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The architecture of membrane proteins in their native environment of the phospholipid bilayer is critical for understanding physiological function, but has been difficult to realize experimentally. In this communication we describe the incorporation of a membrane-anchored protein into a supported phospholipid bilayer. Cytochrome P450 2B4 solubilized and purified from the hepatic endoplasmic reticulum was incorporated into phospholipid bilayer nanostructures and oriented on a surface for visualization by atomic force microscopy. Individual P450 molecules were observed protruding from the bilayer surface. Problems associated with deformation of the protein by the atomic force microscopy probe were avoided by analyzing force-dependent height measurements to quantitate the height of the protein above the bilayer surface. Measurements of the atomic force microscopy cantilever deflection as a function of probe-sample separation reveal that the top of the P450 opposite the N-terminal membrane anchor region sits 3.5 nanometers above the phospholipid-water boundary. Models of the orientation of the enzyme are presented and discussed in relation to membrane interactions and interaction with cytochrome P450 reductase.

In the presence of a low pH environment, the channel-forming T domain of diphtheria toxin undergoes a conformational change that allows for both its own insertion into planar lipid bilayers and the translocation of the toxin's catalytic domain across them. Given that the T domain contributes only three transmembrane segments, and the channel is permeable to ions as large as [glucosamine.sup.+] and [NAD.sup.-], it would appear that the channel must be a multimer. Yet, there is substantial circumstantial evidence that the channel may be formed from a single subunit. To test the hypothesis that the channel formed by the T domain of diphtheria toxin is monomeric, we made mixtures of two T domain constructs whose voltage-gating characteristics differ, and then observed the gating behavior of the mixture's single channels in planar lipid bilayers. One of these constructs contained an [NH.sub.2]-terminal hexahistidine (H6) tag that blocks the channel at negative voltages; the other contained a COOH-terminal H6 tag that blocks the channel at positive voltages. If the channel is constructed from multiple T domain subunits, one expects to see a population of single channels from this mixture that are blocked at both positive and negative voltages. The observed single channels were blocked at either negative or positive voltages, but never both. Therefore, we conclude that the T domain channel is monomeric. KEY WORDS: planar lipid bilayers * histidine tags * voltage gating * single channels * monomer

The extent of incorporation of membrane proteins is reduced when there is a hydrophobic mismatch between membrane lipids and membrane proteins. As the mismatch increases, smaller amounts of the protein are incorporated into the lipid bilayer and larger amounts are found outside the membrane.

Cell membrane recognition is examined in terms of the structure of push-pull rods in lipid bilayer membranes.

The alpha-helical transmembrane peptide Ac-K(sub 2)L(sub 24)K(sub 2)-amide produces inverted nonlamellar phases and this effect is not affected by structural changes. It also does not appear to involve lipid-peptide hydrophobic mismatch.

Membrane proteins present a hydrophobic surface to the surrounding lipid, whereas portions protruding into the aqueous milieu expose a polar surface. But how have proteins evolved to deal with the complex environment at the membrane-water interface? Some insights have been provided by high-resolution structures of membrane proteins, and recent studies of the role of individual amino acids in mediating protein-lipid contacts have shed further light on this issue. It now appears clear that the polar-aromatic residues Trp and Tyr have a specific affinity for a region near the lipid carbonyls, whereas positively charged residues extend into the lipid phosphate region.

The EPR spectroscopic characterization of a new magnetically oriented spin-labeled model membrane system is reported.

Research into foam films stabilized by mixtures of dimyristoyl phosphatidyl chlorine and the charge lipid dimyristoyl phosphatidyl glycerol (DMPG) is presented. A rise in the film thickness with rising content of DMPG in the lipid mixture at constant ionic strength and with falling concentration of sodium chloride suggests an influence of the electrical double layer interaction in the system.

Research designed to measure the long-range self-diffusion of lipo-fullerenes intercalated in oriented multilayers of dipalmitoylphosphatidylcholine (DPPC) between 27 degrees C and 70 degrees C is presented. It was established that the lipo-fullerene diffusion was an order of magnitude slower than that of the DPPC in the host bilayer.

Research has been conducted on the mismatch between transmembrane protein hydrophobic thickness and the supporting lipid bilayer. The structural and functional consequences of this mismatch are discussed.

