Incorporation of Blood-Clotting Proteins into Phospholipid Langmuir Monolayers: A Fluorescence Microscopy Study

Phospholipid monolayers adsorbed at an air-water interface are model cell membranes and have been used in this work to study interactions with blood-clotting proteins. Factor I (non-membrane binding) was used as a control protein, and its association with L-alpha-dipalmitoylphosphatidylcholine Langmuir monolayers was compared to factor VII, a membrane-binding protein. Fluorescence micrographs indicated that factor I penetration of the lipid monolayers in the phase transition region occurred extensively, causing condensation of the lipid film. The association of factor I with phospholipid monolayers was deemed nonspecific. Factor VII was shown to associate with the periphery of lipid domains in the absence of calcium ions, causing flattening of domain edges. In the presence of calcium, factor VII induced expansion of the lipid monolayer. This effect is a specific interaction attributed to exposure of hydrophobic residues upon calcium binding, followed by protein association with lipid hydrocarbon chains. Copyright 2001 Academic Press.     

Targeted drug delivery utilizing protein-like molecular architecture

Nanotechnology-based drug delivery systems (nanoDDSs) have seen recent popularity due to their favorable physical, chemical, and biological properties, and great efforts have been made to target nanoDDSs to specific cellular receptors. CD44/chondroitin sulfate proteoglycan (CSPG) is among the receptors overexpressed in metastatic melanoma, and the sequence to which it binds within the type IV collagen triple-helix has been identified. A triple-helical "peptide-amphiphile" (alpha1(IV)1263-1277 PA), which binds CD44/CSPG, has been constructed and incorporated into liposomes of differing lipid compositions. Liposomes containing distearoyl phosphatidylcholine (DSPC) as the major bilayer component, in combination with distearoyl phosphatidylglycerol (DSPG) and cholesterol, were more stable than analogous liposomes containing dipalmitoyl phosphatidylcholine (DPPC) instead of DSPC. When dilauroyl phosphatidylcholine (DLPC):DSPG:cholesterol liposomes were prepared, monotectic behavior was observed. The presence of the alpha1(IV)1263-1277 PA conferred greater stability to the DPPC liposomal systems and did not affect the stability of the DSPC liposomes. A positive correlation was observed for cellular fluorophore delivery by the alpha1(IV)1263-1277 PA liposomes and CD44/CSPG receptor content in metastatic melanoma and fibroblast cell lines. Conversely, nontargeted liposomes delivered minimal fluorophore to these cells regardless of the CD44/CSPG receptor content. When metastatic melanoma cells and fibroblasts were treated with exogeneous alpha1(IV)1263-1277, prior to incubation with alpha1(IV)1263-1277 PA liposomes, to potentially disrupt receptor/liposome interactions, a dose-dependent decrease in the amount of fluorophore delivered was observed. Overall, our results suggest that PA-targeted liposomes can be constructed and rationally fine-tuned for drug delivery applications based on lipid composition. The selectivity of alpha1(IV)1263-1277 PA liposomes for CD44/CSPG-containing cells represents a targeted-nanoDDS with potential for further development and application.     

Scanning probe lithography on fluid lipid membranes

Scanning probe lithography (SPL) is applied to pattern fluid lipid membranes on a solid borosilicate substrate. Grids of metal lines, prepatterned onto the substrate by electron beam lithography, serve to partition the supported membrane into an array of isolated fluid pixels. By toggling the pH of the surrounding solution, the effect of the probe tip on the membrane can be regulated. Alkaline conditions favor membrane removal, while neutral pH favors membrane deposition. Arbitrary membrane patterns with spatial dimensions limited by the underlying grid size can be constructed by sequential SPL membrane removal followed by refill with a different membrane type. In the present study, bilayers of unique composition fill 1 x 1 mum corrals and were positioned 100 nm apart.     

