Simulation of the distribution and diffusion of a rigid amphipathic particle embedded in a model membrane.

We simulate, by Brownian dynamics, the distribution, orientation and diffusion of a rigid molecule, represented as a dumbbell, with amphipathic nature, embedded in a model membrane. The significant features of a biological membrane are reproduced by means of a Maier-Saupe orienting potential, an enclosing potential and a lipophobic potential. We also evaluate the equilibrium quantities, such as order parameter, and dynamic features, such as rotational or translational diffusivity, of the embedded molecule in terms of the system parameters and compare the obtained results with those obtained from model independent theory.     

Brownian dynamics simulation of probe diffusion in DNA: effects of probe size, charge and DNA concentration

We have used Brownian dynamics simulation to study probe diffusion in solutions of short chain DNA using our previously developed simulation algorithm. We have examined the effect of probe size, charge, and DNA concentration on the probe diffusion coefficient, with the aim of gaining insight into the diffusion of proteins in a concentrated DNA environment. In these simulations, DNA was modeled as a worm-like chain of hydrodynamically equivalent spherical frictional elements while probe particles were modeled as spheres of given charge and hydrodynamic radius. The simulations allowed for both short range Lennard-Jones interactions and long ranged electrostatic interactions between charged particles. For uncharged systems, we find that the effects of probe size and DNA concentration on the probe diffusion coefficient are consistent with excluded volume models and we interpret our results in terms of both empirical scaling laws and the predictions of scaled particle theory. For charged systems, we observe that the effects of probe size and charge are most pronounced for the smallest probes and interpret the results in terms of the probe charge density. For an ionic strength of 0.1 M we find that, below a critical probe surface charge density, the probe diffusion coefficient is largely independent of probe charge and only weakly dependent on the DNA charge. These effects are discussed in terms of the interactions between the probe and the DNA matrix and are interpreted in terms of both the underlying physics of transport in concentrated solutions and the assumptions of the simulation model.     

The orientation of eosin-5-maleimide on human erythrocyte band 3 measured by fluorescence polarization microscopy.

The dominant motional mode for membrane proteins is uniaxial rotational diffusion about the membrane normal axis, and investigations of their rotational dynamics can yield insight into both the oligomeric state of the protein and its interactions with other proteins such as the cytoskeleton. However, results from the spectroscopic methods used to study these dynamics are dependent on the orientation of the probe relative to the axis of motion. We have employed polarized fluorescence confocal microscopy to measure the orientation of eosin-5-maleimide covalently reacted with Lys-430 of human erythrocyte band 3. Steady-state polarized fluorescence images showed distinct intensity patterns, which were fit to an orientation distribution of the eosin absorption and emission dipoles relative to the membrane normal axis. This orientation was found to be unchanged by trypsin treatment, which cleaves band 3 between the integral membrane domain and the cytoskeleton-attached domain. this result suggests that phosphorescence anisotropy changes observed after trypsin treatment are due to a rotational constraint change rather than a reorientation of eosin. By coupling time-resolved prompt fluorescence anisotropy with confocal microscopy, we calculated the expected amplitudes of the e-Dt and e-4Dt terms from the uniaxial rotational diffusion model and found that the e-4Dt term should dominate the anisotropy decay. Delayed fluorescence and phosphorescence anisotropy decays of control and trypsin-treated band 3 in ghosts, analyzed as multiple uniaxially rotating populations using the amplitudes predicted by confocal microscopy, were consistent with three motional species with uniaxial correlation times ranging from 7 microseconds to 1.4 ms.     

Conformation and dynamic properties of a saturated hydrocarbon chain confined in a model membrane: a Brownian dynamics simulation

A Brownian dynamics simulation of a saturated hydrocarbon chain with simple mean-field potentials, namely anchorage, orientation and enclosing, reproducing a biological membrane environment is presented. The simulation was performed for a time equivalent to 1.4 micros thanks to the simplicity of our model. The results are compared with those obtained for a hydrocarbon chain simulated in the absence of the membrane potentials but with confinement. With the appropriate choice of parameters, equilibrium properties, such as deuterium order parameter, chain length, tilt angle and geometry, and dynamic properties, such as dihedral angle transition rate, rotational and translational diffusion, recovered from our simulations, correctly reproduced, are consistent with hydrocarbon-derived molecule experimental results and simulation results obtained from other more complex studies.     

