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Therapeutic peptides offer potential advantages over small molecules in terms of selectivity, affinity, and their ability to target “undruggable” proteins that are associated with a wide range of pathologies. Despite their importance, there are currently no adequate molecular design capabilities that inform medicinal chemistry decisions on peptide programs. More specifically, SAR (Structure-Activity Relationship) analysis and visualization of linear, cyclic, and cross-linked peptides containing non-natural motifs, which are widely used in drug discovery. To bridge this gap, we developed PepSeA (Peptide Sequence Alignment and Visualization), an open-source, freely available package of sequence-based tools (https://github.com/Merck/PepSeA). PepSeA enables multi-sequence alignment of non-natural amino acids and enhanced HELM (Hierarchical Editing Language for Macromolecules) visualization. Via stepwise SAR analysis of a ChEMBL peptide dataset, we demonstrate PepSeA’s power to accelerate decision making in lead optimization campaigns in pharmaceutical settings. PepSeA represents an initial attempt to expand cheminformatics capabilities for therapeutic peptides and to enable rapid and more efficient design–make–test cycles.

Suppression of endothelial ERK1/1 pathways results in multi-organ failure, similar to what is observed in patients treated with anti-VEGF therapies., The mechanism of maintaining vascular endothelial identity and integrity is largely unknown. In this issue of JEM, Ricard et al. (https://doi.org/10.1084/jem.20182151) discover essential roles of ERK1/2 in maintaining endothelial homeostasis and its deletion-related serious defects in multiple tissues and organs.

The endocannabinoid system is a promising target to mitigate pain as the endocannabinoids are endogenous ligands of the pain-mediating receptors—cannabinoid receptors 1 and 2 (CB1 and CB2) and TRPV1. Herein, we report on a class of lipids formed by the epoxidation of N-arachidonoyl-dopamine (NADA) and N-arachidonoyl-serotonin (NA5HT) by epoxygenases. EpoNADA and epoNA5HT are dual-functional rheostat modulators of the endocannabinoid-TRPV1 axis. EpoNADA and epoNA5HT are stronger modulators of TRPV1 than either NADA or NA5HT, and epoNA5HT displays a significantly stronger inhibition on TRPV1-mediated responses in primary afferent neurons. Moreover, epoNA5HT is a full CB1 agonist. These epoxides reduce the pro-inflammatory biomarkers IL-6, IL-1β, TNF-α and nitrous oxide and raise anti-inflammatory IL-10 cytokine in activated microglial cells. The epoxides are spontaneously generated by activated microglia cells and their formation is potentiated in the presence of anandamide. Detailed kinetics and molecular dynamics simulation studies provide evidence for this potentiation using the epoxygenase human CYP2J2. Taken together, inflammation leads to an increase in the metabolism of NADA, NA5HT and other eCBs by epoxygenases to form the corresponding epoxides. The epoxide metabolites are bioactive lipids that are potent, multi-faceted molecules, capable of influencing the activity of CB1, CB2 and TRPV1 receptors., Endocannabinoids are ligands of cannabinoid receptors and a promising target for pain management. Here, the authors report a class of lipids formed by the epoxidation of N-arachidonoyl dopamine and N-arachidonoyl serotonin by cytochrome P450 epoxygenases, which reciprocally regulate canabinoid receptors and display anti-inflammatory activity.

Heterotropic interactions between atorvastatin (ARVS) and dronedarone (DND) have been deciphered using global analysis of the results of binding and turnover experiments for pure drugs and their mixtures. The in vivo presence of atorvastatin lactone (ARVL) was explicitly taken into account by using pure ARVL in analogous experiments. Both ARVL and ARVS inhibit DND binding and metabolism, while a significantly higher affinity of CYP3A4 for ARVL makes the latter the main modulator of activity (effector) in this system. Molecular dynamics simulations reveal significantly different modes of interactions of DND and ARVL with the substrate binding pocket and with a peripheral allosteric site. Interactions of both substrates with residues F213 and F219 at the allosteric site play a critical role in the communication of conformational changes induced by effector binding to productive binding of the substrate at the catalytic site.