Incorporation of blood clotting factor X into phospholipid model membranes: fluorescence microscopy imaging and subphase effects surrounding the lipid phase transition region

Factor X is a blood clotting protein that associates at membrane surfaces to become activated during the coagulation cascade. A molecular level understanding of the protein-membrane phospholipid interactions has not been reached, although it is thought that the protein binds to phospholipids in the presence of calcium through a bridge with the Gla (gamma-carboxyglutamic acid) domain on the protein. In this work, phospholipid Langmuir monolayers have been utilized as model membranes to study factor X association with phospholipid membrane components. Surface pressure measurements indicate that subphase addition of sodium, magnesium, and calcium ions enhances protein penetration of the lipid monolayer, with the largest association found with calcium ions in the subphase. Fluorescence microscopy images collected after protein penetration of lipid monolayers indicate monolayer condensation in the presence of sodium and magnesium ions. Aggregation of lipid domains is induced when calcium is in the subphase, indicating binding-induced flocculation of surface lipid aggregates. Calcium binding to factor X likely causes a conformational change which allows protein-membrane interaction via hydrophobic association with lipid molecules.     

Chemical transduction with fluorescent lipid membranes using selective interactions of acetylcholine receptor with agonist/antagonist and acetylcholine with substrate

Alterations in the physical structure of vesicles and monolayers of phospholipids and soybean lecithin were monitored by measurement on the average fluorescence intensity changes from N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)dipalmitoyl-L-a-phosphatidyl ethanolamine (NBD-PE) located in the lipid matrices. This probe was intimately dispersed at a concentration of 1-2 mol-% in lipid membranes and had an emission sensitive to local environmental structure. Alterations in the structure of soybean lecithin vesicles were induced by the selective interaction of acetylcholine receptor with the agonist carbamylcholine and the antagonist alpha-bungarotoxin. Structural changes in vesicles with a 7:3 mole ratio of dipalmitoylphosphatidyl choline to dipalmitoylphosphatidic acid were observed for selective interactions between acetylcholinesterase and acetylcholine. Enhancement of fluorescence emission from the lipid membranes provided transduction of the selective binding events of the receptor and enzyme. A maximum sensitivity of about a 30% enhancement per micromole of carbamylcholine and a detection limit for the toxin of 10 nM were observed for the receptor. Fluorescence microscopy was used to establish that protein could be incorporated in monolayer lipid membranes and to provide information about potential mechanisms of fluorescence enhancement. These studies show that lipid membranes containing NBD-PE can be used as generic transducers of protein-ligand interactions.     

Design,synthesis and characterization of artificial phospholipids as model membrane receptors for specific binding of rabbit C-reactive protein

C-reactive protein (CRP) is an acute phase reactive protein, which has been shown to specifically bind to phosphorylcholine (PC) and phosphorylethanoamine (PE) moieties in the presence of calcium. In order to investigate the effect of steric hindrance on the specific binding of CRP to membranes, we designed and synthesized six phospholipids, each containing a long-arm spacer of 3, 6 or 8 atoms between the head group and hydrophobic tail. By mixing synthesized lipids and natural lipids the ligand-containing monolayers were prepared, which have PC or PE groups protruding out of the membrane surface. To characterize of the synthesized phospholipids, the thickness of the lipid monolayers was measured by surface plasmon resonance (SPR) technique, the phase behavior of the lipid monolayer at air/water interface was studied by pressure–area analysis, and the specific binding of rabbit C-reactive protein to the synthesized lipid containing membranes was studied by imaging ellipsometry.     

Modeling of biological reactions

Cell membranes consist of lipid bilayers in which proteins are embedded. Many cell functions are carried out at the cell boundary which interface with water. Here we describe the response to an anesthetic of a lipid bilayer and of an enzyme separately. While both systems are markedly affected by anesthetics at appropriately high concentrations, the result at the clinical concentrations seems best explained as principally an effect on the protein somewhat accentuated by its presence in the lipid bilayer. Thus the lipoprotein complex seems to have the properties of the protein alone, except with somewhat greater induced sensitivity due to the lipid matrix in which it is immersed.     