Analysis of saturation transfer electron paramagnetic resonance spectra of a spin-labeled integral membrane protein, band 3, in terms of the uniaxial rotational diffusion model.

Algorithms have been developed for the calculation of saturation transfer electron paramagnetic resonance (ST-EPR) spectra of a nitroxide spin-label assuming uniaxial rotational diffusion, a model that is frequently used to describe the global rotational dynamics of large integral membrane proteins. One algorithm explicitly includes terms describing Zeeman overmodulation effects, whereas the second more rapid algorithm treats these effects approximately using modified electron spin-lattice and spin-spin relaxation times. Simulations are presented to demonstrate the sensitivity of X-band ST-EPR spectra to the rate of uniaxial rotational diffusion and the orientation of the nitroxide probe with respect to the diffusion axis. Results obtained by using the algorithms presented, which are based on the transition-rate formalism, are in close agreement with those obtained by using an eigenfunction expansion approach. The effects of various approximations used in the simulation algorithms are considered in detail. Optimizing the transition-rate formalism to model uniaxial rotational diffusion results in over an order of magnitude reduction in computation time while allowing treatment of nonaxial A- and g-tensors. The algorithms presented here are used to perform nonlinear least-squares analyses of ST-EPR spectra of the anion exchange protein of the human erythrocyte membrane, band 3, which has been affinity spin-labeled with a recently developed dihydrostilbene disulfonate derivative, [15N,2H13]-SL-H2DADS-MAL. These results suggest that all copies of band 3 present in intact erythrocytes undergo rotational diffusion about the membrane normal axis at a rate consistent with a band 3 dimer.     

Molecular dynamics simulation of a dye molecule in the interior of a bilayer: 1,6-diphenyl-1,3,5-hexatriene in dipalmitoylphosphatidylcholine

A molecular dynamics simulation was carried out for a dipalmitoylphosphatidylcholine (DPPC) membrane in its liquid crystalline state containing different concentrations of the dye molecule 1,6-diphenyl-1,3,5-hexatriene (DPH). From a numerical analysis of the trajectories, we obtained information concerning structural changes of the membrane due to the presence of the probe and some hydrodynamic information concerning the probe itself. The hydrodynamic properties regarding dye molecules that have been reported in this article are: rotational and translational diffusion coefficient and relaxation times. From this analysis, we estimated a range of values of 0.6-0.9 cP for the micro-viscosity in the mid-membrane. These simulations also afforded us some information regarding structural changes in the membrane as a consequence of the presence of the fluorescent dyes at different concentrations. Thus, the disorder inside the membrane, the surface area per lipid and thickness of the membrane were also investigated.     

Membrane Orientation and Lateral Diffusion of BODIPY-Cholesterol as a Function of Probe Structure

Cholesterol tagged with the BODIPY fluorophore via the central difluoroboron moiety of the dye (B-Chol) is a promising probe for studying intracellular cholesterol dynamics. We synthesized a new BODIPY-cholesterol probe (B-P-Chol) with the fluorophore attached via one of its pyrrole rings to carbon-24 of cholesterol (B-P-Chol). Using two-photon fluorescence polarimetry in giant unilamellar vesicles and in the plasma membrane (PM) of living intact and actin-disrupted cells, we show that the BODIPY-groups in B-Chol and B-P-Chol are oriented perpendicular and almost parallel to the bilayer normal, respectively. B-Chol is in all three membrane systems much stronger oriented than B-P-Chol. Interestingly, we found that the lateral diffusion in the PM was two times slower for B-Chol than for B-P-Chol, although we found no difference in lateral diffusion in model membranes. Stimulated emission depletion microscopy, performed for the first time, to our knowledge, with fluorescent sterols, revealed that the difference in lateral diffusion of the BODIPY-cholesterol probes was not caused by anomalous subdiffusion, because diffusion of both analogs in the PM was free but not hindered. Our combined measurements show that the position and orientation of the BODIPY moiety in cholesterol analogs have a severe influence on lateral diffusion specifically in the PM of living cells.     