Doxorubicin (DOX) is a chemotherapeutic that is used in the treatment of a wide variety of cancers. However, it causes cardiotoxicity partly due to the formation of reactive oxygen species (ROS). CYP2J2 is a human cytochrome P450 that is highly expressed in cardiomyocytes. It converts arachidonic acid (AA) into four different regioisomers of epoxyeicosatrienoic acids (EETs). Using kinetic analyses we show that AA metabolism by CYP2J2 is modulated by DOX. We show that cytochrome P450 reductase (CPR), the redox partner of CYP2J2, metabolizes DOX to 7-deoxydoxorubicin aglycone (7-de-aDOX). This metabolite then binds to CYP2J2, and inhibits and alters the preferred site of metabolism of AA leading to change in the ratio of the EET regioisomers. Furthermore, molecular dynamics (MD) simulations indicate that 7-de-aDOX and AA can concurrently bind to the CYP2J2 active site to produce these changes in the site of AA metabolism. To see if these observations are unique to DOX/7-de-aDOX, we use non-cardiotoxic DOX analogues, zorubicin (ZRN) and 5-iminodaunorubicin (IDN). ZRN and 5-IDN inhibit CYP2J2-mediated AA metabolism, but does not change the ratio of EET regioisomers. Taken together, we demonstrate that DOX and 7-de-aDOX inhibit CYP2J2-mediated AA metabolism and 7-de-aDOX binds close to the active site to alter the ratio of cardioprotective EETs. These mechanistic studies of CYP2J2 can aid in the design of new alternative DOX derivatives.

The Escherichia coli uracil:proton symporter UraA is a prototypical member of the nucleobase/ascorbate transporter (NAT) or nucleobase/cation symporter 2 (NCS2) family, which corresponds to the human solute carrier family SLC23. UraA consists of 14 transmembrane segments (TMs) that are organized into two distinct domains, the core domain and the gate domain, a structural fold that is also shared by the SLC4 and SLC26 transporters. Here we present the crystal structure of UraA bound to uracil in an occluded state at 2.5 Å resolution. Structural comparison with the previously reported inward-open UraA reveals pronounced relative motions between the core domain and the gate domain as well as intra-domain rearrangement of the gate domain. The occluded UraA forms a dimer in the structure wherein the gate domains are sandwiched by two core domains. In vitro and in vivo biochemical characterizations show that UraA is at equilibrium between dimer and monomer in all tested detergent micelles, while dimer formation is necessary for the transport activity. Structural comparison between the dimeric UraA and the recently reported inward-facing dimeric UapA provides important insight into the transport mechanism of SLC23 transporters.

Cytochrome P450 (CYP) 2J2 is the primary epoxygenase in the heart and is responsible for the epoxidation of arachidonic acid (AA), an ω-6 polyunsaturated fatty acid (PUFA), into anti-inflammatory epoxide metabolites. It also epoxidizes other PUFAs such as docosahexaenoic acid (DHA), linoleic acid (LA), and eicosapentaenoic acid (EPA). Herein, we have performed detailed thermodynamic and kinetic analyses to determine how DHA, LA and EPA modulate AA metabolism by CYP2J2. We use the Nanodisc (ND) system to stabilize CYP2J2 and its redox partner CYP reductase (CPR). We observe that DHA strongly inhibits CYP2J2-mediated AA metabolism, while LA only moderately inhibits and EPA exhibits insignificant inhibition. We also characterized the binding of these molecules using ebastine competitive binding assays and show that DHA binds significantly tighter to CYP2J2 as compared to AA, EPA, or LA. Furthermore, we utilize a combined approach of molecular dynamics (MD) simulations and docking to predict key residues mediating the tight binding of DHA. We show that although all the tested fatty acids form similar contacts to the active site residues, the affinity of DHA binding to CYP2J2 is tighter due to the interaction of DHA with residues Arg-321, Thr-318 and Ser-493. To demonstrate the importance of these residues in binding, we mutated these residues to make two mutant variants—CYP2J2-T318A and CYP2J2-T318V/S493A. Both of these variants showed weaker binding affinity to DHA and AA compared to the WT and the stronger inhibition of AA by DHA in the WT is mitigated in these mutants. Therefore, using a combined experimental and MD simulations approach, we establish that CYP2J2 inhibition of AA metabolism by DHA, EPA and LA is asymmetric due to tighter binding of DHA to select residues in the active site.