Association of Acenaphthoporphyrins with Liposomes for the Photodynamic Treatment of Leishmaniasis

Acenaphthoporphyrins are potential photosensitizers for photodynamic therapy, but their hydrophobicity limits their potential. Liposomes have been widely investigated as delivery vehicles that can transport hydrophobic drugs in biological systems. Here we study the association of acenaphthoporphyrins with liposomes made up of dimyristoyl phosphatidylcholine (DMPC), and to liposomes made up of a mixture of DMPC, cholesterol (Chol) and distearoyl phosphatidylglycerol (DSPG) in a 2:1:0.8 molar ratio to evaluate how liposome composition affects association constants. In liposomes consisting only of DMPC, the smaller monoacenaphthoporphyrin had the largest association constant of 5.5 × 10⁴ m ⁻¹ while the larger adj-diacenaphthoporphyrin and opp-diacenaphthoporphyrin (ODP) had smaller association constants at 1.8 × 10⁴ and 1.5 × 10⁴ m ⁻¹, respectively. The addition of liposomal Chol and DSPG has little effect on the magnitudes of the association constants. Polarization studies show that the acenaphthoporphyrins are driven far into the lipid bilayer to minimize polar–nonpolar interactions. Confocal microscopy confirms that the DMPC liposomes transport the porphyrins into promastigotes of Leishmania tarentolae. The compounds associated with DMPC:Chol:DSPG liposomes are effective in vitro against axenic and intracellular amastigotes of the pathogenic Leishmania panamensis. The effectiveness of the compounds is enhanced upon exposure of cultures to visible light.     

Aqueous self-assembly of phytantriol in ternary systems: effect of monoolein, distearoylphosphatidylglycerol and three water-miscible solvents

The aqueous phase behavior of phytantriol (PT) in mixtures of monoolein (MO), distearoylphosphatidylglycerol (DSPG), propylene glycol (PG), polyethylene glycol 400 (PEG 400) and 2-methyl-2,4-pentanediol (MPD) was investigated by visual inspection, polarized light microscopy and small angle X-ray diffraction at room temperature. The phase diagrams of PT and MO in water are qualitatively very similar and PT/MO mixtures in excess water form one cubic phase of space group Pn3m irrespective of mixing ratio. The addition of the charged membrane lipid DSPG to the PT system gives rise to a considerable water swelling of the cubic phases as well as the occurrence of a cubic phase of space group Im3m. Whereas all three solvents studied give rise to a sponge (L3) phase in the MO-water system, this phase was only found when MPD was added to the PT-water system. The results are discussed with respect to the chemical differences between PT and MO.     

Ion transport across model lipid membranes containing light-harvesting complex II: an effect of light

The effect of light on proton transport across lipid membranes of small unilamellar liposomes containing incorporated major light-harvesting pigment-protein complex of Photosystem II (LHCII) has been studied with the application of pH-sensitive dyes entrapped inside vesicles. Proton permeability coefficient for LHCII-modified membranes was found to be about twice as high as in the case of the control pure lipid vesicles. Illumination of the samples with light absorbed by the LHCII-bound photosynthetic pigments considerably affects the kinetics of proton transport: it increases the rate and decreases the steady-state level of proton gradient across the membranes. The effect was interpreted in terms of heat-induced conformational changes of LHCII molecular structures that affect proton buffering capacity of this protein. Both the control and the LHCII-modified lipid membranes have been found to be practically impermeable to Ca(++) ions, as demonstrated by fluorescence of liposome-entrapped calcium-sensitive probe calcium crimson. The slight differences in the proton transport across the LHCII-containing membranes under the presence of Ca(++) suggest calcium binding to this antenna protein.     