Accelerating membrane insertion of peripheral proteins with a novel membrane mimetic model

Characterizing atomic details of membrane binding of peripheral membrane proteins by molecular dynamics (MD) has been significantly hindered by the slow dynamics of membrane reorganization associated with the phenomena. To expedite lateral diffusion of lipid molecules without sacrificing the atomic details of such interactions, we have developed a novel membrane representation, to our knowledge, termed the highly mobile membrane-mimetic (HMMM) model to study binding and insertion of various molecular species into the membrane. The HMMM model takes advantage of an organic solvent layer to represent the hydrophobic core of the membrane and short-tailed phospholipids for the headgroup region. We demonstrate that using these components, bilayer structures are formed spontaneously and rapidly, regardless of the initial position and orientation of the lipids. In the HMMM membrane, lipid molecules exhibit one to two orders of magnitude enhancement in lateral diffusion. At the same time, the membrane atomic density profile of the headgroup region produced by the HMMM model is essentially identical to those obtained for full-membrane models, indicating the faithful representation of the membrane surface by the model. We demonstrate the efficiency of the model in capturing spontaneous binding and insertion of peripheral proteins by using the membrane anchor (γ-carboxyglutamic-acid-rich domain; GLA domain) of human coagulation factor VII as a test model. Achieving full insertion of the GLA domain consistently in 10 independent unbiased simulations within short simulation times clearly indicates the robustness of the HMMM model in capturing membrane association of peripheral proteins very efficiently and reproducibly. The HMMM model will provide significant improvements to the current all-atom models by accelerating lipid dynamics to examine protein-membrane interactions more efficiently.     

Membrane Orientation and Lateral Diffusion of BODIPY-Cholesterol as a Function of Probe Structure

Cholesterol tagged with the BODIPY fluorophore via the central difluoroboron moiety of the dye (B-Chol) is a promising probe for studying intracellular cholesterol dynamics. We synthesized a new BODIPY-cholesterol probe (B-P-Chol) with the fluorophore attached via one of its pyrrole rings to carbon-24 of cholesterol (B-P-Chol). Using two-photon fluorescence polarimetry in giant unilamellar vesicles and in the plasma membrane (PM) of living intact and actin-disrupted cells, we show that the BODIPY-groups in B-Chol and B-P-Chol are oriented perpendicular and almost parallel to the bilayer normal, respectively. B-Chol is in all three membrane systems much stronger oriented than B-P-Chol. Interestingly, we found that the lateral diffusion in the PM was two times slower for B-Chol than for B-P-Chol, although we found no difference in lateral diffusion in model membranes. Stimulated emission depletion microscopy, performed for the first time, to our knowledge, with fluorescent sterols, revealed that the difference in lateral diffusion of the BODIPY-cholesterol probes was not caused by anomalous subdiffusion, because diffusion of both analogs in the PM was free but not hindered. Our combined measurements show that the position and orientation of the BODIPY moiety in cholesterol analogs have a severe influence on lateral diffusion specifically in the PM of living cells.     

Interpretation of H-NMR Experiments on the Orientation of the Transmembrane Helix WALP23 by Computer Simulations

Orientation, dynamics, and packing of transmembrane helical peptides are important determinants of membrane protein structure, dynamics, and function. Because it is difficult to investigate these aspects by studying real membrane proteins, model transmembrane helical peptides are widely used. NMR experiments provide information on both orientation and dynamics of peptides, but they require that motional models be interpreted. Different motional models yield different interpretations of quadrupolar splittings (QS) in terms of helix orientation and dynamics. Here, we use coarse-grained (CG) molecular dynamics (MD) simulations to investigate the behavior of a well-known model transmembrane peptide, WALP23, under different hydrophobic matching/mismatching conditions. We compare experimental ²H-NMR QS (directly measured in experiments), as well as helix tilt angle and azimuthal rotation (not directly measured), with CG MD simulation results. For QS, the agreement is significantly better than previously obtained with atomistic simulations, indicating that equilibrium sampling is more important than atomistic details for reproducing experimental QS. Calculations of helix orientation confirm that the interpretation of QS depends on the motional model used. Our simulations suggest that WALP23 can form dimers, which are more stable in an antiparallel arrangement. The origin of the preference for the antiparallel orientation lies not only in electrostatic interactions but also in better surface complementarity. In most cases, a mixture of monomers and antiparallel dimers provides better agreement with NMR data compared to the monomer and the parallel dimer. CG MD simulations allow predictions of helix orientation and dynamics and interpretation of QS data without requiring any assumption about the motional model.     

