Langevin dynamics of the choline head group in a membrane environment.

Computer simulations of dipalmitoylphosphatidylcholine (DPPC) have been performed using Langevin dynamics and a Marcelja-type mean field. This work has focused on the dynamics of the choline head group to parameterize the empirical constraints against phosphorus-carbon dipolar couplings (Dp-c) as measured by nuclear magnetic resonance (13C-NMR). The results show good agreement with experimental values at constraints equivalent to the choline tilt observed in joint refinement of x-ray diffraction and neutron diffraction scatterings. Quadrupolar splittings for the alpha and beta positions are also calculated and compared with 2H-NMR experiments. The model predicts torsional transition rates around the alpha-beta bonds and for the two C-O-P-O torsions. It also predicts T1 relaxation times for the alpha and beta carbons.     

Oxidation of unsaturated phospholipids in membrane bilayer mixtures is accompanied by membrane fluidity changes

Steady-state and time-resolved fluorescence spectroscopy has been used to obtain information on oxidation processes and associated dynamical and structural changes in model membrane bilayers made from single unilamellar vesicles (SUV's) of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) mixed with increasing amounts of 1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (SAPC). The highly unsaturated arachidonoyl chain containing four double bonds is prone to oxidation. Lipid oxidation was initiated chemically by a proper oxidant and could be followed on line via the fluorescence changes of an incorporated fluorescent lipophilic fatty acid: 4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoic acid (BP-C11). The oxidation rate increases with an increasing amount of SAPC. Size measurements of different SUV's incorporated with a trace amount of a phosphatidylcholine analogue of BP-C11 using fluorescence correlation spectroscopy have demonstrated that an increase of lipid unsaturation results in smaller sized SUV's and therefore to a larger curvature of the outer bilayer leaflet. This suggests that the lipid-lipid spacing has increased and that the unsaturated fatty acyl chains are better accessible for the oxidant. Oxidation results in some characteristic physical changes in membrane dynamics and structure, as indicated by the use of specific fluorescence probes. Fluorescence measurements of both dipyrenyl- and diphenylhexatriene-labelled PC introduced in non-oxidised and oxidised DOPC-SAPC membranes clearly show that the microfluidity (local fluidity at the very site of the probes) significantly decreases when the oxidised SAPC content increases in the lipid mixture. A similar effect is observed from the lateral diffusion experiments using monopyrenyl PC in the same membrane systems: the lateral diffusion is distinctly slower in oxidised membranes.     

Langevin dynamics simulation of DNA ejection from a phage

We have performed Langevin dynamics simulations of a coarse-grained model of ejection of dsDNA from Φ29 phage. Our simulation results show significant variations in the local ejection speed, consistent with experimental observations reported in the literature for both in vivo and in vitro systems. In efforts to understand the origin of such variations in the local speed of ejection, we have investigated the correlations between the local ejection kinetics and the packaged structures created at various motor forces and chain flexibility. At lower motor forces, the packaged DNA length is shorter with better organization. On the other hand, at higher motor forces typical of realistic situations, the DNA organization inside the capsid suffers from significant orientational disorder, but yet with long orientational correlation times. This in turn leads to lack of registry between the direction of the DNA segments just to be ejected and the direction of exit. As a result, a significant amount of momentum transfer is required locally for successful exit. Consequently, the DNA ejection temporarily slows down exhibiting pauses. This slowing down occurs at random times during the ejection process, completely determined by the particular starting conformation created by prescribed motor forces. In order to augment our inference, we have additionally investigated the ejection of chains with deliberately changed persistence length. For less inflexible chains, the demand on the occurrence of large momentum transfer for successful ejection is weaker, resulting in more uniform ejection kinetics. While being consistent with experimental observations, our results show the nonergodic nature of the ejection kinetics and call for better theoretical models to portray the kinetics of genome ejection from phages.     

