty acids (FA), as already known for UCPs. It depends on FA chain length and saturation, implying that FA's transport is confined to the lipid-protein interface. Purine nucleotides with the preference for ATP and ADP inhibited H+ transport. Specific inhibitors of ATP/ADP transport, carboxyatractyloside or bongkrekic acid, also decreased proton transport. The H+ turnover number was calculated based on ANT1 concentration determined by fluorescence correlation spectroscopy and is equal to 14.6 ± 2.5 s-1. Molecular dynamic simulations revealed a large positively charged area at the protein/lipid interface that might facilitate FA anion's transport across the membrane. ANT's dual function-ADP/ATP and H+ transport in the presence of FA-may be important for the regulation of mitochondrial membrane potential and thus for potential-dependent processes in mitochondria. Moreover, the expansion of proton-transport modulating drug targets to ANT1 may improve the therapy of obesity, cancer, steatosis, cardiovascular and neurodegenerative diseases.
Assuntos
Translocador 1 do Nucleotídeo Adenina/química , Translocador 1 do Nucleotídeo Adenina/metabolismo , Ácidos Graxos/metabolismo , Mitocôndrias/metabolismo , Prótons , Animais , Transporte de Íons , Potencial da Membrana Mitocondrial , Camundongos , Conformação ProteicaRESUMO
We revisit the mechanism of reaction between a model lysine side chain and reactive aldehyde 4-hydroxynonenal in different solvents with an increasing water content. We show by model organic reactions and qualitative spectrometric analysis that a nonpolar pyrrole adduct is dominantly formed in non-aqueous solvents dichloromethane and acetonitrile. On the other hand, in aqueous acetonitrile and neat water, other polar products are also isolated, including Michael adducts, hemiacetal adducts, and pyridinium salt adducts, at the same time as the ratio of nonpolar products to polar products is decreasing. The experiments are supported by detailed quantum chemical calculations of the reaction mechanism with different computational setups showing that the pyrrole adduct is the most thermodynamically stable product compared to Michael adducts and hemiacetal adducts and also indicating that water molecules released along the reaction pathway are catalyzing reaction steps involving proton transfer. Finally, we also identify the mechanism of the pyridinium salt adduct that is formed only in aqueous solutions.
RESUMO
We performed all-atom molecular dynamics simulations and calculated free energy profiles and apparent pKa values for neutral and anionic forms of single myristic (C14:0), palmitic (C16:0) and stearic (C18:0) fatty acid embedded in a DOPC bilayer and explicit water solvent. We showed that the neutral forms of the fatty acids are stabilized inside the bilayer by hydrogen bonding of a fatty acid carboxylic group with DOPC phosphate and carbonyl groups. In contrast to the neutral form, the anionic forms of the fatty acids are shifted towards the water-membrane interface and are instead stabilized by hydrogen bonding to interfacial water. By using umbrella sampling simulations, we calculated free energies of stabilization and revealed that the free energy of stabilization inside the bilayer increases with the chain length for both the neutral and deprotonated forms. On the other hand, the free energies of flip-flop of both the neutral and anionic forms are constant upon the prolongation of the fatty acid. Based on the free energy curves, we also calculated apparent fatty acid pKa,app values in the bilayer, which are 7.0, 7.2 and 6.3 for myristic, palmitic and stearic acid and are increased by several pKa units compared to the corresponding pKa values in water. By further analysis of the calculated curves we found that spontaneous protonation of fatty acid anions takes place in the bilayer interior at ca. 1.4 nm from the bilayer center for all studied fatty acids.
Assuntos
Ácidos Graxos/química , Bicamadas Lipídicas/química , Fosfatidilcolinas/química , Concentração de Íons de Hidrogênio , Simulação de Dinâmica MolecularRESUMO
Peroxidases are essential elements in many biotechnological applications. An especially interesting concept involves split enzymes, where the enzyme is separated into two smaller and inactive proteins that can dimerize into a fully active enzyme. Such split forms were developed for the horseradish peroxidase (HRP) and ascorbate peroxidase (APX) already. Both peroxidases have a high potential for biotechnology applications. In the present study, we performed biophysical comparisons of these two peroxidases and their split analogues. The active site availability is similar for all four structures. The split enzymes are comparable in stability with their native analogues, meaning that they can be used for further biotechnology applications. Also, the tertiary structures of the two peroxidases are similar. However, differences that might help in choosing one system over another for biotechnology applications were noticed. The main difference between the two systems is glycosylation which is not present in the case of APX/sAPEX2, while it has a high impact on the HRP/sHRP stability. Further differences are calcium ions and cysteine bridges that are present only in the case of HRP/sHRP. Finally, computational results identified sAPEX2 as the systems with the smallest structural variations during molecular dynamics simulations showing its dominant stability comparing to other simulated proteins. Taken all together, the sAPEX2 system has a high potential for biotechnological applications due to the lack of glycans and cysteines, as well as due to high stability.
