Istvan P. Sugar

Monte Carlo Simulation of Two-Component Bilayers: DMPC/DSPC Mixtures*

In this paper, we describe a relatively simple lattice model of a two-component, two-state phospholipid bilayer. Application of Monte Carlo methods to this model permits simulation of the observed excess heat capacity versus temperature curves of dimyristoylphosphatidylcholine (DMPC)/distearoylphosphatidylcholine (DSPC) mixtures as well as the lateral distributions of the components and properties related to these distributions. The analysis of the bilayer energy distribution functions reveals that the gel-fluid transition is a continuous transition for DMPC, DSPC, and all DMPC/DSPC mixtures. A comparison of the thermodynamic properties of DMPC/DSPC mixtures with the configurational properties shows that the temperatures characteristics of the configurational properties correlate well with the maxima in the excess heat capacity curves rather than with the onset and completion temperatures of the gel-fluid transition. In the gel-fluid coexistence region, we also found excellent agreement between the threshold temperatures at different system compositions detected in fluorescence recovery after photobleaching experiments and the temperatures at which the percolation probability of the gel clusters is 0.36. At every composition, the calculated mole fraction of gel state molecules at the fluorescence recovery after photobleaching threshold is 0.34 and, at the percolation threshold of gel clusters, it is 0.24. The percolation threshold mole fraction of gel or fluid lipid depends on the packing geometry of the molecules and the interchain interactions. However, it is independent of temperature, system composition, and state of the percolating cluster.

Monte Carlo simulations of membranes: phase transition of small unilamellar dipalmitoylphosphatidylcholine vesicles.

Publisher Summary Monte Carlo (MC) methods have proved to be useful with model membrane systems, making it possible to simulate the fluctuating membrane conformations and calculate membrane properties and relate them to experimental observables. Correlations between particular conformational properties and complex membrane functions would aid in the interpretation of the functions and provide the basis for further experimental work. In any case, quantitative agreement between calculation and experiment is essential. This chapter describes a simple model for the gel-to-liquid crystalline transition of lipid bilayers. Bilayer membranes composed of a single lipid undergo a gel-to-liquid crystalline transition at a temperature defined by the chemical nature of the lipid, the degree of hydration, and basic structure of the membrane. This transition has been described extensively for bilayer membranes made of dipalmitoylphosphatidylcholine (DPPC). The approach is to introduce as much experimental information as possible into the calculation, assume values for a minimal number of additional parameters, and then test the results quantitatively with experimental data. The chapter reviews other MC simulations of one- and two-component membrane systems and discusses the computational methods applied.

Cholesterol superlattice modulates the activity of cholesterol oxidase in lipid membranes.

Here, the interplay between membrane cholesterol lateral organization and the activity of membrane surface-acting enzymes was addressed using soil bacteria cholesterol oxidase (COD) as a model. Specifically, the effect of the membrane cholesterol mole fraction on the initial rate of cholesterol oxidation catalyzed by COD was investigated at 37 degrees C using cholesterol/1-palmitoyl-2-oleoyl-l-alpha-phosphatidylcholine (POPC) large unilamellar vesicles (LUVs, approximately 800 nm in diameter). In the three concentration ranges examined (18.8-21.2, 23.6-26.3, and 32.2-34.5 mol % cholesterol), the initial activity of COD changed with cholesterol mole fraction in a biphasic manner, exhibiting a local maximum at 19.7, 25.0, and 33.4 mol %. Within the experimental errors, these mole fractions agree with the critical cholesterol mole fractions (C(r)) (20.0, 25.0, and 33.3) theoretically predicted for maximal superlattice formation. The activity variation with cholesterol content was correlated well with the area of regular distribution (A(reg)) in the plane of the membrane as determined by nystatin fluorescence. A similar biphasic change in COD activity was detected at the critical sterol mole fraction 20 mol % in dehydroergosterol (DHE)/POPC LUVs (approximately 168 nm in diameter). These results indicate that the activity of COD is regulated by the extent of sterol superlattice for both sterols (DHE and cholesterol) and for a wide range of vesicle sizes (approximately 168-800 nm). The present work on COD and the previous study on phospholipase A(2) (sPLA(2)) [Liu and Chong (1999) Biochemistry 38, 3867-3873] suggest that the activities of some surface-acting enzymes may be regulated by the extent of sterol superlattice in the membrane in a substrate-dependent manner. When the substrate is a sterol, as it is with COD, the enzyme activity reaches a local maximum at C(r). When phospholipid is the substrate, the minimum activity is at C(r), as is the case with sPLA(2). Both phenomena are in accordance with the sterol superlattice model and manifest the functional importance of membrane cholesterol content.