Classification of Dynamical Diffusion States in Single Molecule Tracking Microscopy

Single molecule tracking of membrane proteins by fluorescence microscopy is a promising method to investigate dynamic processes in live cells. Translating the trajectories of proteins to biological implications, such as protein interactions, requires the classification of protein motion within the trajectories. Spatial information of protein motion may reveal where the protein interacts with cellular structures, because binding of proteins to such structures often alters their diffusion speed. For dynamic diffusion systems, we provide an analytical framework to determine in which diffusion state a molecule is residing during the course of its trajectory. We compare different methods for the quantification of motion to utilize this framework for the classification of two diffusion states (two populations with different diffusion speed). We found that a gyration quantification method and a Bayesian statistics-based method are the most accurate in diffusion-state classification for realistic experimentally obtained datasets, of which the gyration method is much less computationally demanding. After classification of the diffusion, the lifetime of the states can be determined, and images of the diffusion states can be reconstructed at high resolution. Simulations validate these applications. We apply the classification and its applications to experimental data to demonstrate the potential of this approach to obtain further insights into the dynamics of cell membrane proteins.     

Classification of Dynamical Diffusion States in Single Molecule Tracking Microscopy

Single molecule tracking of membrane proteins by fluorescence microscopy is a promising method to investigate dynamic processes in live cells. Translating the trajectories of proteins to biological implications, such as protein interactions, requires the classification of protein motion within the trajectories. Spatial information of protein motion may reveal where the protein interacts with cellular structures, because binding of proteins to such structures often alters their diffusion speed. For dynamic diffusion systems, we provide an analytical framework to determine in which diffusion state a molecule is residing during the course of its trajectory. We compare different methods for the quantification of motion to utilize this framework for the classification of two diffusion states (two populations with different diffusion speed). We found that a gyration quantification method and a Bayesian statistics-based method are the most accurate in diffusion-state classification for realistic experimentally obtained datasets, of which the gyration method is much less computationally demanding. After classification of the diffusion, the lifetime of the states can be determined, and images of the diffusion states can be reconstructed at high resolution. Simulations validate these applications. We apply the classification and its applications to experimental data to demonstrate the potential of this approach to obtain further insights into the dynamics of cell membrane proteins.     

Effects of protein binding on a lipid bilayer containing local anesthetic articaine, and the potential of mean force calculation: a molecular dynamics simulation approach

Articaine, as a local anesthetic drug has been simulated in neutral and charged forms, and its interaction with the dimyristoylphosphatidylcholine (DMPC) lipid bilayer membrane is investigated by molecular dynamics simulation using GROMACS software. In order to obtain the optimum location of the drug molecules, as they penetrate into the membrane, umbrella sampling is applied and the free energy is calculated. The effect of protein binding to DMPC membrane on the process of drug diffusion through the membrane is considered. Five simulation systems are designed and by applying the potential of mean force, the molecular dynamics simulation on the system is performed. In light of the obtained results, the electrostatic potential, variation of lipid bilayer’s order parameter and the diffusion coefficient of drug are discussed.

Intrasequence GFP in class I MHC molecules,a rigid probe for fluorescence anisotropy measurements of the membrane environment

Fluorescence anisotropy measurements can elucidate the microenvironment of a membrane protein in terms of its rotational diffusion, interactions, and proximity to other proteins. However, use of this approach requires a fluorescent probe that is rigidly attached to the protein of interest. Here we describe the use of one such probe, a green fluorescent protein (GFP) expressed and rigidly held within the amino acid sequence of a major histocompatibility complex (MHC) class I molecule, H2L(d). We contrast the anisotropy of this GFP-tagged MHC molecule, H2L(d)GFPout, with that of an H2L(d) that was GFP-tagged at its C-terminus, H2L(d)GFPin. Both molecules fold properly, reach the cell surface, and are recognized by specific antibodies and T-cell receptors. We found that polarized fluorescence images of H2L(d)GFPout in plasma membrane blebs show intensity variations that depend on the relative orientation of the polarizers and the membrane normal, thus demonstrating that the GFP is oriented with respect to the membrane. These variations were not seen for H2L(d)GFPin. Before transport to the membrane surface, MHC class I associates with the transporter associated with antigen processing complex in the endoplasmic reticulum. The intensity-dependent steady-state anisotropy in the ER of H2L(d)GFPout was consistent with FRET homotransfer, which indicates that a significant fraction of these molecules were clustered. After MCMV-peptide loading, which supplies antigenic peptide to the MHC class I releasing it from the antigen processing complex, the anisotropy of H2L(d)GFPout was independent of intensity, suggesting that the MHC proteins were no longer clustered. These results demonstrate the feasibility and usefulness of a GFP moiety rigidly attached to the protein of interest as a probe for molecular motion and proximity in cell membranes.     

