On the impact of nanotube diameter on biomembrane indentation – Computer simulations study

The influence of the single-walled carbon nanotubes on the phospholipid bilayer has been studied using steered molecular dynamics (SMD) simulations. The impact of different nanotubes on the phospholipid bilayer structure is discussed as well as the speed of indentation. Additionally, a series of simulations with pulling out of the nanotubes from the membrane were performed. The deflection of the membrane in both nanoindenation and extraction processes is also discussed. The self-sealing ability of membrane during this process is examined. Complete degradation of the bilayer was not observed even for the most invasive nanoindentation process studied. The obtained results show that carbon nanotubes can be regarded as potential drug carriers for targeted therapy.     

Role of a mutated residue at the entrance of the substrate access channel in cytochrome p450 engineered for vitamin D(3) hydroxylation activity

The cytochrome P450 enzyme engineered for enhancement of vitamin D(3) (VD(3)) hydroxylation activity, Vdh-K1, includes four mutations (T70R, V156L, E216M, and E384R) compared to the wild-type enzyme. Plausible roles for V156L, E216M, and E384R have been suggested by crystal structure analysis (Protein Data Bank 3A50 ), but the role of T70R, which is located at the entrance of the substrate access channel, remained unclear. In this study, the role of the T70R mutation was investigated by using computational approaches. Molecular dynamics (MD) simulations and steered molecular dynamics (SMD) simulations were performed, and differences between R70 and T70 were compared in terms of structural change, binding free energy change (PMF), and interaction force between the enzyme and substrate. MD simulations revealed that R70 forms a salt bridge with D42 and the salt bridge affects the locations and the conformations of VD(3) in the bound state. SMD simulations revealed that the salt bridge tends to be formed strongly when VD(3) passes through the binding pocket. PMFs showed that the T70R mutation leads to energetic stabilization of enzyme-VD(3) binding in the region near the heme active site. Interestingly, these results concluded that the D42-R70 salt bridge at the entrance of the substrate access channel affects the region near the heme active site where the hydroxylation of VD(3) occurs; i.e., it is thought that the T70R mutation plays an important role in enhancing VD(3) hydroxylation activity. A significant future challenge is to compare the hydroxylation activities of R70 and T70 directly by a quantum chemical calculation, and three-dimensional coordinates of the enzyme and VD(3) obtained from MD and SMD simulations will be available for the future challenge.     

The effect of the diameter of cyclic peptide nanotube on its chirality discrimination

In this work, the transport behaviors of the enantiomers of lactic acid (LA) in two cyclic peptide nanotubes (CPNTs) with different diameters were studied using steered molecular dynamic (SMD) simulation to investigate the effect of the diameter of CPNT on the discrimination of the enantiomers of LA. For this purpose, two cyclic peptides with two different sizes ([Ala-_D-Ala-_L]₅ and [Ala-_D-Ala-_L]₄) were used for constructing two CPNTs so that each CPNT was composed of eight cyclic peptide units. The docking calculations were performed to obtain the appropriate position of each enantiomer at the lumen of each CPNT. The variation of the pulling force versus time, exerted on the enantiomers moving in the CPNTs was calculated using the SMD simulations with two different strategies (positional and directional).The obtained results showed that the diameter of CPNT has considerable effect on the discrimination of the LA enantiomers so that the increase of the diameter of CPNT, increased the velocity difference between two enantiomers and improved the performance of CPNT for the chirality discrimination. The SMD simulations indicated that the velocity of S-enantiomer became more than R-enantiomer and its motion became more comfortable than R-enantiomer when the diameter of CNPT increased. The RDFs of the H and O atoms of the LA enantiomers relative to the O atoms of CPNT were calculated and it was found that the increase of the diameter of CPNT creates the significant changes in the RDFs of H1, H2 and H3 atoms of the enantiomers.     