Research has been conducted on the poly(ethylene glycol) (PEG)-stabilized liposomes. The similarities and differences between charged and uncharged PEG-decorated liposomes have been investigated.

A new study uses Brewster angle microscopy and absorption spectroscopy to investigate the phase behavior and molecular structure of one, two, and three component lipid models of the stratum corneum comprising ceramide, hydroxy fatty acid, cholesterol and perdeuterated palmitic acid.

In gel-phase lipid membranes, the paramagnetic metal-ion centers in cytochrome c oxidase are closer vertically to the 5-position of the lipid chains than to the 14-position. They are also close enough to the first-shell lipids to create substantial dipolar interactions.

The D-Leu(super 10) residue creates the optimal orientation of Trp(super 9) in gramicidin channels in lipid bilayers. Gramicidin A has four Trp residues, which are essential for channel assembly and stability and ion transport.

Biochemical methods for determining membrane protein topology and insertion are described. Understanding the mechanisms of insertion will allow scientists to predict the three-dimensional structure of the protein from its amino acid sequence.

Research has been conducted on the passive transbilayer movement. The investigation of this movement on a planar bilayer lipid membranes that contain long-chain fatty acids is presented.

Research has been conducted on the mechanism used by bacteria to transport haem into cytoplasmic membrane. The movement of haem into and across lipid bilayers has been investigated.

A study is presented of the single-molecule dynamics of the membrane probe diIC18 dispersed in a controlled environment of lipid monolayers and bilayers of DPPC.

The adhesion energy of two apposing galactosylceramide bilayers is approximately -1.5 erg/cm(super)2. This is larger than thought and represents a strong interaction between the two layers. This is probably due to hydrogen bond formation and hydrophobic reactions between galactose headgroups.

The electrostatic binding strength of water-soluble proteins to a bilayer membrane requires charge-enriched domains that occur during the transition to the gel state. The charge of the proteins and the membrane itself are less important.

Gramicidin S (GS) generally penetrates most deeply into disordered lipid bilayers with a negative surface charge. GS only penetrates bilayers in the liquid-crystalline state and it is located at the polar/apolar region near the glycerol backbone.

The in-plane and out-of-plane structure of phospholipid monolayers were determined at the air-water interface as a function of hydrophilic lipid headgroup size. Synchrotron grazing-incidence X-ray diffraction and reflectivity were employed. Di-stearoyl-phosphatidyl-ethanolamine (DSPE) lipid monolayers were systematically modified by chemically grafting hydrophilic poly(ethylene glycol) chains to the lipid headgroups. The increased packing stresses from these bulky polymer headgroups distort the unit cell and the in-plane packing modes of the monolayers, leading to large out-of-plane changes and staggering of the lipid molecules.

Experiments with pulsed radio frequency fields have shown influence on the low-frequency behavior of lipid bilayer membranes. In this paper, we present an electromagnetic and thermal analysis of the used exposure device to clarify whether the observed effects have a thermal cause and to determine the fields at the lipid bilayer. In order to model the very thin lipid bilayer (about 5 nm) accurately, the electromagnetic analysis is broken into several steps employing the finite difference time domain technique and a finite element/boundary element hybrid approach. based on the obtained power loss due to the electromagnetic fields, the temperature change is calculated using the finite element method for the solution of the heat conduction equation. Both, the electromagnetic and the thermal analysis are performed for a variety of material parameters of the exposure device. The electromagnetic analysis shows that the exposure device is capable of producing voltages on the order of 1 mV across the lipid bilayer. The combined electromagnetic and thermal calculations reveal that the temperature oscillations due to the pulsed radio frequency fields are too small to directly influence the low-frequency behavior of the lipid bilayer. Index Terms - Bioelectric phenomena, electromagnetic fields, lipid bilayer membrane, numerical analysis, pulsed radio fields, thermal analysis, wireless communication systems.

Research was conducted to examine the effect of physicochemical forces within the lipid bilayer on the folding of the integral membrane protein bacteriorhodopsin. Three different lipid systems were developed, where each system consisted of two lipid species but the same hydrocarbon chains. Results indicate that the increase in lipid chain pressure either hinders insertion of the denatured state of bacterioopsin into the bilayer or slows a folding step within the bilayer, to the extent that an intermediate involved in the bacteriorhodopsin generation is effectively trapped.