Peripheral membrane proteins are structurally diverse proteins that are involved in fundamental cellular processes. Their activity of these proteins is frequently modulated through their interaction with cellular membranes, and as a result techniques to study the interfacial interaction between peripheral proteins and the membrane are in high demand. Due to the fluid nature of the membrane and the reversibility of protein-membrane interactions, the experimental study of these systems remains a challenging task. Molecular dynamics simulations offer a suitable approach to study protein-lipid interactions; however, the slow dynamics of the lipids often prevents sufficient sampling of specific membrane-protein interactions in atomistic simulations. To increase lipid dynamics while preserving the atomistic detail of protein-lipid interactions, in the highly mobile membrane-mimetic (HMMM) model the membrane core is replaced by an organic solvent, while short-tailed lipids provide a nearly complete representation of natural lipids at the organic solvent/water interface. Here, we present a brief introduction and a summary of recent applications of the HMMM to study different membrane proteins, complementing the experimental characterization of the presented systems, and we offer a perspective of future applications of the HMMM to study other classes of membrane proteins. This article is part of a Special Issue entitled: Membrane proteins edited by J.C. Gumbart and Sergei Noskov.

Hemagglutinin (HA) is a protein located on the surface of the influenza virus that mediates viral fusion to the host cellular membrane. During the fusion process the HA fusion peptide (HAfp), formed by the first 23 N-terminal residues of HA and structurally characterized by two alpha helices (Helix A and Helix B) tightly packed in a hairpin-like arrangement, is the only part of the virus in direct contact with the host membrane. After encountering the host cell HAfp is believed to insert into the membrane, thereby initiating the fusion of the viral and host membranes. Detailed characterization of the interactions between the HAfp and cellular membrane is therefore of high relevance to the mechanism of viral entry into the host cell. Employing HMMM membrane representation with enhanced lipid mobility, we have performed a large set of independent simulations of unbiased membrane binding of HAfp. We have been able to capture spontaneous binding and insertion of HAfp consistently in nearly all the simulations. A reproducible membrane-bound configuration emerges from these simulations, despite employing a diverse set of initial configurations. Extension of several of the simulations into full membrane systems confirms the stability of the membrane-bound form obtained from HMMM binding simulations. The resulting model allows for the characterization of important interactions between the peptide and the membrane, and the details of the binding process of the peptide for the first time. Upon membrane binding, Helix A inserts much deeper into the membrane than Helix B, suggesting that the former is responsible for hydrophobic anchoring of the peptide into the membrane. Helix B, in contrast, is found to establish major amphipathic interactions at the interfacial region thereby contributing to binding strength of HAfp.

Biological membranes constitute a critical component in all living cells. In addition to providing a conducive environment to a wide range of cellular processes, including transport and signaling, mounting evidence has established active participation of specific lipids in modulating membrane protein function through various mechanisms. Understanding lipid-protein interactions underlying these mechanisms at a sufficiently high resolution has proven extremely challenging, partly due to the semi-fluid nature of the membrane. In order to address this challenge computationally, multiple methods have been developed, including an alternative membrane representation termed highly mobile membrane mimetic (HMMM) in which lateral lipid diffusion has been significantly enhanced without compromising atomic details. The model allows for efficient sampling of lipid-protein interactions at atomic resolution, thereby significantly enhancing the effectiveness of molecular dynamics simulations in capturing membrane-associated phenomena. In this review, after providing an overview of HMMM model development, we will describe briefly successful application of the model to study a variety of membrane processes, including lipid-dependent binding and insertion of peripheral proteins, the mechanism of phospholipid insertion into lipid bilayers, and characterization of optimal tilt angle of transmembrane helices. We conclude with practical recommendations for proper usage of the model in simulation studies of membrane processes.

