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1.
A systematic study of the influence of the packing density of proteins on their activity is performed with cytochrome c oxidase (CcO) from R. sphaeroides as an example. The protein was incorporated into a protein-tethered bilayer lipid membrane and CcO was genetically engineered with a histidine-tag, attached to Subunit II, and then tethered by an interaction with functionalized thiol compounds bound to a gold electrode. The packing density was varied by diluting the functionalized thiol with a nonfunctionalized thiol that does not bind to the enzyme. After attaching the CcO to the gold surface, a lipid bilayer was formed to incorporate the tethered proteins. The reconstituted protein-lipid bilayer was characterized by surface enhanced infrared reflection absorption spectroscopy (SEIRAS), electrochemical impedance spectroscopy, surface plasmon resonance, and atomic force microscopy. The activity of the proteins within the reconstituted bilayer was probed by direct electrochemical electron injection and was shown to be very sensitive to the packing density of protein molecules. At low surface density of CcO, the bilayer did not effectively form, and protein aggregates were observed, whereas at very high surface density, very little lipid is able to intrude between the closely packed proteins. In both of these cases, redox activity, measured by the efficiency to accept electrons, is low. Redox activity of the enzyme is preserved in the biomimetic structure but only at a moderate surface coverage in which a continuous lipid bilayer is present and the proteins are not forced to aggregate. Electrostatic and other interaction forces between protein molecules are held responsible for these effects.  相似文献   

2.
Membrane‐bound c‐Src non‐receptor tyrosine kinase, unlike other acyl‐modified lipid‐anchored proteins, anchors to the membrane by a myristoyl chain along with a polybasic residue stretch, which is shorter in chain length than its host membrane. The packing defect arising from this mismatched chain length of the host and the lipid anchor significantly affects the lateral organization of heterogeneous membranes. We reveal the mixing of phase domains and formation of novel nanoscale‐clusters upon membrane binding of the Myr‐Src (2–9) peptide. Fluorescence cross correlation spectroscopy was used to explore the nature of these clusters. We show that Myr‐Src (2–9) is able to oligomerize, and the peptide clusters are embedded in a lipid platform generated by lipid sorting. Further, using confocal fluorescence microscopy and FRET assays we show that localized charge enrichment and membrane curvature are able to shift the partition coefficient towards the more ordered lipid phase.  相似文献   

3.
Membrane proteins have evolved to fold and function in a lipid bilayer, so it is generally assumed that their stability should be optimized in a natural membrane environment. Yet optimal stability is not always in accord with optimization of function, so evolutionary pressure, occurring in a complex membrane environment, may favor marginal stability. Here, we find that the transmembrane helix dimer, glycophorin A (GpATM), is actually much less stable in the heterogeneous environment of a natural membrane than it is in model membranes and even common detergents. The primary destabilizing factors are electrostatic interactions between charged lipids and charged GpATM side chains, and nonspecific competition from other membrane proteins. These effects overwhelm stabilizing contributions from lateral packing pressure and excluded volume. Our work illustrates how evolution can employ membrane composition to modulate protein stability.  相似文献   

4.
Amphipathic surface-active helices enable peripheral proteins to perform a variety of important cellular functions such as: lipid association and transport, membrane perturbation and disruption in programmed cell death or antimicrobial activity, and signal transduction. Amphipathic helices that adopt a surface-active membrane location are also found in transmembrane proteins. Since they possess similar amino acid composition and therefore chemical and physical properties, it seems intuitively obvious that the specific role of these surface seeking, or horizontal helices in membrane spanning proteins in some ways parallel those of their cousins in peripheral proteins. This review compares research literature and data from both proteins sets (peripheral proteins and transmembrane) to examine this assumption. Furthermore, since the occurrence of surface-active/seeking helices in transmembrane protein structure is often omitted from comment in the literature, a brief survey of their apparent roles in transmembrane protein/lipid stabilization, microenvironment enclosure and signal transduction is offered here.  相似文献   