Non-empirical quantum chemical studies on electron transfer reactions in trans- and cis-diamminedichloroplatinum(II) complexes

The search was made for theoretical confirmation of hypothesis that mechanism of cisplatin cytotoxicity is based on dissociative electron transfer (ET) processes. Applying quantum chemical calculations based on supermolecular approach, the reactions mimicking presumed steps of cisplatin activation were evaluated. The electronic structure of model systems: cis- and transplatin with free electrons, hydrated electrons, and water, was studied by using density functional (DFT) within the Huzinaga basis set and GAUSSIAN-09 package. The respective energy was evaluated with the use of B3LYP density hybrid functional. The calculations were performed for gas phase and water solution; the solvent effects were studied by using the polarizable continuum model. Analysis of the energetic and structural parameters of cisplatin vs. transplatin behavior in the model systems leads to conclusion: there are two possible ways of cisplatin biotransformation, hydrolysis and hydrated electron impact, dependent on the medium redox state.     

Computer simulation of primary kinetic isotope effects in the proposed rate-limiting step of the glyoxalase I catalyzed reaction

The proposed rate-limiting step of the glyoxalase I catalyzed reaction is the proton abstraction from the C1 carbon of the substrate by Glu(172). Here we examine primary kinetic isotope effects and the influence of quantum dynamics on this process by computer simulations. The calculations utilize the empirical valence bond method in combination with the molecular dynamics free energy perturbation technique and path integral simulations. For the enzyme-catalyzed reaction a H/D kinetic isotope effect of 5.0 +/- 1. 3 is predicted in reasonable agreement with the experimental result of about 3. Furthermore, the magnitude of quantum mechanical effects is found to be very similar for the enzyme reaction and the corresponding uncatalyzed process in solution, in agreement with other studies. The problems associated with attaining the required accuracy in order for the present approach to be useful as a diagnostic tool for the study of enzyme reactions are also discussed.     

晋西黄土区人工林细根与土壤水碳的耦合关系

以晋西黄土区山西离石典型人工林刺槐、侧柏、核桃为研究对象,研究其深剖面（0-500 cm）细根参数、土壤水分和有机碳的分布特征,并以农地为对照,评价各人工林土壤水分亏缺和有机碳积累效应,在此基础上探讨三者的耦合关系。结果表明：（1）3种人工林土壤浅层（0-70 cm）细根累计生物量占整个土层的56%-71%,具有明显表聚性。（2）3种人工林土壤深层（70-500 cm）土壤水分亏缺效应均显著高于浅层（P < 0.05）,与农地相比,其亏缺值表现为：侧柏 > 核桃 > 刺槐。（3）3种人工林深层土壤有机碳密度占整个土层的77%-86%;与农地相比,侧柏和核桃土壤有机碳积累效应总体为正向积累作用,刺槐则相反。（4）在土壤浅层,细根参数与土壤水分和有机碳密度均有显著相关性,而在深层,细根主要与有机碳密度显著相关,与土壤水分的相关性仅在刺槐样地显著。晋西黄土区不同人工林深层细根分布有很大差异,且已对其深层土壤水分和有机碳的分布产生影响。综合来看,刺槐的细根分布已造成深层土壤水分亏缺,同时也不利于深层有机碳的积累。     

Free-energy landscape of glycerol permeation through aquaglyceroporin GlpF determined from steered molecular dynamics simulations

The free-energy landscape of glycerol permeation through the aquaglyceroporin GlpF has been estimated in the literature by the nonequilibrium method of steered molecular dynamics (SMD) simulations and by the equilibrium method of adaptive biasing force (ABF) simulations. However, the ABF results qualitatively disagree with the SMD results that were based on the Jarzynski equality (JE) relating the equilibrium free-energy difference to the nonequilibrium work of the irreversible pulling experiments. In this paper, I present a new SMD study of the glycerol permeation through GlpF to explore the free-energy profile of glycerol along the permeation channel. Instead of the JE in terms of thermodynamic work, I use the fluctuation-dissipation theorem (FDT) of Brownian dynamics (BD), in terms of mechanical work, for extracting the free-energy difference from the nonequilibrium work of irreversible pulling experiments. The results of this new SMD-BD-FDT study are in agreement with the experimental data and with the ABF results.     