Using Nanodiscs, we quantitate the heterotropic interaction between two different drugs mediated by monomeric CYP3A4 incorporated into a native-like membrane environment. The mechanism of this interaction is deciphered by global analysis of multiple turnover experiments performed under identical conditions using the pure substrates progesterone (PGS) and carbamazepine (CBZ) and their mixtures. Activation of CBZ epoxidation and simultaneous inhibition of PGS hydroxylation are measured and quantitated through differences in their respective affinities towards both a remote allosteric site and the productive catalytic site near the heme iron. Preferred binding of PGS at the allosteric site and higher preference of CBZ binding at the productive site give rise to a non-trivial drug-drug interaction. Molecular dynamics simulations indicate functionally important conformational changes caused by PGS binding at the allosteric site and by two CBZ molecules positioned inside the substrate binding pocket. Structural changes involving Phe-213, Phe-219, and Phe-241 are suggested to be responsible for the observed synergetic effects and positive allosteric interactions between these two substrates. Such a mechanism is likely of general relevance to the mutual heterotropic effects caused by biologically active compounds which exhibit different patterns of interaction with the distinct allosteric and productive sites of CYP3A4, as well as other xenobiotic metabolizing cytochromes P450 that are also involved in drug-drug interactions. Importantly, this work demonstrates that a monomeric CYP3A4 can display the full spectrum of activation and cooperative effects that are observed in hepatic membranes.

Cytochrome P450 3A4 (CYP3A4) is the most abundant membrane-associated isoform of the P450 family in humans and is responsible for biotransformation of more than 50% of drugs metabolized in the body. Despite the large number of crystallographic structures available for CYP3A4, no structural information for its membrane-bound state at an atomic level is available. In order to characterize binding, depth of insertion, membrane orientation, and lipid interactions of CYP3A4, we have employed a combined experimental and simulation approach in this study. Taking advantage of a novel membrane representation, highly mobile membrane mimetic (HMMM), with enhanced lipid mobility and dynamics, we have been able to capture spontaneous binding and insertion of the globular domain of the enzyme into the membrane in multiple independent, unbiased simulations. Despite different initial orientations and positions of the protein in solution, all the simulations converged into the same membrane-bound configuration with regard to both the depth of membrane insertion and the orientation of the enzyme on the surface of the membrane. In tandem, linear dichroism measurements performed on CYP3A4 bound to Nanodisc membranes were used to characterize the orientation of the enzyme in its membrane-bound form experimentally. The heme tilt angles measured experimentally are in close agreement with those calculated for the membrane-bound structures resulted from the simulations, thereby verifying the validity of the developed model. Membrane binding of the globular domain in CYP3A4, which appears to be independent of the presence of the transmembrane helix of the full-length enzyme, significantly reshapes the protein at the membrane interface, causing conformational changes relevant to access tunnels leading to the active site of the enzyme.