RESUMO
Within the last decade, the field of bio-nanoengineering has achieved significant advances allowing us to generate, e.g., nanoscaled molecular machineries with arbitrary shapes. To unleash the full potential of novel methods such as DNA origami technology, it is important to functionalise complex molecules and nanostructures precisely. Thus, considerable attention has been given to site-selective modifications of proteins allowing further incorporation of various functionalities. Here, we describe a method for the covalent attachment of oligonucleotides to the glycosylated horseradish peroxidase protein (HRP) with high N-terminus selectivity and significant yield while conserving the enzymatic activity. This two-step process includes a pH-controlled metal-free diazotransfer reaction using imidazole-1-sulfonyl azide hydrogen sulfate, which at pH 8.5 results in an N-terminal azide-functionalized protein, followed by the Cu-free click SPAAC reaction to dibenzocyclooctyne- (DBCO) modified oligonucleotides. The reaction conditions were optimised to achieve maximum yield and the best performance. The resulting protein-oligonucleotide conjugates (HRP-DNA) were characterised by electrophoresis and mass spectrometry (MS). Native-PAGE experiments demonstrated different migration patterns for HRP-DNA and the azido-modified protein allowing zymogram experiments. Structure-activity relationships of novel HRP-DNA conjugates were assessed using molecular dynamics simulations, characterising the molecular interactions that define the structural and dynamical properties of the obtained protein-oligonucleotide conjugates (POC).
Assuntos
DNA , Oligonucleotídeos , Peroxidase do Rábano Silvestre/químicaRESUMO
The knowledge on the sorption behaviour of antibiotics on nanomaterials is limited, especially regarding the reaction mechanism on the surface of carbon nanomaterials, which may determine both the adsorptive capacity and regeneration efficiency of graphene adsorbers. In this work, we used molecular modelling to generate the most comprehensive (to date) adsorption dataset for pristine and functionalised graphene interacting with 8 ß-lactams, 3 macrolide, 12 quinolone, 4 tetracycline, 15 sulphonamide, trimethoprim, 2 lincosamide, 2 phenicole and 4 nitroimidazole antibiotics, and their transformation products in water and n-octanol. Results show that various non-covalent interactions that operate simultaneously, including van der Waals dispersion forces, π-interactions, hydrophobic interaction and hydrogen bonding, facilitate adsorption. The molecular properties of antibiotics and graphene/graphene oxide, as well as the composition of the background solution regulate the magnitude of these interactions. Our findings demonstrate that the most efficient method for the removal of antibiotics from aquatic environments is the use of graphene at environmental pH. The subsequent regeneration of the sorbent is best achieved through washing with slightly basic (pH 8-10) non-polar solvents. The obtained theoretical insights expand and complement experimental observations and provide important information that can contribute to further exploration into the adsorbent properties of graphene-based materials, and towards the development of predictive adsorption models.
Assuntos
Grafite , Poluentes Químicos da Água , Purificação da Água , Adsorção , Antibacterianos/química , Grafite/química , Poluentes Químicos da Água/análise , Purificação da Água/métodosRESUMO
Oxidative stress and ROS are important players in the pathogenesis of numerous diseases. In addition to directly altering proteins, ROS also affects lipids with negative intrinsic curvature such as phosphatidylethanolamine (PE), producing PE adducts and lysolipids. The formation of PE adducts potentiates the protonophoric activity of mitochondrial uncoupling proteins, but the molecular mechanism remains unclear. Here, we linked the ROS-mediated change in lipid shape to the mechanical properties of the membrane and the function of uncoupling protein 1 (UCP1) and adenine nucleotide translocase 1 (ANT1). We show that the increase in the protonophoric activity of both proteins occurs due to the decrease in bending modulus in lipid bilayers in the presence of lysophosphatidylcholines (OPC and MPC) and PE adducts. Moreover, MD simulations showed that modified PEs and lysolipids change the lateral pressure profile of the membrane in the same direction and by the similar amplitude, indicating that modified PEs act as lipids with positive intrinsic curvature. Both results indicate that oxidative stress decreases stored curvature elastic stress (SCES) in the lipid bilayer membrane. We demonstrated that UCP1 and ANT1 sense SCES and proposed a novel regulatory mechanism for the function of these proteins. The new findings should draw the attention of the scientific community to this important and unexplored area of redox biochemistry.