Calorimetric and molecular mechanics studies of the thermotropic phase behavior of membrane phospholipids.

In this review, we summarize the results of recent studies on the main phase transition behavior of phospholipid bilayers using the combined approaches of molecular mechanics simulations and high-resolution differential scanning calorimetry. Following a brief overview of the phase transition phenomenon exhibited by the lipid bilayer, we begin with the review by showing how several structural parameters underlying various phospholipids including phosphatidylcholine, phosphatidylethanolamine, and phosphatidylglycerol are defined and determined. Specifically, these structural parameters are obtained with saturated lipids packed in the gel-state bilayer using computer-based molecular mechanics calculations. Then we proceed to present the calorimetric data obtained with the lipid bilayer composed of saturated phospholipids as it undergoes the gel-to-liquid-crystalline phase transition in excess water. The general equations that can correlate the gel-to-liquid-crystalline phase transition temperature (T(m)) of the lipid bilayer with the structural parameters of the lipid molecule constituting the lipid bilayer are subsequently presented. From these equations, two tables of predicated T(m) values for well over 400 molecular species of saturated phosphatidylcholine and saturated phosphatidylethanolamine are generated. We further review the structure and chain-melting behavior of a large number of sn-1 saturated/sn-2 unsaturated phospholipids. Two T(m)-diagrams are shown, from which the effects of the number and the position of one to five cis carbon-carbon double bonds on T(m) can be viewed simultaneously. Finally, in the last part of this review, simple molecular models that have been invoked to interpret the characteristic T(m) trends exhibited by lipid bilayers composed of unsaturated lipids with different numbers and positions of cis carbon-carbon double bonds as seen in the T(m)-diagram are presented.     

Langevin dynamics simulations of genome packing in bacteriophage

We use Langevin dynamics simulations to study the process by which a coarse-grained DNA chain is packaged within an icosahedral container. We focus our inquiry on three areas of interest in viral packing: the evolving structure of the packaged DNA condensate; the packing velocity; and the internal buildup of energy and resultant forces. Each of these areas has been studied experimentally, and we find that we can qualitatively reproduce experimental results. However, our findings also suggest that the phage genome packing process is fundamentally different than that suggested by the inverse spool model. We suggest that packing in general does not proceed in the deterministic fashion of the inverse-spool model, but rather is stochastic in character. As the chain configuration becomes compressed within the capsid, the structure, energy, and packing velocity all become dependent upon polymer dynamics. That many observed features of the packing process are rooted in condensed-phase polymer dynamics suggests that statistical mechanics, rather than mechanics, should serve as the proper theoretical basis for genome packing. Finally we suggest that, as a result of an internal protein unique to bacteriophage T7, the T7 genome may be significantly more ordered than is true for bacteriophage in general.     

Interactions of annexins with membrane phospholipids.

The annexins are proteins that bind to membranes and can aggregate vesicles and modulate fusion rates in a Ca2(+)-dependent manner. In this study, experiments are presented that utilize a pyrene derivative of phosphatidylcholine to examine the Ca2(+)-dependent membrane binding of soluble human annexin V and other annexins. When annexin V and other annexins were bound to liposomes containing 5 mol % acyl chain labeled 3-palmitoyl-2-(1-pyrenedecanoyl)-L-alpha-phosphatidylcholine, a decrease in the excimer-to-monomer fluorescence ratio was observed, indicating that annexin binding may decrease the lateral mobility of membrane phospholipids without inducing phase separation. The observed increases of monomer fluorescence occurred only with annexins and not with other proteins such as parvalbumin or bovine serum albumin. The extent of the increase of monomer fluorescence was dependent on the protein concentration and was completely and rapidly reversible by EDTA. Annexin V binding to phosphatidylserine liposomes was consistent with a binding surface area of 59 phospholipid molecules per protein. Binding required Ca2+ concentrations ranging between approximately 10 and 100 microM, where there was no significant aggregation or fusion of liposomes on the time scale of the experiments. The polycation spermine also displaced bound annexins, suggesting that binding is largely ionic in nature under these conditions.     

