Lipid mobility and molecular binding in fluid lipid membranes

The lateral mobility of dilute concentrations of fluorescently labeled lipids doped into supported membranes is found to change upon receptor ligand binding at the membrane surface, even when the lipid is not directly involved in the binding event. Experiments using membrane microarrays are performed that illustrate the use of lipid mobility measurements as an effectively label-free strategy of detecting binding on membrane surfaces.     

A model for binding of inhalation anesthetics to membranes: two dose-dependent distinctly different binding modes

Inhalation anesthetics currently in clinical use, such as halothane, methoxyflurane, enflurane, isoflurane, etc., are polar hydrophobic molecules, except nitrous oxide, which is an apolar and weak anesthetic, incapable of inducing surgical stage anesthesia. Experimental data are accumulating that these potent amphipathic inhalation anesthetics preferentially bind membranes and macromolecules on the surface at clinical concentrations. The anesthetic binding to lipid membranes in the low concentration range is characterized by a saturable curve approaching to a limiting value. When the anesthetic concentration s greatly increased above the clinical range, the binding starts to exceed the limiting saturation value. Our model for anesthetic binding to membranes consists of two parts: Langmuir-type adsorption to the membrane surface at the low concentration range and penetration into the hydrophobic core at the high concentration range. The present communication provides a statistical-thermodynamic basis to analyze this twostep interaction. An expression is derived for membrane capacitance as a function of anesthetic concentration, which explains the experimental data well. Binding parameters of anesthetics are estimated according to the theory.This study was supported by NIH grants GM 25716 and GM 26950, and by the Medical Research Service of the Veterans Administration.     

Recent theoretical advances in elasticity of membranes following Helfrich's spontaneous curvature model

Recent theoretical advances in elasticity of membranes following Helfrich's famous spontaneous curvature model are summarized in this review. The governing equations describing equilibrium configurations of lipid vesicles, lipid membranes with free edges, and chiral lipid membranes are presented. Several analytic solutions to these equations and their corresponding configurations are demonstrated.     

A theoretical study of micro-domain formation in mixed lipid membranes

The thermodynamic stability of micro-clusters in a membrane built-up by charged and uncharged lipid molecules is discussed. A simple variational function is proposed in order to describe the essential structure of such lipid domains. Solvent-screened electrostatic repulsion between the lipid ionic head groups, short-range forces between the lipid hydrophobic taisl and entropic effects are taken into account. The stability conditions as well as the composition and the size of the lipid micro-domains are calculated and expressed as a function of molecular parameters for the membrane and its environment (for example, short-range forces, surface charge density of the lipid bilayer, ion concentration of the electrolyte solution in contact with the lipid membrane and temperature). As an application, the effect of micro-domain formation on the number of adsorbed ions on a charged lipid membrane has been calculated.     

Peptide mobility and peptide mapping in capillary zone electrophoresis. Experimental determination and theoretical simulation.

The electrophoretic mobilities of 58 peptides that varied in size from 2 to 39 amino acids and varied in charge from 0.65 to 7.82 are presented. The measurements were conducted at 22 degrees C using a 10% linear polyacrylamide-coated column and a 50 mM phosphate buffer at pH 2.5. Excellent separation of peptides and highly reliable peptide maps of protein digests are routinely obtained using these experimental conditions. The electrophoretic data were used to test existing theoretical models that correlate electrophoretic mobility with physical parameters. The results indicate that the Offord model that correlates electrophoretic mobility with the charge-to-size parameter q/M2/3 offers the best fit of our reliable experimental data. Furthermore, we also obtained the capillary zone electrophoretic profile of the endoproteinase Lys-C digests of a peptide sequencing standard, melittin, and horse myoglobin under the same experimental conditions as described above. The resulting peptide maps were compared with corresponding theoretical simulation.     