RESUMO
The effect of different branching types of glycosylation on the structure and dynamics of the horseradish peroxidase (HRP) and an engineered split horseradish peroxidase (sHRP) was studied using all-atom molecular dynamics (MD) simulations. Although tertiary structures of both proteins are stable in the presence, as well as in the absence of glycans, differences in the dynamical properties regarding the presence of glycans were noticed. Fluctuations in the protein structure along both proteins are decreased when glycosylation is introduced. We identified two main regions that are affected the most. The peripheral region is impacted directly by glycans and the central region within the active site with a propagated effect of glycans. Since the mentioned central region in the glycoprotein is not surrounded by glycans and is close to the heme, it is easily approachable to the solvent and substrate. An influence of the glycan presence on the electrostatic potential of the protein and on the heme cofactor was also observed. Altogether, this work presents a global and local analysis of the glycosylation influence on HRP protein's structural and dynamical properties at a molecular level.
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Among many reactive oxygen species (ROS), which are constantly generated during oxidative stress in cellular membranes, the formation and subsequent reactivity of ubiquitous 4-hydroxy-2- nonenal (HNE) with nearby amino acids and lipids represent one of the main research targets in cell physiology in the last decades. Starting from the first synthesis of HNE in 1967, the chemistry and reactivity of HNE are constantly under intense scrutiny. This review shows recent advances in the field, which are discussed with the special emphasis on revealing intricate details of numerous reaction mechanisms of HNE with lipids and amino acids, with the goal of understanding the reactivity of HNE at the molecular level.
Assuntos
Aldeídos/química , Aminoácidos/química , Humanos , Lipídeos/químicaRESUMO
2,4-Dinitrophenol (DNP) is a classic uncoupler of oxidative phosphorylation in mitochondria which is still used in "diet pills", despite its high toxicity and lack of antidotes. DNP increases the proton current through pure lipid membranes, similar to other chemical uncouplers. However, the molecular mechanism of its action in the mitochondria is far from being understood. The sensitivity of DNP's uncoupling action in mitochondria to carboxyatractyloside, a specific inhibitor of adenine nucleotide translocase (ANT), suggests the involvement of ANT and probably other mitochondrial proton-transporting proteins in the DNP's protonophoric activity. To test this hypothesis, we investigated the contribution of recombinant ANT1 and the uncoupling proteins UCP1-UCP3 to DNP-mediated proton leakage using the well-defined model of planar bilayer lipid membranes. All four proteins significantly enhanced the protonophoric effect of DNP. Notably, only long-chain free fatty acids were previously shown to be co-factors of UCPs and ANT1. Using site-directed mutagenesis and molecular dynamics simulations, we showed that arginine 79 of ANT1 is crucial for the DNP-mediated increase of membrane conductance, implying that this amino acid participates in DNP binding to ANT1.
Assuntos
2,4-Dinitrofenol/farmacologia , Bicamadas Lipídicas/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Mitocôndrias Hepáticas/metabolismo , Translocases Mitocondriais de ADP e ATP/metabolismo , Proteínas de Desacoplamento Mitocondrial/metabolismo , Animais , Camundongos , RatosRESUMO
Reactive aldehydes (RAs), such as 4-hydroxy-2-nonenal (HNE) and 4-oxo-2-nonenal (ONE), produced by cells under conditions of oxidative stress, were shown to react with phosphatidylethanolamine (PE) in biological and artificial membranes. They form RA-PE adducts, which affect the function of membrane proteins by modifying various biophysical properties of the membrane. The ratio of protein to lipid in biological membranes is different, but can reach 0.25 in the membranes of oligodendrocytes. However, the impact of RA-PE adducts on permeability (P) of the neat lipid phase and molecular mechanism of their action are poorly understood. In this study, we showed that HNE increased the membrane P for ions, and in particular for sodium. This effect depended on the presence of DOPE, and was not recorded for the more toxic compound, ONE. Molecular dynamics simulations suggested that HNE-PE and ONE-PE adducts anchored different positions in the lipid bilayer, and thus changed the membrane lipid area and bilayer thickness in different ways. Sodium permeability, calculated in the presence of double HNE-PE adducts, was increased by three to four orders of magnitude when compared to PNa in adduct - free membranes. A novel mechanism by which HNE alters permeability of the lipid membrane may explain the multiple toxic or regulative effects of HNE on the function of excitable cells, such as neurons, cardiomyocytes and neurosensory cells under conditions of oxidative stress.