The dynamic nature of lipid membranes presents significant challenges with respect to understanding the molecular basis of protein/membrane interactions. Consequently, there is relatively little known about the structural mechanisms by which membrane-binding proteins might distinguish subtle variations in lipid membrane composition and/or structure. We have previously developed a multidisciplinary approach that combines molecular dynamics simulation with interfacial x-ray scattering experiments to produce an atomistic model for phosphatidylserine recognition by the immune receptor Tim4. However, this approach requires a previously determined protein crystal structure in a membrane-bound conformation. Tim1, a Tim4 homolog with distinct differences in both immunological function and sensitivity to membrane composition, was crystalized in a closed-loop conformation that is unlikely to support membrane binding. Here we have used a previously described highly mobile membrane mimetic membrane in combination with a conventional lipid bilayer model to generate a membrane-bound configuration of Tim1 in silico. This refined structure provided a significantly improved fit of experimental x-ray reflectivity data. Moreover, the coupling of the x-ray reflectivity analysis with both highly mobile membrane mimetic membranes and conventional lipid bilayer molecular dynamics simulations yielded a dynamic model of phosphatidylserine membrane recognition by Tim1 with atomic-level detail. In addition to providing, to our knowledge, new insights into the molecular mechanisms that distinguish the various Tim receptors, these results demonstrate that in silico membrane-binding simulations can remove the requirement that the existing crystal structure be in the membrane-bound conformation for effective x-ray reflectivity analysis. Consequently, this refined methodology has the potential for much broader applicability with respect to defining the atomistic details of membrane-binding proteins.

The cellular membrane constitutes the first element that encounters a wide variety of molecular species to which a cell might be exposed. Hosting a large number of structurally and functionally diverse proteins associated with this key metabolic compartment, the membrane not only directly controls the traffic of various molecules in and out of the cell, it also participates in such diverse and important processes as signal transduction and chemical processing of incoming molecular species. In this article, we present a number of cases where details of interaction of small molecular species such as drugs with the membrane, which are often experimentally inaccessible, have been studied using advanced molecular simulation techniques. We have selected systems in which partitioning of the small molecule with the membrane constitutes a key step for its final biological function, often binding to and interacting with a protein associated with the membrane. These examples demonstrate that membrane partitioning is not only important for the overall distribution of drugs and other small molecules into different compartments of the body, it may also play a key role in determining the efficiency and the mode of interaction of the drug with its target protein. This article is part of a Special Issue entitled: Biosimulations edited by Ilpo Vattulainen and Tomasz Rog.

Dielectrophoresis (DEP) is the motion of particles due to polarization effects in nonuniform electric fields. DEP has great potential for handling cells and is a non-destructive phenomenon. It has been utilized for different cell analysis, from viability assessments to concentration enrichment and separation. Insulator-based DEP (iDEP) provides an attractive alternative to conventional electrode-based systems; in iDEP, insulating structures are used to generate nonuniform electric fields, resulting in simpler and more robust devices. Despite the rapid development of iDEP microdevices for applications with cells, the fundamentals behind the dielectrophoretic behavior of cells has not been fully elucidated. Understanding the theory behind iDEP is necessary to continue the progress in this field. This work presents the manipulation and separation of bacterial and yeast cells with iDEP. A computational model in COMSOL Multiphysics was employed to predict the effect of direct current-iDEP on cells suspended in a microchannel containing an array of insulating structures. The model allowed predicting particle behavior, pathlines and the regions where dielectrophoretic immobilization should occur. Experimental work was performed at the same operating conditions employed with the model and results were compared, obtaining good agreement. This is the first report on the mathematical modeling of the dielectrophoretic response of yeast and bacterial cells in a DC-iDEP microdevice.

A lab-on-a-chip device is described for continuous sorting of fluorescent polystyrene microparticles utilizing direct current insulating dielectrophoresis (DC-iDEP) at lower voltages than previously reported. Particles were sorted by combining electrokinetics and dielectrophoresis in a 250 μm wide PDMS microchannel containing a rectangular insulating obstacle and four outlet channels. The DC-iDEP particle flow behaviors were investigated with 3.18, 6.20 and 10 μm fluorescent polystyrene particles which experience negative DEP forces depending on particle size, DC electric field magnitude and medium conductivity. Due to negative DEP effects, particles are deflected into different outlet streams as they pass the region of high electric field density around the obstacle. Particles suspended in dextrose added phosphate buffer saline (PBS) at conductivities ranging from 0.50 to 8.50 mS/cm at pH 7.0 were compared at 6.85 and 17.1 V/cm. Simulations of electrokinetic and dielectrophoretic forces were conducted with COMSOL Multiphysics® to predict particle pathlines. Experimental and simulation results show the effect of medium and voltage operating conditions on particle sorting. Further, smaller particles experience smaller iDEP forces and are more susceptible to competing nonlinear electrostatic effects, whereas larger particles experience greater iDEP forces and prefer channels 1 and 2. This work demonstrates that 6.20 and 10 μm particles can be independently sorted into specific outlet streams by tuning medium conductivity even at low operating voltages. This work is an essential step forward in employing DC-iDEP for multiparticle sorting in a continuous flow, multiple outlet lab-on-a-chip device.