Research was conducted to examine the calcium dependence of C-type natriuretic peptide-formed fast K+ channel using a synthetic 17-amino acid segment of a 39-amino acid C-type natriuretic peptide found in platypus venom. The dependence of the channel on high (Ca2+)(sub cis) indicates that it may become active under physiological conditions where Ca2+ levels are high and under pathological conditions that results in a CA2+ overload.

Research was conducted to examine the fragmentation reactions of n-butoxychlorocarbene, isobutoxychlorocarbene and benzyloxychlorocarbene using laser flash photolysis and product analysis studies. The rate constants for fragmentations of the carbenes were shown to increase linearly with the concentration of added Bu4NCl in pyridine-MeCN. Results are in agreement with a S(sub N)2-like mechanism for the carbene fragmentations in which the benzyl case involves the competitive incursion of S(sub N)1-like fragmentation.

Research was conducted to examine the transbilayer movement of molecular umbrellas in large unilamellar vesicles obtained from 1-palmitoyl-2-oleyol-sn-glycero-3-phosphatidylglycerol. Results cast serious doubt on the notion of a ca. 750 Da molecular weight cutoff for rapid transbilayer movement which has so far discouraged the exploration of large molecules as carriers of drugs for cellular delivery through passive transport.

Research was conducted to examine the effects of glycosylation on the structure and dynamics of eel calcitonin (CT) in micelles and lipid bilayers. Using nuclear magnetic resonance spectroscopy, the three-dimensional structures of eel CT and two glycosylated CT derivatives in micelles were determined. The unmodified and the glycosylated eel CT addresses the conformational changes and the stabilization of the peptide by the glycosylation, the topology of the peptide molecule and the orientation of the sugar moiety relative to the peptide in micelles.

A study demonstrates the use of the micro-patterning of thiol-terminated lipophilic self-assembled monolayers to support planar lipid bilayer membranes which satisfy the critical criteria required for use as potential biosensors. This new method involving the integration of microcontact printing and self-assembly of lipid vesicles enables the construction of a tethered biomimetic membrane. Experimental methods and results are discussed.

The orientation in lipid bilayers of the signal sequence of the bacterial protein LamB was examined using binding, circular dichroism and fluorescence quenching experiments. Measurements were made of binding changes due to the incorporation of lipid probes utilized in the parallax fluorescence quenching technique of determining peptide penetration depth. The signal peptide bound to a similar extent to neutral bilayers composed of either egg phosphatidylcholine or phosphatidylcholines brominated at various positions on their acyl chains.

A study on gram-negative bacterial outer membrane phospholipase A (OMPLA) activity investigated whether or not a dimerization process is responsible for triggering OMPLA activity in membranes using fluorescence resonance energy transfer. Results of the study were analyzed within the context of the regulation of OMPLA activity by the surrounding lipid phase. Experimental methodology, results and conclusions are presented.

A new membrane-spanning dimeric phosphatidyl ethanolamine molecule which readily forms stable vesicles of uniform size and a flat, thin lamellar film with very slight variation in surface features has been synthesized. The acyl chains at the 2-position of glycerol in the lipid are joined at their termini by a carbon-carbon bond, resulting in a trans-membrane type fatty acid linkage. The lipid has good thermal stability and high phase transition temperature. Results are significant in that phosphatidyl ethanolamine does not form vesicles on its own.

The aqueous-membrane partitioning of alamethicin, a voltage-gated channel-forming peptide, was assessed as a function of the membrane spontaneous curvature. The partitioning of the peptide in lipid compositions produced from dioleoylphosphatidylcholine (DOPC) and varied percentages of dioleoylphosphatidylethanolamine (DOPE), dioleoylphosphatidylethanolamine-N-methyl (DOPE-Me) or dioleoylphosphatidylethanolamine-N,N-dimethyl (DOPE-Me2). Results showed that the binding free energy for the peptide is linearly related to the spontaneous curvature.

A study was conducted to analyze the molecular mechanism of the delivery process of benzene derivatives from water to the lipid bilayer phase. A site-selective nuclear magnetic resonance method was utilized to carry out the analysis. Results indicated that the benzene derivatives penetrate into the bilayer within a few minutes after injection and are delivered deeply into the bilayer interior in the order PrBe > BzOH.