A theoretical analysis of micelle-mediated transport through porous membranes

A novel method is proposed for the purpose of controlled release of a sparingly water soluble compound. The solubility of a sparingly water soluble compound can be increased by addition of a sufficient amount of surfactant to form micelles. The flux of the compound across a porous membrane can be enhanced if the membrane has pores larger than the micelle size so that the compound-loaded micelles can diffuse simultaneously, and micelle-mediated transport occurs. The membrane permeability of the micelle is a monotonically decreasing function of the ratio of the size of the micelle to the membrane pore size (R_m/R_p). However, the solubilizing capacity of the micelle increases with increasing size of the micelle. These opposing effects influence the transport and may result in an optimum flux of the solubilizate at a particular size of the micelle. In the determination of the optimum surfactant molecule, the concept of the hydrophilic-lipophilic balance (HLB) is employed in order to have stable aqueous solutions of the surfactants. For a family of nonionic surfactants solubilizing the hydrophobic and hydrocarbon substance n-heptane, it is shown that there exists a maximum flux of the solubilizate at a value of R_m/R_p within the limitation of the HLB. The release rates over a long period of time are nearly constant for micelles close to the optimum size for a given pore size.     

A unified and quantitative receptor model for the microtubule binding of paclitaxel and epothilone

[formula: see text] Paclitaxel and epothilone represent the two major classes of antimicrotubule agents that promote tubulin polymerization and, presumably, mitotic arrest during cell division. A common minireceptor binding site model at beta-tubulin has been constructed for these structurally divergent compounds. Utilizing 20 amino acids identified in photoaffinity labeling experiments, the 3-D model correlates measured and predicted Ki's with r = 0.99 and rms(delta Gcalc-delta Gexp) = 0.2 kcal/mol. In addition, the model predicts the affinity of compounds not used in the training set and explains much of the SAR for the paclitaxel and epothilone families.     

A model calculation of protonic mobility in ice

The protonic conductivity in ice crystals is considered within the framework of strong coupling developed earlier by the present authors for semiconductors with narrow bands and strong coupling of electrons with phonons. The generalization for a two-band model is developed. The model used is the one described by Gosar and Pintar, the principal feature of which is the concept of the protonic conduction bands. The analytical expression for the protonic mobility is given. The results of the theory are in qualitative agreement with the experimental data.     

Effect of competing binding modes on retention in chromatography and capillary electrophoresis. A theoretical consideration

A mathematical formalism has been developed to describe equilibrium in a system involving a single selector and a selectand molecule capable of binding to this selector by different modes. The generalization of this model to a multiselector system has also been considered. Applications of the developed equations in chromatography and CE are discussed.     

Analysis of a theoretical model for anisotropic enzyme membranes application to enzyme electrodes

A theoretical model of diffusion and reaction in an anisotropic enzyme membrane is presented with particular emphasis on the application of such membranes in enzyme electrodes. The dynamic response of systems in which the kinetics are linear, which comprises the practical operating regime for enzyme electrodes in analysis, is investigated via an analytic solution of the governing differential equations. The response is presented as a function of a single dimensionless group, Μ, that is the membrane modulus.     

Interfacial mobility and bonding strength in nanocomposite thin film membranes

The interfacial interaction strength and transition properties in a reverse selective thin film nanocomposite system, silica-poly[(trimethylsilyl)propyne] (SiO(x)-PTMSP), are investigated locally by heated tip atomic force microscopy. SiO(x)-PTMSP has recently been introduced as a new class of reverse selective membrane materials with extraordinarily high permeability and selectivity (reverse selectivity). Here, we examine the thermal transition properties of the polymer matrix and the debonding strength between PTMSP and silica. Transitions at 330 degrees C were identified as degradation processes. Criteria for debonding were found to include polymer viscoelastic responses, particle size, embedding depth, scan speed, and frequency of impact. Probe-particle impact forces revealed a debonding energy of 2.6 J/m(2) and an impact force transition that occurs 30 degrees C below the degradation temperature in the neat polymer, confirming the presence of enhanced polymer mobility at the SiO(x)-PTMSP interface.     

A model of interdomain mobility in a multidomain protein

Domain mobility plays an essential role in the biological function of multidomain systems. The characteristic times of domain motions fall into the interval from nano- to milliseconds, amenable to NMR studies. Proper analysis of NMR relaxation data for these systems in solution has to account for interdomain motions, in addition to the overall tumbling and local intradomain dynamics. Here we propose a model of interdomain mobility in a multidomain protein, which considers domain reorientations as exchange/interconversion between two distinct conformational states of the molecule, combined with fully anisotropic overall tumbling. Analysis of 15N-relaxation data for Lys48-linked diubiquitin at pH 4.5 and 6.8 showed that this model adequately fits the experimental data and allows characterization of both structural and motional properties of diubiquitin, thus providing information about the relative orientation of ubiquitin domains in both interconverting states. The analysis revealed that the two domains reorient on a time scale of 9-30 ns, with the amplitudes sufficient for allowing a protein ligand access to the binding sites sequestered at the interface in the closed conformation. The analysis of a possible mechanism controlling the equilibrium between the interconverting states in diubiquitin points toward protonation of His68, which results in three different charged states of the molecule, with zero, +e, and +2e net charge. Only two of the three states are noticeably populated at pH 4.5 or 6.8, which assures applicability of the two-state model to diubiquitin at these conditions. We also compare our model with the "extended model-free" approach and discuss possible future developments of the model.     