Insulator-based dielectrophoresis (iDEP) is a dielectrophoretic mode where non-uniform electric fields are created employing insulating structures. This article presents an evaluation of the selectivity of an iDEP microdevice by employing DC electric fields with mixtures of polystyrene nano and microparticles. Experimental and mathematical modeling work was carried out in order to assess the selectivity of an iDEP microdevice to immobilize only the large particles from a binary mixture. Negative dielectrophoretic trapping was observed and the effects of particle concentration, concentration ratio, size ratio, and magnitude of the applied electric potential (200 to 600 V) on the microdevice selectivity were studied. The results demonstrated that high selectivity can be obtained with iDEP.

Insulator-based dielectrophoresis (iDEP), an efficient technique with great potential for miniaturization, has been successfully applied for the manipulation of a wide variety of bioparticles. When iDEP is applied employing direct current (DC) electric fields, other electrokinetic transport mechanisms are present: electrophoresis and electroosmotic flow. In order to concentrate particles, iDEP has to overcome electrokinetics. This study presents the characterization of electrokinetic flow under the operating conditions employed with iDEP; in order to identify the optimal conditions for particle concentration employing DC-iDEP, microparticle image velocimetry (microPIV) was employed to measure the velocity of 1-microm-diameter inert polystyrene particles suspended inside a microchannel made from glass. Experiments were carried out by varying the properties of the suspending medium (conductivity from 25 to 100 microS/cm and pH from 6 to 9) and the strength of the applied electric field (50-300 V/cm); the velocities values obtained ranged from 100 to 700 microm/s. These showed that higher conductivity and lower pH values for the suspending medium produced the lowest electrokinetic flow, improving iDEP concentration of particles, which decreases voltage requirements. These ideal conditions for iDEP trapping (pH = 6 and sigma(m) = 100 microS/cm) were tested experimentally and with the aid of mathematical modeling. The microPIV measurements allowed obtaining values for the electrokinetic mobilities of the particles and the zeta potential of the glass surface; these values were used with a mathematical model built with COMSOL Multiphysics software in order to predict the dielectrophoretic and electrokinetic forces exerted on the particles; the modeling results confirmed the microPIV findings. Experiments with iDEP were carried out employing the same microparticles and a glass microchannel that contained an array of cylindrical insulating structures. By applying DC electric fields across the insulating structures array, it was seen that the dielectrophoretic trapping was improved when the electrokinetic force was the lowest; as predicted by microPIV measurements and the mathematical model. The results of this study provide guidelines for the selection of optimal operating conditions for improving insulator-based dielectrophoretic separations and have the potential to be extended to bioparticle applications.