Visualization of early events in acetic acid denaturation of HIV-1 protease: a molecular dynamics study

Protein denaturation plays a crucial role in cellular processes. In this study, denaturation of HIV-1 Protease (PR) was investigated by all-atom MD simulations in explicit solvent. The PR dimer and monomer were simulated separately in 9 M acetic acid (9 M AcOH) solution and water to study the denaturation process of PR in acetic acid environment. Direct visualization of the denaturation dynamics that is readily available from such simulations has been presented. Our simulations in 9 M AcOH reveal that the PR denaturation begins by separation of dimer into intact monomers and it is only after this separation that the monomer units start denaturing. The denaturation of the monomers is flagged off by the loss of crucial interactions between the α-helix at C-terminal and surrounding β-strands. This causes the structure to transit from the equilibrium dynamics to random non-equilibrating dynamics. Residence time calculations indicate that denaturation occurs via direct interaction of the acetic acid molecules with certain regions of the protein in 9 M AcOH. All these observations have helped to decipher a picture of the early events in acetic acid denaturation of PR and have illustrated that the α-helix and the β-sheet at the C-terminus of a native and functional PR dimer should maintain both the stability and the function of the enzyme and thus present newer targets for blocking PR function.     

Computational investigation of the effect of thermal perturbation on the mechanical unfolding of titin I27

The emergence of single-molecule force measurement experiments has facilitated a better understanding of protein folding pathways and the thermodynamics involved. Computational methods such as steered molecular dynamics (SMD) simulations are helpful in providing atomistic level information on the unfolding pathways. Recent experimental studies have showed that combinations of single-molecule experiments with traditional methods such as chemical and/or thermal denaturation yield additional insights into the folding phenomenon. In this study, we report results from extensive computations (a total of about 60 SMD simulations with a total length of about 0.4 μs) that address the effect of thermal perturbation on the mechanical stability of the I27 domain of the protein titin. A wide range of temperatures (280-340 K) were considered for the pulling, which was done at both constant velocity and constant force using SMD simulations. Good agreement with experimental data, such as for the trends in changes in average force and the maximum force with respect to the temperature, was obtained. This study identifies two competing pathways for the mechanical unfolding of I27, and illustrates the significance of combining various techniques to examine protein folding.     

Exploration of the chlorpyrifos escape pathway from acylpeptide hydrolases using steered molecular dynamics simulations

Acylpeptide hydrolases (APH) catalyze the removal of an N-acylated amino acid from blocked peptides. APH is significantly more sensitive than acetylcholinesterase, a target of Alzheimer’s disease, to inhibition by organophosphorus (OP) compounds. Thus, OP compounds can be used as a tool to probe the physiological functions of APH. Here, we report the results of a computational study of molecular dynamics simulations of APH bound to the OP compounds and an exploration of the chlorpyrifos escape pathway using steered molecular dynamics (SMD) simulations. In addition, we apply SMD simulations to identify potential escape routes of chlorpyrifos from hydrolase hydrophobic cavities in the APH-inhibitor complex. Two previously proposed APH pathways were reliably identified by CAVER 3.0, with the estimated relative importance of P1 > P2 for its size. We identify the major pathway, P2, using SMD simulations, and Arg526, Glu88, Gly86, and Asn65 are identified as important residues for the ligand leaving via P2. These results may help in the design of APH-targeting drugs with improved efficacy, as well as in understanding APH selectivity of the inhibitor binding in the prolyl oligopeptidase family.     