Copper(II) tetraphenylporphine (CuTPP) partitions favorably into dimyristoylphosphatidylcholine (DMPC) bilayer membranes. This was gleaned from an electron paramagnetic resonance spectra of oriented liquid-crystalline DMPC membranes, which imply an orientation of CuTPP with the plane of the molecule parallel to the bilayer surface. The change of the CuTPP orientation at a high CuTPP/DMPC molar ratio is accompanied by self-association via planar stacking of CuTPP molecules near the lipid bilayer center.

Research was conducted to examine the hypothesis that the interactions between amyloid beta-peptides (A-beta) and neuronal membranes have an important role to play in the neuropathology of Alzheimer's disease. The interactions of A-beta with ganglioside-containing membranes were analyzed using Fourier transform infrared-polarized attenuated total reflection spectroscopy and circular dichroism. Results indicate that A-beta (1-40) disturbs the function and structure of the membranes through the imposition of negative curvature strain on ganglioside-containing lipid bilayers.

Research was conducted to examine the effects of the electrostatic iberiotoxin (IbTX) mutants, namely K27N, K27Q, K27R, R34N and R34D on block of single calcium-activated (maxi-K) channels incorporated into planar lipid bilayers. The objective was to gain an insight into the electrostatic structure of the maxi-K-channel vestibule. Results demonstrate that the dissociation kinetics for IbTX K27R were enhanced by depolarizing membrane potentials.

A study relating to research on the chemistry and physics of fullerene and fullerene films was conducted to investigate the combination of fullerene chemistry and the chemistry of lipid bilayer membranes. The study describes the results of a quantitative thermodynamic approach to the binding of electrolyte cations, specifically tetraalkylammonium ions, to the electrogenerated radical anion and the dianion of the C60 moieties in a film cast on an electrode surface in aqueous media.

Research was conducted to examine the hypothesis that the polymerization of the lipid headgroups inhibits calcium-induced fusion of small unilamellar vesicles of the lipid di-n-dodecyloxypropyl beta-nitrostyryl phosphate but does not affect vesicle aggregation. A comparison of titrations of vesicles into CaCl2 in the absence and presence of LMPAM provided an accurate microcalorimetric determination of the enthalpies of vesicle aggregation and fusion. Results reveal that the driving force of membrane fusion is of entropic origin.

The properties of the beta-type antimicrobial peptide protegrin-1 were investigated. Protegrin-1 was analyzed in aligned lipid bilayers using oriented circular dichroism (OCD) and shown to exhibit two significantly distinct OCD spectra as a function of lipid composition, hydration condition and peptide concentration. These two distinct spectra allow the easy determination of the state of the peptide in aligned multilayers. However, the structural basis of the spectra cannot be determined due to lack of a theoretical approach.

An experimental protocol that can simultaneously measure spectroscopic and physiological signals from ion channels in a planar lipid bilayer was developed in order to investigate the relationship between structure and function of ion channels. A synthetic channel homologous to the S4 segment of the domain IV of the sodium channel of the electric eel was constructed and inserted in a lipid bilayer to evaluate the utility of the protocol.

The cytoplasmic membrane in Gram-positive bacteria is enveloped by a thick murein sacculus, containing as many as 100 peptidoglycan layers, preventing cells from osmotic lysis and warding off enzyme attack. Channels occur in the mycolic acid layer of the mycobacterial cell wall, and a study investigates the channel from the cell wal portion of the envelope of Corynebacterium glutamicum. A protein responsible for channel-formation layer in C. glutamicum was identified. Cell wall porin analysis showed that the cell wall channel is wide and filled with water.

Fluorescent-labeled analogues of phosphatidylcholine and phosphatidylserine were incorporated in the inner leaf of human and murine erythrocytes and their outward movement or flop was monitored in the presence of albumin. Inhibition studies and the use of erythrocytes from mutant mice revealed that the multidrug resistance protein 1 is involved in the translocation of the labeled phospholipids from the inner leaf of the membrane outwards.

Research was conducted to examine the interaction of the antimicrobial peptides Ala19-magainin 2 amide and magainin 2 amide with lipid using two lipid photolabels, azidobenzoylamido capryloyl galactosylceramide and azidobenzoyl galactosylceramide. The hydrophobic photolabeling method was used to detect magainin molecules which are inserted deeply into the bilayer. Results indicate that the use of the photolabeling method has allowed the detection of peptide monomers which inserted into the bilayer and/or formed a channel at some time during the labeling procedure.