A variable target intensity-restrained global optimization (VARTIGO) procedure for determining three-dimensional structures of polypeptides from NOESY data: application to gramicidin-S.

SummaryA global optimization method for intensity-restrained structure refinement, based on variable target function (VTF) analysis, is illustrated using experimental data on a model peptide, gramicidin-S (GS) dissolved in DMSO. The method (referred to as VARTIGO for variable target intensity-restrained global optimization) involves minimization of a target function in which the range of NOE contacts is gradually increased in successive cycles of optimization in dihedral angle space. Several different starting conformations (including all-trans) have been tested to establish the validity of the method. Not all optimizations were successful, but these were readily identifiable from their large NOE R-factors. We also show that it is possible to simultaneously optimize the rotational correlation time along with the dihedral angles. The structural features of GS thus obtained from the successful optimizations are in excellent agreement with the available experimental data. A comparison is made with structures generated from an intensity-restrained single target function (STF) analysis. The results on GS suggest that VARTIGO refinement is capable of yielding better quality structures. Our work also underscores the need for a simultaneous analysis of different NOE R-factors in judging the quality of optimized structures. The NOESY data on GS in DMSO appear to provide evidence for the presence of two orientations for the ornithine side chain, in fast exchange. The NOESY spectra for this case were analyzed using a relaxation rate matrix which is a weighted average of the relaxation rate matrices for the individual conformations.

Fluorescence studies of lipid regular distribution in membranes.

This article reviews the use of fluorescent lipids and free probes in the studies of lipid regular distribution in model membranes. The first part of this article summarizes the evidence and physical properties for lipid regular distribution in pyrene-labeled phosphatidylcholine (PC)/unlabeled PC binary mixtures as revealed by the fluorescence of pyrene-labeled PC. The original and the extended hexagonal superlattice model are discussed. The second part focuses on the fluorescence studies of sterol regular distributions in membranes. The experimental evidence for sterol superlattice formation obtained from the fluorescent sterol (i.e. dehydroergosterol) and non-sterol fluorescent probes (e.g. DPH and Laurdan) are evaluated. Prospects and concerns are given with regard to the sterol regular distribution. The third part deals briefly with the evidence for polar headgroup superlattices. The emphasis of this article is placed on the new concept that membrane properties and activities, including the activities of surface acting enzymes, drug partitioning, and membrane free volume, are fine-tuned by minute changes in the concentration of bulky lipids (e.g. sterols and pyrene-containing acyl chains) in the vicinities of the critical mole fractions for superlattice formation.

Misty Mountain clustering: application to fast unsupervised flow cytometry gating