Proton-transport mechanisms in cytochrome c oxidase revealed by studies of kinetic isotope effects

Cytochrome c oxidase (CytcO) is a membrane-bound enzyme, which catalyzes the reduction of di-oxygen to water and uses a major part of the free energy released in this reaction to pump protons across the membrane. In the Rhodobacter sphaeroides aa? CytcO all protons that are pumped across the membrane, as well as one half of the protons that are used for O? reduction, are transferred through one specific intraprotein proton pathway, which holds a highly conserved Glu286 residue. Key questions that need to be addressed in order to understand the function of CytcO at a molecular level are related to the timing of proton transfers from Glu286 to a "pump site" and the catalytic site, respectively. Here, we have investigated the temperature dependencies of the H/D kinetic-isotope effects of intramolecular proton-transfer reactions in the wild-type CytcO as well as in two structural CytcO variants, one in which proton uptake from solution is delayed and one in which proton pumping is uncoupled from O? reduction. These processes were studied for two specific reaction steps linked to transmembrane proton pumping, one that involves only proton transfer (peroxy-ferryl, P→F, transition) and one in which the same sequence of proton transfers is also linked to electron transfer to the catalytic site (ferryl-oxidized, F→O, transition). An analysis of these reactions in the framework of theory indicates that that the simpler, P→F reaction is rate-limited by proton transfer from Glu286 to the catalytic site. When the same proton-transfer events are also linked to electron transfer to the catalytic site (F→O), the proton-transfer reactions might well be gated by a protein structural change, which presumably ensures that the proton-pumping stoichiometry is maintained also in the presence of a transmembrane electrochemical gradient. Furthermore, the present study indicates that a careful analysis of the temperature dependence of the isotope effect should help us in gaining mechanistic insights about CytcO.     

Defluorination of 4-fluorophenol by cytochrome P450BM3-F87G: activation by long chain fatty aldehydes

Cytochrome P450(BM3)-F87G catalyzed the oxidative defluorination of 4-fluorophenol, followed by reduction of the resulting benzoquinone to hydroquinone via the NADPH P450-reductase activity of the enzyme. The k (cat) and K (m) for this reaction were 71?±?5?min(-1) and 9.5?±?1.3?mM, respectively. Co-incubation of the reaction mixture with long chain aldehydes stimulated the defluorination reaction, with the 2,3-unsaturated aldehyde, 2-decenal producing a 12-fold increase in catalytic efficiency. At 150?μM aldehyde, k (cat) increased to 158?±?4, while K (m) decreased to 1.8?±?0.2. The effects of catalase, glutathione and ascorbate on the reaction were all consistent with a direct oxygen insertion mechanism, as opposed to a radical mechanism. The study demonstrates the potential use of P450(BM3) mutants in oxidative defluorination reactions, and characterizes the novel stimulatory action of straight chain aldehydes on this activity.     

Steered molecular dynamics studies of titin I1 domain unfolding

The cardiac muscle protein titin, responsible for developing passive elasticity and extensibility of muscle, possesses about 40 immunoglobulin-like (Ig) domains in its I-band region. Atomic force microscopy (AFM) and steered molecular dynamics (SMD) have been successfully combined to investigate the reversible unfolding of individual Ig domains. However, previous SMD studies of titin I-band modules have been restricted to I27, the only structurally known Ig domain from the distal region of the titin I-band. In this paper we report SMD simulations unfolding I1, the first structurally available Ig domain from the proximal region of the titin I-band. The simulations are carried out with a view toward upcoming atomic force microscopy experiments. Both constant velocity and constant force stretching have been employed to model mechanical unfolding of oxidized I1, which has a disulfide bond bridging beta-strands C and E, as well as reduced I1, in which the disulfide bridge is absent. The simulations reveal that I1 is protected against external stress mainly through six interstrand hydrogen bonds between its A and B beta-strands. The disulfide bond enhances the mechanical stability of oxidized I1 domains by restricting the rupture of backbone hydrogen bonds between the A'- and G-strands. The disulfide bond also limits the maximum extension of I1 to approximately 220 A. Comparison of the unfolding pathways of I1 and I27 are provided and implications to AFM experiments are discussed.     