A study was conducted to analyze the electrostatic characteristics of the dipalmitoylphosphatidylcholine bilayer in water based on molecular dynamics computations in the isothermal-isobaric ensemble. Five independent molecular dynamic computations were carried out on the bilayer system at two water concentrations at 353 K. Experimental results indicated that a polarization of the system supported anisotropic fluctuations.

A study was conducted to determine direct electrochemical proof for photoinduced porphyrin anion-cation charge separation between Mg-tetrakis(pentafluorophenyl)porphyrin and Mg-magnesium octaethylporphyrin. Experimental results indicated that photoinduced intermolecular electron transfer from the polyalkyl to polyfluoro-metalloporphyrins in the lipid bilayers correlated with a second-order rate constant.

Cryo-transmission electron microscopy and high performance liquid chromatography are used for the first time to study the dynamic interaction between phospholipase A2 (PLA2) activity and the physico-chemical properties of lipid bilayers. The characterization of PLA2 hydrolysis of phospholipid vesicles related to changes in lipid composition and morphology is studied in particular. Cryo-TEM revealed, among others, that starting in the lag phase, vesicles become perforated and degrade into open vesicles, bilayer fragments and micelles.

Activation energies for the individual steps of secretory and viral fusion are reported to be large [Oberhauser, A. F., Monck, J. R. & Fernandez, J. M. (1992) Biophys. J. 61, 800-809; Clague, M. J., Schoch, C., Zech, L. & Blumenthal, R. (1990) Biochemistry 29, 1303-1308]. Understanding the cause for these large activation energies is crucial to defining the mechanisms of these two types of biological membrane fusion. We showed recently that the fusion of protein-free model lipid bilayers mimics the sequence of steps observed during secretory and viral fusion, suggesting that these processes may involve common lipid, rather than protein, rearrangements. To test for this possibility, we determined the activation energies for the three steps that we were able to distinguish as contributing to the fusion of protein-free model lipid bilayers. Activation energies for lipid rearrangements associated with formation of the reversible first intermediate, with conversion of this to a semi-stable second intermediate, and with irreversible fusion pore formation were 37 kcal/mol, 27 kcal/mol, and 22 kcal/mol, respectively. The first and last of these were comparable to the activation energies observed for membrane lipid exchange (42 kcal/mol) during viral fusion and for the rate of fusion pore opening during secretory granule release (23 kcal/mol). This striking similarity suggests strongly that the basic molecular processes involved in secretory and viral fusion involve a set of lipid molecule rearrangements that also are involved in model membrane fusion.

Research was conducted to demonstrate that the calcium-dependent binding of annexin V to phospholipid vesicles influences the bilayer inner fluidity gradient. Phospholipids spin labeled at positions C-5, C-12 and C-16 were used to study the response of the phospholipids involved in the interaction. Results indicate that membrane rigidification seemed to parallel the crystallizing tendency of vesicle-bound annexin V but may not be involved in the calcium channeling activity of the protein.

A new and sensitive molecular probe, 2-(2[prime]hydroxyphenyl)imidazo[1,2-a] pyridine (HPIP), for monitoring structural changes in lipid bilayers is presented. Migration of HPIP from water into vesicles involves rupture of hydrogen (H) bonds with water and formation of an internal H bond once the probe is inside the vesicle. These structural changes of the dye allow the occurrence of a photoinduced intramolecular proton-transfer reaction and a subsequent twisting/rotational process upon electronic excitation of the probe. The resulting large Stokes-shifted fluorescence band depends on the twisting motion of the zwitterionic phototautomer and is characterized in vesicles of dimyristoylphosphatidylcholine and in dipalmitoyl-phosphatidylcholine at the temperature range of interest and in the presence of cholesterol. Because the fluorescence of aqueous HPIP does not interfere in the emission of the probe within the vesicles, HPIP proton-transfer/twisting motion fluorescence directly allows us to monitor and quantify structural changes within bilayers. The static and dynamic fluorescence parameters are sensitive enough to such changes to suggest this photostable dye as a potential molecular probe of the physical properties of lipid bilayers.