Subdiffusive-Brownian crossover in membrane proteins: a generalized Langevin equation-based approach

In this work, we propose a generalized Langevin equation-based model to describe the lateral diffusion of a protein in a lipid bilayer. The memory kernel is represented in terms of a viscous (instantaneous) and an elastic (noninstantaneous) component modeled through a Dirac δ function and a three-parameter Mittag-Leffler type function, respectively. By imposing a specific relationship between the parameters of the three-parameter Mittag-Leffler function, the different dynamical regimes—namely ballistic, subdiffusive, and Brownian, as well as the crossover from one regime to another—are retrieved. Within this approach, the transition time from the ballistic to the subdiffusive regime and the spectrum of relaxation times underlying the transition from the subdiffusive to the Brownian regime are given. The reliability of the model is tested by comparing the mean-square displacement derived in the framework of this model and the mean-square displacement of a protein diffusing in a membrane calculated through molecular dynamics simulations.     

Inhibitor studies with adenohypophyseal granule membrane ATPase. Evidence for a membrane environment which modulates sensitivity to inhibitors

The limiting membranes of pituitary growth hormone and prolactin secretory granules contain a Mg2+-ATPase sensitive to anions. This enzyme is in many ways similar to mitochondrial ATPase. The enzyme was potently inhibited by oligomycin (Ki 6.5 X 10(-9) M), and was much more sensitive to the inhibitor than pituitary mitochondrial ATPase (Ki 2.7 X 10(-7) M). In contrast, the enzyme activity of intact secretory granules was only sparingly inhibited by oligomycin (maximal inhibition close to 30% at 5 X 10(-4) M). However, oligomycin (5 microM) did diminish to basal levels the enhanced granule ATPase activity observed in the presence of a stimulatory anion (25 mM sodium sulfite). Other compounds known to inhibit the proton translocating mitochondrial ATPase were also tested for their ability to inhibit the secretory granule ATPase. A similar pattern of limited inhibition in granules and greater sensitivity in isolated membranes was seen with the inhibitors N,N-dicyclohexylcarbodiimide and efrapeptin. In contrast, tri-n-butyltin chloride was a potent inhibitor of the ATPase of intact granules, and the susceptibility of the enzyme to inhibition by this compound was less after isolation of membranes. These observations suggest that pituitary secretory granule membrane ATPase may have a proton pumping function similar to that of the mitochondrial enzyme. In addition, the data imply that the inhibitor binding site(s) may be masked, inaccessible, or ineffective in intact granules, but exposed (or activated) in isolated membranes. The greater sensitivity of granule ATPase to tri-n-butyltin chloride, in contrast to the greater sensitivity of membrane ATPase to the other inhibitors, indicates that the tin compound may be effective at a membrane site(s) distinct from the others, or that the mechanism of inhibition is different.     

Accessibility of phospholipids in the chromaffin granule membrane.

1. The accessibility of phospholipids in the membrane of the adrenomedullary storage vesicles (chromaffin granules) has been studied. 2. The reaction of 2,4,6-trinitrobenzenesulphonic acid with both intact granules and their ghosts, results in the labelling of 70% of the phosphatidylethanolamine. 3. The action of phospholipase A2 (from bee venom), phospholipase C (from Bacillus cereus) and sphingomyelinase C (from Staphylococcus aureus) on granules and their ghosts was followed as a function of time. No significant difference was observed between the intact granules and their ghosts. 4. In the intact granules the various treatments led to varying amounts of lysis although again no evidence was obtained that such lysis in any way increased the amount of accessible phospholipid. 5. Highly purified granule preparations were also compared with the so-called "large granule" fraction and no significant differences were detected. 6. Approx. 67% of phosphatidylethanolamine + phosphatidic acid, 50% of phosphatidylserine + phosphatidylinositol, 65% of phosphatidylcholine and 20% of sphingomyelin is accessible to enzymatic degradation. In total, approx. 50% of all the phospholipids reacted. 7. It is also shown that, unlike in enzymatic treatment, all the phosphatidylcholine can be exchanged in the presence of a phospholipid exchange protein (prepared from beef liver). 8. It is concluded that transmembrane movement of phosphatidylcholine is slow in isolated membranes of chromaffin granules. The presence of the exchange protein, however, in conjunction with membrane proteins and specific phospholipid arrangements may catalyse this transmembrane movement.     