A nonradioactive electrophoretic mobility shift assay for measurement of pregnane X receptor binding activity to CYP3A4 response element

The electrophoretic mobility shift assay (EMSA) is a method for the study of specific DNA–protein interactions in vitro. The pregnane X receptor (PRX) is a key xenobiotic sensor that regulates the expression of drug‐metabolizing enzymes and many other genes. Radiolabeled ³²P‐DNA‐probes had been used in studies of PXR‐DNA interactions. There is an increasing need for nonradioactive assays, due to the health, safety and environmental issues. In the current study, we present a protocol for the nonradioactive electrophoretic mobility shift assay, allowing studying interactions between human PXR with promoter DNA sequences.     

A simple theoretical model for dual phosphorescence

A simple theoretical model is presented to explain the observed anomalous dual phosphorescences of certain aromatic carbonyl compounds in some rigid media. The phenomenon of dual phosphorescence for large molecules violates the well-known Kasha rule stating that the emission can occur only from the lowest excited electronic state of a given multiplicity. For a small energy gap between the second triplet state (T₂) and the first triplet state (T₁), the sparse density of T₁ vibronic levels, isoenergetic with the T₂ vibrationless level, leads to a rather slow T₂ → T₁ radiationless process which is unable to quench the T₂ emission completely. Two cases of T₁ = ³nπ^*, T₂ = ³ππ^* and T₁ = ³ππ^*, T₂ = ³nπ^* are discussed at both the low-temperature and the high-temperature limits.     

Environments of ethidium binding to allosteric Dna: II. Accessibility, mobility and mode of binding

DNA binding compounds were previously shown to bind to the right-handed DNA forms and hybrid B-Z forms in a highly cooperative manner and indicate that structural specificity plays a key role in a ligand binding to DNA. In this study, the modes of binding and structural specificity of agents to unusual DNA are examined by a variety of fluorescence techniques (intensity, polarization and quenching, etc.) to explore a reliable method to detect the association environment of ligands to deoxyoligonucleotides initially containing a B-Z junction between the left-handed Z-DNA and right-handed B-DNA. The results of fluorescence energy transfer measurement demonstrated that the ligand molecules bind to the allosterically converted DNA structures by intercalation. In the absence of high-resolution structural data, this fluorescence energy transfer measurement allowed reliable measures and infer the binding environment of ligands to the allosteric DNA structures.     

Hydroxycamptothecin deactivation rates and binding to model membranes and HSA determined by fluorescence spectra analysis

Camptothecins (CPTs) are fluorescent compounds exhibiting anticancer activity. They can exist in two forms, a lactone and a carboxylate. In neutral and base solution, lactone forms hydrolyse and convert into carboxylates. Only the lactone forms of CPTs are biologically active. Because of strong affinity of the carboxylate form of the parent drug camptothecin to human serum albumin (HSA), this protein promotes the deactivation of this compound. On the other hand, the lactone forms of camptothecins do not hydrolyse and are stabilized when bound to membranes. The following three hydroxycamptothecins, 10 hydroxycamptothecin (10-OH-CPT), 7-ethyl-10-hydroxy-camptothecin (SN-38) and 7-tert-butyldimethylsil-10-hydroxycamptothecin (DB-67) were studied. Factor analysis of a set of fluorescence excitation spectra recorded during lactone hydrolysis facilitated the high-throughput determination of the deactivation rates of camptothecin and each hydroxycamptothecin in phosphate buffered saline. The fluorescence spectra of hydroxycamptothecins diluted in HSA solution or suspended in DMPC liposomes were recorded, and the association constants of these drugs to membranes and plasma proteins were calculated. Among the analysed agents, DB-67 exhibited the most desirable properties including low affinity of the carboxylate form for albumin and high affinity of its lactone form for model membranes.