Pore structure, thinning effect, and lateral diffusive dynamics of oriented lipid membranes interacting with antimicrobial peptide protegrin-1: 31P and 2H solid-state NMR study

Membrane pores that are induced in oriented membranes by an antimicrobial peptide (AMP), protegrin-1 (PG-1), are investigated by (31)P and (2)H solid state NMR spectroscopy. We incorporated a well-studied peptide, protegrin-1 (PG-1), a beta-sheet AMP, to investigate AMP-induced dynamic supramolecular lipid assemblies at different peptide concentrations and membrane compositions. Anisotropic NMR line shapes specifying toroidal pores and thinned membranes, which are formed in membrane bilayers by the binding of AMPs, have been analyzed for the first time. Theoretical NMR line shapes of lipids distributed on the surface of toroidal pores and thinned membranes reproduce reasonably well the line shape characteristics of our experimentally measured (31)P and (2)H solid-state NMR spectra of oriented lipids binding with PG-1. The lateral diffusions of lipids are also analyzed from the motionally averaged one- and two-dimensional (31)P and (2)H solid-state NMR spectra of oriented lipids that are binding with AMPs.     

Probing the reaction mechanism of spore photoproduct lyase (SPL) via diastereoselectively labeled dinucleotide SP TpT substrates

5-Thyminyl-5,6-dihydrothymine (commonly called spore photoproduct or SP) is the exclusive DNA photodamage product in bacterial endospores. It is generated in the bacterial sporulation phase and repaired by a radical SAM enzyme, spore photoproduct lyase (SPL), at the early germination phase. SPL utilizes a special [4Fe-4S] cluster to reductively cleave S-adenosylmethionine (SAM) to generate a reactive 5'-dA radical. The 5'-dA radical is proposed to abstract one of the two H-atoms at the C6 carbon of SP to initiate the repair process. Via organic synthesis and DNA photochemistry, we selectively labeled the 6-H(proS) or 6-H(proR) position with a deuterium in a dinucleotide SP TpT substrate. Monitoring the deuterium migration in enzyme catalysis (employing Bacillus subtilis SPL) revealed that it is the 6-H(proR) atom of SP that is abstracted by the 5'-dA radical. Surprisingly, the abstracted deuterium was not returned to the resulting TpT after enzymatic catalysis; an H-atom from the aqueous buffer was incorporated into TpT instead. This result questions the currently hypothesized SPL mechanism which excludes the involvement of protein residue(s) in SPL reaction, suggesting that some protein residue(s), which is capable of exchanging a proton with the aqueous buffer, is involved in the enzyme catalysis. Moreover, evidence has been obtained for a possible SAM regeneration after each catalytic cycle; however, such a regeneration process is more complex than currently thought, with one or even more protein residues involved as well. These observations have enabled us to propose a modified reaction mechanism for this intriguing DNA repair enzyme.     

Relative retention of the fibroblast growth factors FGF-1 and FGF-2 on strong cation-exchange sorbents

The isocratic retention of two heparin-binding fibroblast growth factors, FGF-1 (acidic FGF) and FGF-2 (basic FGF), was compared on a set of six preparative strong cation-exchange adsorbents. The FGFs comprise a solute pair that are structurally equivalent, yet differ in protein parameters of potential importance in cation-exchange chromatography, such as isoelectric point, net charge, and the number and distribution of basic amino acids. The cation-exchange adsorbents comprise a diverse set of materials in common use for protein purification, with physical and chemical properties that have been characterized and described previously. Isocratic k' values for the two proteins obtained on each adsorbent at several different [NaCl] are compared with one another and with corresponding data for hen egg lysozyme, which is also strongly retained on cation-exchangers. Of the six adsorbents examined, three showed strong retention of both FGFs, with equivalent k' values for FGF-1 and FGF-2. Three others, which showed weaker overall retention for the FGF pair, showed much larger retention differences between FGF-1 and FGF-2. The trends in retention order among the stationary phases are very similar to those seen previously with other unrelated proteins. However, retention differences between the two FGFs, and between the FGFs and lysozyme, do not correlate well with simple charge properties such as net charge, indicating, as in some previous studies, the importance of local regions on the protein surface in determining retention. These observations are interpreted in terms of the structural features of the proteins and the physicochemical properties of the adsorbents.     