BackgroundThere are many important clustering questions in computational biology for which no satisfactory method exists. Automated clustering algorithms, when applied to large, multidimensional datasets, such as flow cytometry data, prove unsatisfactory in terms of speed, problems with local minima or cluster shape bias. Model-based approaches are restricted by the assumptions of the fitting functions. Furthermore, model based clustering requires serial clustering for all cluster numbers within a user defined interval. The final cluster number is then selected by various criteria. These supervised serial clustering methods are time consuming and frequently different criteria result in different optimal cluster numbers. Various unsupervised heuristic approaches that have been developed such as affinity propagation are too expensive to be applied to datasets on the order of 106 points that are often generated by high throughput experiments.ResultsTo circumvent these limitations, we developed a new, unsupervised density contour clustering algorithm, called Misty Mountain, that is based on percolation theory and that efficiently analyzes large data sets. The approach can be envisioned as a progressive top-down removal of clouds covering a data histogram relief map to identify clusters by the appearance of statistically distinct peaks and ridges. This is a parallel clustering method that finds every cluster after analyzing only once the cross sections of the histogram. The overall run time for the composite steps of the algorithm increases linearly by the number of data points. The clustering of 106 data points in 2D data space takes place within about 15 seconds on a standard laptop PC. Comparison of the performance of this algorithm with other state of the art automated flow cytometry gating methods indicate that Misty Mountain provides substantial improvements in both run time and in the accuracy of cluster assignment.ConclusionsMisty Mountain is fast, unbiased for cluster shape, identifies stable clusters and is robust to noise. It provides a useful, general solution for multidimensional clustering problems. We demonstrate its suitability for automated gating of flow cytometry data.

Component and State Separation in DMPC/DSPC Lipid Bilayers:A Monte Carlo Simulation Study

In this paper a two-state, two-component, Ising-type model is used to simulate the lateral distribution of the components and gel/fluid state acyl chains in dimyristoylphosphatidylcholine/distearoylphosphatidylcholine (DMPC/DSPC) lipid bilayers. The same model has been successful in calculating the excess heat capacity curves, the fluorescence recovery after photobleaching (FRAP) threshold temperatures, the most frequent center-to-center distances between DSPC clusters, and the fractal dimensions of gel clusters (Sugar, I. P., T. E. Thompson, and R. L. Biltonen, 1999. Biophys. J. 76:2099-2110). Depending on the temperature and mole fraction the population of the cluster size is either homogeneous or inhomogeneous. In the inhomogeneous population the size of the largest cluster scales with the size of the system, while the rest of the clusters remain small with increasing system size. In a homogeneous population, however, every cluster remains small with increasing system size. For both compositional and fluid/gel state clusters, threshold temperatures-the so-called percolation threshold temperatures-are determined where change in the type of the population takes place. At a given mole fraction, the number of percolation threshold temperatures can be 0, 1, 2, or 3. By plotting these percolation threshold temperatures on the temperature/mole fraction plane, the diagrams of component and state separation of DMPC/DSPC bilayers are constructed. In agreement with the small-angle neutron scattering measurements, the component separation diagram shows nonrandom lateral distribution of the components not only in the gel-fluid mixed phase region, but also in the pure gel and pure fluid regions. A combined diagram of component and state separation is constructed to characterize the lateral distribution of lipid components and gel/fluid state acyl chains in DMPC/DSPC mixtures. While theoretical phase diagrams of two component mixtures can be constructed only in the case of first-order transitions, state and component separation diagrams can be constructed whether or not the system is involved in first-order transition. The effects of interchain interactions on the component and state separation diagrams are demonstrated on three different models. The influences of state and component separation on the in-plane and off-plane membrane reactions are discussed.

Lipid Lateral Organization in Fluid Interfaces Controls the Rate of Colipase Association

Colipase, a cofactor of pancreatic triacylglycerol lipase, binds to surfaces of lipolysis reactants, like fatty acid and diacylglycerol, but not to the nonsubstrate phosphatidylcholine. The initial rate of colipase binding to fluid, single-phase lipid monolayers was used to characterize the interfacial requirements for its adsorption. Colipase adsorption rates to phosphatidylcholine/reactant mixed monolayers depended strongly on lipid composition and packing. Paradoxically, reactants lowered colipase adsorption rates only if phosphatidylcholine was present. This suggests that interactions between phosphatidylcholine and reactants create dynamic complexes that impede colipase adsorption. Complex formation was independently verified by physical measurements. Colipase binding rate depends nonlinearly on the two-dimensional concentration of phosphatidylcholine. This suggests that binding is initiated by a cluster of nonexcluded surface sites smaller than the area occupied by a bound colipase. Binding rates are mathematically consistent with this mechanism. Moreover, for each phosphatidylcholine-reactant pair, the complex area obtained from the analysis of binding rates agrees well with the independently measured collapse area of the complex. The dynamic complexes between phosphatidylcholine and lipids, like diacylglycerols, exist independently of the presence of colipase. Thus, our results suggest that lipid complexes may regulate the fluxes of other proteins to membranes during, for example, lipid-mediated signaling events in cells.