Accumulating evidence suggests that an immune response can be triggered by the presence of abnormal lipid components, such as phosphatidylserine (PS), in the outer leaflet of the cellular membrane. The T-cell immunoglobulin and mucin domain (TIM) family of proteins recognize PS exposed on the surface of the membrane. TIM proteins are expressed by numerous cell types, and despite their close structural similarity, they are involved in triggering different immune responses. These specific roles have been attributed to different factors, including differential binding modes of TIM proteins to anionic membranes and their variable sensitivity to lipid composition of the membrane.In order to study the mechanism of membrane binding by TIM proteins, we have performed MD simulations of TIM1 and TIM3 employing our enhanced-dynamics HMMM membrane model. For each protein, we have performed a total of 30 independent simulations with different lipid compositions (1:1 PC:PS and 7:3 PC:PS), providing robust statistics to characterize the membrane-bound form of these proteins. The results suggest that, despite the overall structural similarity, TIM1 and TIM3 establish different interactions with the membrane upon binding. Moreover, simulations show that in addition to the PS-binding pocket found in TIM proteins, other specific protein-membrane ionic interactions can be formed in each case, suggesting a molecular basis for their different biological roles.In addition to MD simulations, the orientation of TIM1 and TIM3 in model PS-containing membranes has been characterized using X-ray scattering. The agreement between the X-ray experiments and the MD simulations provide a detailed description of the membrane-binding mechanism of TIM proteins.

Cytochrome P450 (CYP) enzymes constitute a large family of enzymes present in a wide variety of organisms that are involved in the metabolism of xenobiotics. In humans, CYP3A4 is the most abundant isoform in the liver and is responsible for the metabolism of a large variety of drugs. CYP enzymes are anchored in the cellular membrane by a transmembrane alpha helix and by the insertion of an unknown hydrophobic region from their globular domain into the lipid bilayer. Despite its high relevance to drug entry and binding, an experimental membrane-bound structure of CYP3A4 has not been reported to this date, and only soluble structures are currently available. Molecular dynamics (MD) simulations of other soluble CYP structures have suggested that the presence of the lipid bilayer might initiate important conformational changes in CYPs, but due to the limited lipid motion in such studies, the nature of these changes are still largely uncharacterized. In order to study the interaction of CYP3A4 with a membrane, we performed MD simulations employing a highly mobile membrane mimetic (HMMM) model developed by our group. The HMMM model allows the unbiased association of CYP3A4 with a phosphatidylcholine (PC) bilayer, providing an all-atom description of this process for the first time. The enhanced lipid mobility achieved by the HMMM model allows for a detailed description of the dynamics of CYP3A4, reveling the mechanism of opening and closing of the tunnels from the active site upon membrane binding. In particular, it is observed that the presence of the PC bilayer induces the closing of the access tunnel going through the BC loop of the globular domain. The resulting membrane-bound model exhibits an orientation that is in close agreement with experimental data.

Low frequency alternating current insulator-based dielectrophoresis is a novel technique that allows for highly controlled manipulation of particles. By varying the shape of an AC voltage applied across a microchannel containing an array of insulating cylindrical structures it was possible to concentrate and immobilize microparticles in bands; and then, move the bands of particles to a different location. Mathematical modeling was performed to analyze the distribution of the electric field and electric field gradient as function of the shape of the AC applied potential, employing frequencies in the 0.2–1.25 Hz range. Three different signals were tested: sinusoidal, half sinusoidal and sawtooth. Experimental results demonstrated that this novel dielectrophoretic mode allows highly controlled particle manipulation.

This paper is the first report on the characterization of the hydrodynamic conditions in a flow cell designed to study adsorption processes by spectroscopic ellipsometry. The resulting cell enables combining the advantages of in situ spectroscopic ellipsometry with stagnation point flow conditions. An additional advantage is that the proposed cell features a fixed position of the " inlet tube" with respect to the substrate, thus facilitating the alignment of multiple substrates. Theoretical calculations were performed by computational fluid dynamics and compared with experimental data (adsorption kinetics) obtained for the adsorption of polyethylene glycol to silica under a variety of experimental conditions. Additionally, a simple methodology to correct experimental data for errors associated with the size of the measured spot and for variations of mass transfer in the vicinity of the stagnation point is herein introduced. The proposed correction method would allow researchers to reasonably estimate the adsorption kinetics at the stagnation point and quantitatively compare their results, even when using different experimental setups. The applicability of the proposed correction function was verified by evaluating the kinetics of protein adsorption under different experimental conditions. © 2010. Fil: Mora, Maria F.. The University of Texas at San Antonio; Estados Unidos Fil: Reza Nejadnik, M.. The University of Texas at San Antonio; Estados Unidos Fil: Baylon Cardiel, Javier L.. Tecnológico de Monterrey; México Fil: Giacomelli, Carla Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Físico-química de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Instituto de Investigaciones en Físico-química de Córdoba; Argentina Fil: Garcia, Carlos D.. The University of Texas at San Antonio; Estados Unidos