Investigation of finite system-size effects in molecular dynamics simulations of lipid bilayers

In the absence of external stress, the surface tension of a lipid membrane vanishes at equilibrium, and the membrane exhibits long wavelength undulations that can be described as elastic (as opposed to tension-dominated) deformations. These long wavelength fluctuations are generally suppressed in molecular dynamics simulations of membranes, which have typically been carried out on membrane patches with areas <100 nm2 that are replicated by periodic boundary conditions. As a result, finite system-size effects in molecular dynamics simulations of lipid bilayers have been subject to much discussion in the membrane simulation community for several years, and it has been argued that it is necessary to simulate small membrane patches under tension to properly model the tension-free state of macroscopic membranes. Recent hardware and software advances have made it possible to simulate larger, all-atom systems allowing us to directly address the question of whether the relatively small size of current membrane simulations affects their physical characteristics compared to real macroscopic bilayer systems. In this work, system-size effects on the structure of a DOPC bilayer at 5.4 H2O/lipid are investigated by performing molecular dynamics simulations at constant temperature and isotropic pressure (i.e., vanishing surface tension) of small and large single bilayer patches (72 and 288 lipids, respectively), as well as an explicitly multilamellar system consisting of a stack of five 72-lipid bilayers, all replicated in three dimensions by using periodic boundary conditions. The simulation results are compared to X-ray and neutron diffraction data by using a model-free, reciprocal space approach developed recently in our laboratories. Our analysis demonstrates that finite-size effects are negligible in simulations of DOPC bilayers at low hydration, and suggests that refinements are needed in the simulation force fields.     

pH-sensitive polymer-assisted refolding of urea-denatured fibroblast growth factor

A pH-responsive polymer Eudragit S-100 has been found to assist in correct folding of FGF-2(fibroblast growth factor-2) denatured with 8 mol/L urea and 10 mmol/L dithiothreitol at pH 7.2.The refolding of FGF-2 was performed by directly diluting denatured FGF-2 into a refolding buffer containing Eudragit S-100.The ability of Eudragit S-100 to enhance protein refolding level was investigated using MTr method,fluorescence emission spectroscopy and reverse phase HPLC.On the other hand,the result shows the ab...     

Potential of mean force and pKa profile calculation for a lipid membrane-exposed arginine side chain

The issue of ionizable protein side chains interacting with lipid membranes has been the focus of much attention since the proposal of the paddle model of voltage-gated ion channels, which suggested multiple arginine (Arg) side chains may move through the hydrocarbon core of a lipid membrane. Recent cell biology experiments have also been interpreted to suggest that these side chains would face only small free energy penalties to cross membranes, challenging a long-standing view in membrane biophysics. Here, we employ side chain analog and transmembrane helix models to determine the free energy of an Arg side chain, as a function of protonation state, across a membrane. We observe high free energy barriers for both the charged and neutral states that would prohibit lipid-exposed movement. The mechanisms for charged and neutral Arg transport are, however, very different, with the neutral state experiencing simple dehydration, whereas the charged state experiences a complex mechanism involving connections to the bilayer interfaces that deform the local membrane structure. We employ special methods to ensure sampling of these interfacial connections and decompose the free energy to shed light on the mechanisms. These deformations are found to preferentially stabilize the protonated form, such that the Arg side chain remains almost exclusively charged inside the membrane, with a pKa shift of 相似文献   

High fluidity and soft elasticity of the inner membrane of Escherichia coli revealed by the surface rheology of model Langmuir monolayers

We have studied the equilibrium and linear mechanical properties of model membranes of Escherichia coli built up as Langmuir monolayers of a native lipid extract using surface thermodynamics, fluorescence microscopy, and surface rheology measurements. The experimental study has been carried out at different temperatures across the physiological operative range 15-37 degrees C. Lipid phase coexistence has been revealed over a broad pressure range by fluorescence microscopy. The presence of ordered domains has been invoked to explain the emergence of shear elasticity accompanying the hydrostatic compression elasticity typical of fluid monolayers. The surface rheology measurements point out the soft character of E. coli membranes; i.e., upon deformation they react as a near-ideal compliant body with minimal energy dissipation, thus optimizing the effectiveness of external stresses in producing membrane deformations. These mechanical features appear to be independent of temperature, suggesting the existence of a passive thermoregulation mechanism.     