Glycolipid Acquisition by Human Glycolipid Transfer Protein Dramatically Alters Intrinsic Tryptophan Fluorescence INSIGHTS INTO GLYCOLIPID BINDING AFFINITY

Glycolipid transfer proteins (GLTPs) are small, soluble proteins that selectively accelerate the intermembrane transfer of glycolipids. The GLTP fold is conformationally unique among lipid binding/transfer proteins and serves as the prototype and founding member of the new GLTP superfamily. In the present study, changes in human GLTP tryptophan fluorescence, induced by membrane vesicles containing glycolipid, are shown to reflect glycolipid binding when vesicle concentrations are low. Characterization of the glycolipid-induced “signature response,” i.e. ∼40% decrease in Trp intensity and ∼12-nm blue shift in emission wavelength maximum, involved various modes of glycolipid presentation, i.e. microinjection/dilution of lipid-ethanol solutions or phosphatidylcholine vesicles, prepared by sonication or extrusion and containing embedded glycolipids. High resolution x-ray structures of apo- and holo-GLTP indicate that major conformational alterations are not responsible for the glycolipid-induced GLTP signature response. Instead, glycolipid binding alters the local environment of Trp-96, which accounts for ∼70% of total emission intensity of three Trp residues in GLTP and provides a stacking platform that aids formation of a hydrogen bond network with the ceramide-linked sugar of the glycolipid headgroup. The changes in Trp signal were used to quantitatively assess human GLTP binding affinity for various lipids including glycolipids containing different sugar headgroups and homogenous acyl chains. The presence of the glycolipid acyl chain and at least one sugar were essential for achieving a low-to-submicromolar dissociation constant that was only slightly altered by increased sugar headgroup complexity.

Fluorescence evidence for cholesterol regular distribution in phosphatidylcholine and in sphingomyelin lipid bilayers.

Our previous studies indicated that sterols (including cholesterol and dehydroergosterol) can be regularly distributed into hexagonal superlattices in the plane of liquid-crystalline phosphatidylcholine bilayers. It was suggested that regular and irregular regions coexist in the membrane. In the present study, we report supporting evidence for our sterol regular distribution model. We have examined the fractional concentration dependencies of dehydroergosterol (a naturally occurring cholesterol analogue) fluorescence intensity and lifetime in various phosphatidylcholine and sphingomyelin bilayers. Fluorescence intensity and lifetime dips have been observed at specific sterol mole fractions. At those mole fractions, the acrylamide quenching rate constant of dehydroergosterol fluorescence reaches a local maximum. Those mole fractions match the critical sterol mole fractions at which sterol molecules are expected to be regularly distributed into hexagonal superlattices. The results support the idea that the sterols in the regular region are embedded in the bilayer less deep than those in the irregular regions. We have also examined the fractional cholesterol concentration dependencies of diphenylhexatriene (DPH) fluorescence intensity, lifetime, and polarization in DMPC vesicles. DPH fluorescence intensity and polarization also exhibit distinct dips and peaks, respectively, at critical sterol mole fractions for hexagonal superlattices. However, DPH lifetime changes little with sterol mole fraction. As a comparison, the fluorescence properties of DHE and DPH behave differently in response to the formation of sterol regular distribution. Furthermore, finding evidence for sterol regular distribution in both phosphatidylcholine and sphingomyelin membranes raises the possibility that sterol regular distribution may occur within phospholipid/cholesterol enriched domains of real biological membranes.