Brownian dynamics simulation of the unsaturated lipidic molecules oleic and docosahexaenoic acid confined in a cellular membrane

A Brownian dynamics (BD) simulation of two unsaturated molecules, oleic and docosahexaenoic acid, in an environment that reproduces a cellular membrane, is presented. The results of the simulations, performed using mean-field potentials, were calibrated with experimental results obtained for oleic acid in a cellular membrane. The agreement between simulation and experimental results is excellent which validates subsequent simulation outcome for docosahexaenoic acid. This molecule is a major component of several cellular membranes thought to be involved in specific biological functions that require conformational changes of membrane components. The results for docosahexaenoic acid indicate that it is minimally influenced by temperature changes and that it presents great conformational variability.     

Brownian dynamics simulation of the unsaturated lipidic molecules oleic and docosahexaenoic acid confined in a cellular membrane

A Brownian dynamics (BD) simulation of two unsaturated molecules, oleic and docosahexaenoic acid, in an environment that reproduces a cellular membrane, is presented. The results of the simulations, performed using mean-field potentials, were calibrated with experimental results obtained for oleic acid in a cellular membrane. The agreement between simulation and experimental results is excellent which validates subsequent simulation outcome for docosahexaenoic acid. This molecule is a major component of several cellular membranes thought to be involved in specific biological functions that require conformational changes of membrane components. The results for docosahexaenoic acid indicate that it is minimally influenced by temperature changes and that it presents great conformational variability.     

Fatty acid composition and unsaturated of intracellular membrane phospholipids from rat liver and hepatoma 27]

The fatty acid composition of phospholipids of mitochondria and microsomes from rat liver and hepatoma 27 was investigated. Basing on the fatty acid and phospholipid composition the unsaturation of the lipid bilayer of the intracellular membranes was calculated. The unsaturation of the phospholipids of the hepatoma mitochondria and microsomes was found to be much lower than that of the corresponding rat liver membranes. The lipid bilayer of the rat liver and hepatoma plasma membranes was shown to be more saturated than that of the intracellular membranes.     

Calcium-dependent activation of a multifunctional protein kinase by membrane phospholipids.

The proenzyme of a Ca2+-dependent protease-activated protein kinase previously obtained from mammalian tissues (Inoue, M., Kishimoto, A., Takai, Y., and Nishizuka, Y. (1977) J. Biol. Chem. 252, 7610-7616) was enzymatically fully active without limited proteolysis when Ca2+ and a membrane-associated factor were simultaneously present in the reaction mixture. The activation process was reversed by removing Ca2+ with ethylene glycol bis(beta-aminoethyl ether)N,N,N',N'-tetraacetic acid. An apparent Ka value for Ca2+ was less than 5 x 10(-5) M. Other divalent cations were inactive except for Sr2+, which was 5% as active as Ca2+. The factor was almost exclusively localized in membrane fractions of various tissues including brain, liver, kidney, skeletal muscle, blood cells, and adipose tissue. It was easily extractable with chloroform/methanol (2:1), and was recovered in the phospholipid fraction. In fact, this membrane factor could be replaced by chromatographically pure phosphatidylinositol, phosphatidylserine, phosphatidic acid, or diphosphatidylglycerol. Phosphatidylethanolamine, phosphatidylcholine, and sphingomyelin were far less effective under the comparable conditions. Ca2+-dependent modulator protein was unable to support enzymatic activity. The enzyme thus activated showed an ability to phosphorylate five histone fractions and muscle phosphorylase kinase, and appeared to possess multifunctional catalytic activities.     