Lateral organization of a membrane protein in a supported binary lipid domain: direct observation of the organization of bacterial light-harvesting complex 2 by total internal reflection fluorescence microscopy

A unique method is described for directly observing the lateral organization of a membrane protein (bacterial light-harvesting complex LH2) in a supported lipid bilayer using total internal reflection fluorescence (TIRF) microscopy. The supported lipid bilayer consisted of anionic 1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1'-glycerol)] (DOPG) and 1,2-distearoly-sn-3-[phospho-rac-(1'-glycerol)] (DSPG) and was formed through the rupture of a giant vesicle on a positively charged coverslip. TIRF microscopy revealed that the bilayer was composed of phase-separated domains. When a suspension of cationic phospholipid (1,2-dioleoyl-sn-glycero-3-ethylphosphocholine: EDOPC) vesicles (approximately 400 nm in diameter), containing LH2 complexes (EDOPC/LH2 = 1000/1), was put into contact with the supported lipid bilayer, the cationic vesicles immediately began to fuse and did so specifically with the fluid phase (DOPG-rich domain) of the supported bilayer. Fluorescence from the incorporated LH2 complexes gradually (over approximately 20 min) spread from the domain boundary into the gel domain (DSPG-rich domain). Similar diffusion into the domain-structured supported lipid membrane was observed when the fluorescent lipid (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-lissamine-rhodamine B sulfonyl: N-Rh-DOPE) was incorporated into the vesicles instead of LH2. These results indicate that vesicles containing LH2 and lipids preferentially fuse with the fluid domain, after which they laterally diffuse into the gel domain. This report describes for first time the lateral organization of a membrane protein, LH2, via vesicle fusion and subsequent lateral diffusion of the LH2 from the fluid to the gel domains in the supported lipid bilayer. The biological implications and applications of the present study are briefly discussed.     

Chemically induced phospholipid translocation across biological membranes

Chemical means of manipulating the distribution of lipids across biological membranes is of considerable interest for many biomedical applications as a characteristic lipid distribution is vital for numerous cellular functions. Here we employ atomic-scale molecular simulations to shed light on the ability of certain amphiphilic compounds to promote lipid translocation (flip-flops) across membranes. We show that chemically induced lipid flip-flops are most likely pore-mediated: the actual flip-flop event is a very fast process (time scales of tens of nanoseconds) once a transient water defect has been induced by the amphiphilic chemical (dimethylsulfoxide in this instance). Our findings are consistent with available experimental observations and further emphasize the importance of transient membrane defects for chemical control of lipid distribution across cell membranes.     

Coupled membrane fluctuations and protein mobility in supported intermembrane junctions

Junctions between lipid membranes make possible cell-free explorations of physical mechanisms that can contribute to protein and lipid organization at a variety of biophysical interfaces. Recent studies of mobile antibodies sandwiched between lipid bilayer membranes have shown that strong intermembrane adhesion and protein mobility alone are sufficient to drive inert proteins into micron-scale patterns of dense and sparse zones. Though the length scale of these patterns was suspected to be related to membrane rigidity, a quantitative understanding has so far been unavailable. We introduce data showing radially structured protein patterns that also demonstrate micron-scale organization. We then provide a simple model that relates the spectrum of membrane fluctuations to the observed protein distributions; in brief, only membrane modes that are slow enough to couple to the protein mobility drive intermembrane protein patterns.