Mechanism of CDK5 activation revealed by steered molecular dynamics simulations and energy calculations

In the current work, CDK5/p25 complexes were pulled apart by applying external forces with steered molecular dynamics (SMD) simulations. The crucial interactions between the kinase and the activation protein were investigated and the SMD simulations showed that several activation-relevant motifs of CDK5 leave p25 in sequence during the pulling and lead to an apo-CDK2 like CDK5 structure after separation. Based on systematic examination of hydrogen bond breaking and classical MD/molecular mechanics-generalized Born/surface area) (MM-GBSA) calculations, a CDK5 activation mechanism by p25 is suggested. This is the first step towards the systemic development of CDK inhibitors and the mechanism proposed could lead to a better understanding of the protein–protein recognition characteristics between the kinase and its activator.     

Relationship between enantioselective transformation of racemic quizalofop‐ethyl and soil bacterial diversity: A destructive approach

This study investigated the resilience of bacterial diversity in soils restored after autoclaving, in terms of richness, evenness and community structure, and its feedback on the enantioselective transformation of racemic quizalofop‐ethyl (rac‐QE). Microbial biomass carbon (MBC) and bacterial richness (indexed by operational taxonomic units [OTUs]) in restored soil recovered to approximately 50% and 29%, respectively, of the native soil within 43 days. Bacterial evenness was much lower in restored soil than in native soil. The relative proportions of dominant bacterial genera differed significantly (P < .05) between restored and native soils. Importantly, two major bacterial genera that recolonized restored soil were not detected in native soil. Highly enantioselective transformation of rac‐QE was observed in restored soils, whereas QE enantiomers exhibited comparable transformation rates in native soils. The second‐round enantioselective transformation of rac‐QE was altered by the first‐round transformation of enantiopure quizalofop‐P‐ethyl (R‐P‐QE) in restored and native soils through selective effects of R‐P‐QE on the bacterial community. The transformation rate of rac‐QE was predominantly determined by bacterial abundance and richness, while the enantioselectivity was correlated more with bacterial structure.     

Kalanchoe pinnata inhibits mast cell activation and prevents allergic airway disease

Aqueous extract of Kalanchoe pinnata (Kp) have been found effective in models to reduce acute anaphylactic reactions. In the present study, we investigate the effect of Kp and the flavonoid quercetin (QE) and quercitrin (QI) on mast cell activation in vitro and in a model of allergic airway disease in vivo. Treatment with Kp and QE in vitro inhibited degranulation and cytokine production of bone marrow-derived mast cells following IgE/Fc?RI crosslinking, whereas treatment with QI had no effect. Similarly, in vivo treatment with Kp and QE decreased development of airway hyperresponsiveness, airway inflammation, goblet cell metaplasia and production of IL-5, IL-13 and TNF. In contrast, treatment with QI had no effect on these parameters. These findings demonstrate that treatment with Kp or QE is effective in treatment of allergic airway disease, providing new insights to the immunomodulatory functions of this plant.     

General method of analysis of kinetic equations for multistep reversible mechanisms in the single-exponential regime: application to kinetics of open complex formation between Esigma70 RNA polymerase and lambdaP(R) promoter DNA

A novel analytical method based on the exact solution of equations of kinetics of unbranched first- and pseudofirst-order mechanisms is developed for application to the process of Esigma70 RNA polymerase (R)-lambdaPR promoter (P) open complex formation, which is described by the minimal three-step mechanism with two kinetically significant intermediates (I1, I2), [equation: see text], where the final product is an open complex RPo. The kinetics of reversible and irreversible association (pseudofirst order, [R] > [P]) to form long-lived complexes (RPo and I2) and the kinetics of dissociation of long-lived complexes both exhibit single exponential behavior. In this situation, the analytical method provides explicit expressions relating observed rate constants to the microscopic rate constants of mechanism steps without use of rapid equilibrium or steady-state approximations, and thereby provides a basis for interpreting the composite rate constants of association (ka), isomerization (ki), and dissociation (kd) obtained from experiment for this or any other sequential mechanism of any number of steps. In subsequent papers, we apply this formalism to analyze kinetic data obtained in the reversible and irreversible binding regimes of Esigma70 RNA polymerase (R)-lambdaP(R) promoter (P) open complex formation.     