A mathematical model is implemented to study the performance of an insulator-based dielectrophoretic device. The geometry of the device was captured in a computational model that solves Laplace equation within an array of cylindrical insulating structures. From the mathematical model it was possible to predict the location and magnitude of the zones of dielectrophoretic trapping of microparticles. Simulation and experimental results of trapping zones are compared for different operating conditions.

CYP2J2 epoxygenase is an extrahepatic, membrane bound cytochrome P450 (CYP) that is primarily found in the heart and mediates endogenous fatty acid metabolism. CYP2J2 interacts with membranes through an N-terminal anchor and various non-contiguous hydrophobic residues. The molecular details of the motifs that mediate membrane interactions are complex and not fully understood. To gain better insights of these complex protein–lipid interactions, we employed molecular dynamics (MD) simulations using a highly mobile membrane mimetic (HMMM) model that enabled multiple independent spontaneous membrane binding events to be captured. Simulations revealed that CYP2J2 engages with the membrane at the F-G loop through hydrophobic residues Trp-235, Ille-236, and Phe-239. To explore the role of these residues, three F-G loop mutants were modeled from the truncated CYP2J2 construct (Δ34) which included Δ34-I236D, Δ34-F239H and Δ34-I236D/F239H. Using the HMMM coordinates of CYP2J2, the simulations were extended to a full POPC membrane which showed a significant decrease in the depth of insertion for each of the F-G loop mutants. The CYP2J2 F-G loop mutants were expressed in E. coli and were shown to be localized to the cytosolic fraction at a greater percentage relative to construct Δ34. Notably, the functional data demonstrated that the double mutant, Δ34-I236D/F239H, maintained native-like enzymatic activity. The membrane insertion characteristics were examined by monitoring CYP2J2 Trp-quenching fluorescence spectroscopy upon binding nanodiscs containing pyrene phospholipids. Relative to the Δ34 construct, the F-G loop mutants exhibited lower Trp quenching and membrane insertion. Taken together, the results suggest that the mutants exhibit a different membrane topology in agreement with the MD simulations and provide important evidence towards the involvement of key residues in the F-G loop of CYP2J2.

Cytochrome P450 proteins (CYPs) constitute a large group of enzymes involved in the metabolism of a large number of endogenous compounds and xenobiotics. In humans, CYP3A4 is the most abundant isoform, which is responsible for the metabolism of more than 50% of clinically used drugs. It has been suggested that the interaction of CYPs with the membrane is crucial for the binding of liposoluble substrates to the active site of the enzyme.Molecular dynamics simulations using a HMMM membrane model have captured spontaneous insertion of the globular domain of CYP3A4 into the membrane and revealed a significant reconfiguration of the access tunnels leading to the active site upon membrane binding. We have identified that the binding to the membrane favors the opening of several access tunnels to the active site not observed in the crystallographic structures. Rearrangement of a Phe-cluster appears to be the main membrane-induced event resulting in the opening of the access tunnels. Given the location of these tunnels, they might be involved in leading compounds in or out of the active site. In order to characterize the role of these access tunnels in access/egress of compounds, we have also performed steered molecular dynamics simulations to pull progesterone from the active site of the membrane-bound CYP3A4. Parameters for progesterone were optimized employing the Force Field Toolkit recently developed by our group. We tested 5 different tunnels, previously identified to be open when CYP3A4 is bound to the membrane. To complement the SMD simulations, the partitioning of progesterone into the membrane was also studied. The results of these simulations allow us to elucidate the role of the membrane in the access/egress of compounds to the active site of CYP3A4.