High spatial resolution observation of single-molecule dynamics in living cell membranes

Self-organized lipid bilayers together with proteins are the essential building blocks of biological membranes. Membranes are associated with all living systems as they make up cell boundaries and provide basic barriers to cellular organelles. It is of interest to study the dynamics of individual molecules in cell membranes as the mechanism of how biological membranes function at the single molecule remains to be elucidated. In this letter we describe a study in which we incubate rat basophilic leukemia cells with a fluorescently labeled cell membrane component on a surface containing zero-mode waveguides (ZMWs). We used the ZMW to confine fluorescent excitation to an approximately 100-nm region of the membrane to monitor lipid diffusion along the cellular membrane. We showed that confinement with a ZMW largely reduced fluorescent contributions from the cytosolic pool that is present when using a more standard technique such as laser-induced confocal microscopy. We show that optical confinement with ZMWs is a facile way to probe dynamic processes on the membrane surface.     

Molecular dynamics simulation of interaction between phospholipid membrane and pyrazine and its derivatives

We have performed a comparative molecular dynamics simulation of the diffusion process of the heterocyclic compound pyrazine and its methylated derivatives into the model membrane phospholipid bilayer. Several structural and dynamical bilayer parameters were measured, and qualitative interrelations between parameter changes and the substituted pyrazine structure were studied. The simulation results support the hypothesis that molecular mechanisms of biological effects of substituted pyrazines involve dissolution of the effector molecule in the membrane bilayer and subsequent changes in bilayer properties. This stage can provide the means for pyrazine molecules to interact with integral membrane proteins, directly or indirectly through the changed lipid environment of the protein.     

Effects of cholesterol concentration on the interaction of cytarabine with lipid membranes: a molecular dynamics simulation study

Liposomal cytarabine, DepoCyt, is a chemotherapy agent which is used in cancer treatment. This form of cytarabine has more efficacy and fewer side effects relative to the other forms. Since DepoCyt contains the cytarabine encapsulated within phosphatidylcholine and the sterol molecules, we modeled dioleoylphosphatidylcholine (DOPC)/cholesterol bilayer membrane as a carrier for cytarabine to study drug–bilayer interactions. For this purpose, we performed a series of united-atom molecular dynamics (MD) simulations for 25?ns to investigate the interactions between cytarabine and cholesterol-containing DOPC lipid bilayers. Only the uncharged form of cytarabine molecule was investigated. In this study, different levels of the cholesterol content (0, 20, and 40%) were used. MD simulations allowed us to determine dynamical and structural properties of the bilayer membrane and to estimate the preferred location and orientation of the cytarabine molecule inside the bilayer membrane. Properties such as membrane thickness, area per lipid, diffusion coefficient, mass density, bilayer packing, order parameters, and intermolecular interactions were examined. The results show that by increasing the cholesterol concentration in the lipid bilayers, the bilayer thickness increases and area per lipid decreases. Moreover, in accordance with the experiments, our calculations show that cholesterol molecules have ordering effect on the hydrocarbon acyl chains. Furthermore, the cytarabine molecule preferentially occupies the polar region of the lipid head groups to form specific interactions (hydrogen bonds). Our results fully support the experimental data. Our finding about drug–bilayer interaction is crucial for the liposomal drug design.     

Measurement of diffusion potentials in liposomes. Origin and properties of the threshold level in the oxonol VI response

A model is presented for the response of the membrane potential probe oxonol VI on diffusion potentials in liposomes. In this model the dependence of the probe response on the initial ion gradient is explained in terms of internal volume, internal ion concentration, membrane capacity and initial membrane potential. It is found that in the presence of an initial membrane potential (positive outside) there is a threshold value of the ion gradient needed for a probe response, which increases when the internal volume or the internal ion concentration decrease. The model is confirmed by experiments with liposomes of different sizes and internal KCl concentrations, prepared from asolectin or lipids isolated from the thermophilic cyanobacterium Synechococcus 6716. The significance of the model for threshold values observed in other energy-dependent phenomena is discussed.