Structural and dynamic effects of oxidatively modified phospholipids in unsaturated lipid membranes.

Phospholipid hydroperoxides and phospholipid alcohols are two of the major forms of oxidatively modified phospholipids produced during oxidant stress and lipid peroxidation. The process of lipid peroxidation is known to affect the physiological function of membranes. We, therefore, investigated the effects of lipid peroxidation products on the molecular interactions in membranes. Our study was specifically focused on the effects of lipid peroxidation products on static membrane structure (molecular orientational order) and on the reorientational dynamics of the probe molecules in lipid bilayers. The study was done by performing angle-resolved fluorescence depolarization measurements (AFD) on the fluorescent probe diphenylhexatriene (DPH) and by performing angle-resolved electron spin resonance (A-ESR) measurements on cholestane (CSL) nitroxide spin probes embedded in macroscopically oriented planar bilayers consisting of 2-10% 1-palmitoyl-2-(9/13-hydroperoxylinoleoyl)phosphatidylcholine (PLPC-OOH) or 1-palmitoyl-2-(9/13-hydroxylinoleoyl)phosphatidylcholine (PLPC-OH) in 1-palmitoyl-2-linoleoylphosphatidylcholine (PLPC) or dilinoleoylphosphatidylcholine (DLPC). Both probe molecules have rigid cylindrical geometries and report on the overall molecular order and dynamics. However, being more polar, the nitroxide spin probe CSL is preferentially located near the surface of the membrane, while the less polar fluorescent probe DPH reports preferentially near the central hydrophobic region of the lipid bilayers. The results show that the presence of relatively small amounts of oxidatively modified phospholipids within the PLPC or DLPC membranes causes pronounced structural effects as the molecular orientational order of the probe molecules is strongly decreased. In contrast, the effect on membrane reorientational dynamics is minimal.     

Molecular dynamics studies of the interface between a model membrane and an aqueous solution.

Molecular Dynamics (MD) computer simulation studies are reported for a system consisting of two model membranes in contact with an aqueous solution. The influence of the membrane on the adjacent liquid is of main interest in the present study. It is therefore attempted to make the system sufficiently large to encompass the entire region between bulk liquid and the membranes. The latter are modeled by two-dimensional arrays of COO- groups with rotational and translational degrees of freedom. The water molecules are represented by the well-tested TIP4P model. The intermolecular potentials are parametrized in terms of Coulomb interactions between partial charges on the molecular frames and empirical, mostly Lennard-Jones (12-6), interactions centered at the atomic positions. A strong layering of the liquid accompanied by an increase in average water density is found in the vicinity of the membrane. The structural perturbation reaches approximately 8 A into the liquid. We discuss the static structure in these layers in terms of atom-atom distance distribution functions and study the average orientation of the water molecule dipoles with respect to the membrane. From the distribution of the ions, we find that less than 50% of the surface charge of the membrane is neutralized by Na+ ions in the first layer above the membrane. A simplified model of the adsorption site of the ion on the membrane is developed from the distance distributions. Finally the hydration of the Na+ in the first adsorbed layer is discussed.     

Photolabelling of membrane proteins with photoactive phospholipids.

Photoactive probes have been introduced recently to study the hydrophobic sector of integral membrane proteins. A simple procedure to synthesize a new series of highly radioactive aryl azido-phospholipids is presented. They effectively exchange with the boundary lipids and, on illumination, the cross-link to several membrane proteins with high efficiency. The procedure and analysis of labelling of ATPase from sarcoplasmic reticulum is reported as an example. The advantages in using these photoactive phospholipids are discussed together with some information obtained on their use.