Insights into membrane translocation of the cell-penetrating peptide pVEC from molecular dynamics calculations

Discovery of cargo carrying cell-penetrating peptides has opened a new gate in the development of peptide-based drugs that can effectively target intracellular enzymes. Success in application and development of cell-penetrating peptides in drug design depends on understanding their translocation mechanisms. In this study, our aim was to examine the bacterial translocation mechanism of the cell-penetrating pVEC peptide (LLIILRRRIRKQAHAHSK) using steered molecular dynamics (SMD) simulations. The significance of specific residues or regions for translocation was studied by performing SMD simulations on the alanine mutants and other variants of pVEC. Residue-based analysis showed that positively charged residues contribute to adsorption to the lipid bilayer and to electrostatic interactions with the lipid bilayer as peptides are translocated. Translocation takes place in three main stages; the insertion of the N-terminus into the bilayer, the inclusion of the whole peptide inside the membrane and the exit of the N-terminus from the bilayer. These three stages mirror the three regions on pVEC; namely, the hydrophobic N-terminus, the cationic midsection, and the hydrophilic C-terminus. The N-terminal truncated pVEC, I3A, L5A, R7A mutants and scramble-pVEC make weaker interactions with the lipids during translocation highlighting the contribution of the N-terminal residues and the sequence of the structural regions to the translocation mechanism. This study provides atomistic detail about the mechanism of pVEC peptide translocation and can guide future peptide-based drug design efforts.     

Photochromic agents as tools for protein structure study: lapachenole is a photoaffinity ligand of cytochrome P450 3A4

Cytochrome P450 3A4 is a drug-metabolizing enzyme of extraordinarily broad substrate specificity. This quality imparts upon the enzyme special importance in understanding its determinants of activity and substrate recognition. Limited successes in P450 3A4 active-site structure studies have been achieved by use of mechanism-based inactivators and photoaffinity ligands. We report here the potential of photochromic agents, compounds with the ability to undergo light-induced, reversible reactions, to be used as effective photoaffinity ligands. Four such compounds of the chromene family were shown by ultraviolet and visible spectroscopy to undergo photoinduced rearrangements to highly conjugated and reactive products in buffered aqueous solution. While some of these intermediates were very long-lived (>12 h, photoactivated lapachenole), others existed for milliseconds in their opened forms (precocene I and 2,2-dimethyl-5,6-benzo-2H-chromene) and were observed by laser flash photolysis. Each of the tricyclic structures studied rapidly underwent Michael addition reactions with the test nucleophile glutathione upon irradiation to form single conjugated products. The smaller precocene I reacted more extensively to form multiple products. These attributes of the chromenes inspired testing of their potential to label cytochrome P450 3A4 in a light-dependent fashion. Access to the protein active site by lapachenole was demonstrated with the molecule's ability to competitively inhibit P450 3A4-mediated oxidative metabolism of midazolam with an IC(50) value of 71 microM. This inhibition became irreversible upon irradiation of the enzyme-ligand complex with ultraviolet light. These results clearly demonstrate that chromenes are effective photoaffinity reagents for the cytochrome P450 superfamily of enzymes and probably other proteins as well.     

Kinetics of photoconversion of protochlorophyllide 649 to chlorophyllide 676 at low temperature in etiolated cotyledons of Pharbitis nil

The kinetics of the photoconversion of protochlorophyllide 649 to chlorophyllide 676 were studied spectrophotometrically over the temperature range of ?15 – ?80°C under light-saturating conditions in etiolated cotyledons of Pharbitis nil. Photoconversion obeyed the sum of two first-order kinetics over this low temperature range. Activation energies obtained from the rate constants were about 5000 cal; this suggests that these two processes may be physical processes not chemical reactions. The results indicate that photoconversion involves two main steps. One is the step dependent on both light intensity and temperature that has been well studied. The other, which is concerned in this study, is the step dependent on temperature only, which may be the requisite for photoconversion. This latter step seems to be related to the binding mode of protochlorophyllide to a holochrome protein or to conformational changes in the protochlorophyllide-holochrome.