5.
Lateral lipid phase separation of titratable PS or PA lipids and their assembly in domains induced by changes in pH are significant in liposome-based drug delivery: environmentally responsive lipid heterogeneities can be tuned to alter collective membrane properties such as permeability (altering drug release) and surface topography (altering drug carrier reactivity) impacting, therefore, the therapeutic outcomes. At the micrometer scale fluorescence microscopy on giant unilamellar fluid vesicles (GUVs) shows that lowering pH (from 7.0 to 5.0) promotes condensation of titratable PS or PA lipids into beautiful floret-shaped domains in which lipids are tightly packed via hydrogen-bonding and van der Waals interactions. The order of lipid packing within domains increases radially toward the domain center. Lowering pH enhances the lipid packing order, and at pH 5.0 domains appear to be entirely in the solid (gel) phase. Domains phenomenologically comprise a circular "core" cap beyond which interfacial instabilities emerge resembling leaf-like stripes. At pH 5.0 stripes are of almost vanishing Gaussian curvature independent of GUVs' preparation path and in agreement with a general condensation mechanism. Increasing incompressibility of domains is strongly correlated with a larger number of thinner stripes per domain and increasing relative rigidity of domains with smaller core cap areas. Line tension drives domain ripening; however, the final domain shape is a result of enhanced incompressibility and rigidity maximized by domain coupling across the bilayer. Introduction of a transmembrane osmotic gradient (hyperosmotic on the outer lipid leaflet) allows the domain condensation process to reach its maximum extent which, however, is limited by the minimal expansivity of the continuous fluid membrane.  相似文献   

6.
The synthetic peptide acetyl-K(2)-G-L(24)-K(2)-A-amide (P(24)) and its analogs have been successfully utilized as models of the hydrophobic transmembrane alpha-helical segments of integral membrane proteins. The central polyleucine region of these peptides was designed to form a maximally stable, very hydrophobic alpha-helix which will partition strongly into the hydrophobic environment of the lipid bilayer core, while the dilysine caps were designed to anchor the ends of these peptides to the polar surface of the lipid bilayer and to inhibit the lateral aggregation of these peptides. Moreover, the normally positively charged N-terminus and the negatively charged C-terminus have both been blocked in order to provide a symmetrical tetracationic peptide, which will more faithfully mimic the transbilayer region of natural membrane proteins and preclude favorable electrostatic interactions. In fact, P(24) adopts a very stable alpha-helical conformation and transbilayer orientation in lipid model membranes. The results of our recent studies of the interaction of this family of alpha-helical transmembrane peptides with phospholipid bilayers are summarized here.  相似文献   

7.
Lipid membrane nanotechnology can play a key role in preserving the function of transmembrane proteins on biofunctional substrates. We show here that rational nanoscopic actuation of a polymer-tethered lipid bilayer can be achieved by modulating the dielectric environment at the membrane-substrate interface. This provides a hydrated platform with increased lipid mobility compared to bilayers supported directly onto silica. We suggest that this construct may be used for promoting the functional reconstitution of transmembrane proteins on planar surfaces for bioanalytical devices.  相似文献   

8.
Despite the rapid and enormous progress in biotechnologies, the biochemical analysis of membrane proteins is still a difficult task. The presence of the large hydrophobic region buried in the lipid bilayer membrane (transmembrane domain) makes it difficult to analyze membrane proteins in standard assays developed for water-soluble proteins. To handle membrane proteins, the lipid bilayer membrane may be used as a platform to sustain their functionalities. Relatively slow progress in developing micro total analysis systems (μTAS) for membrane protein analysis directly reflects the difficulty of handling lipid membranes, which is a common problem in bulk measurement technologies. Nonetheless, researchers are continuing to develop efficient and sensitive analytical microsystems for the study of membrane proteins. Here, we review the latest developments, which enable detection of events caused by membrane proteins, such as ion channel current, membrane transport, and receptor/ligand interaction, by utilizing microfabricated structures. High-throughput and highly sensitive detection systems for membrane proteins are now becoming a realistic goal. Although most of these systems are still in the early stages of development, we believe this field will become one of the most important applications of μTAS for pharmaceutical and clinical screenings as well as for basic biochemical research.  相似文献   

9.
Design and characterization of helical ribbon assemblies of a bolaamphiphilic conjugated polymer and their color-coded transformation into nanofibers are described. An L-glutamic acid modified bolaamphiphilic diacetylene lipid was synthesized and self-assembled into right-handed helical ribbons with micron scale length and nano scale thickness under mild conditions. The ribbon structures were further stabilized by polymerizing well-aligned diacetylene units to form bisfunctional polydiacetylenes (PDAs). Transitions from flat sheets to helical ribbons and tubes were observed by transmission electron microscopy. The helical ribbons appear to originate from the rupture of flat sheets along domain edges and the peeling off between stacked lipid layers. These results point to the applicability of chiral packing theory in bolaamphiphilic supramolecular assemblies. Contact mode atomic force microscopy observations revealed that high order existed in the surface packing arrangement. Hexagonal and pseudorectangular packings were observed in flat and twisted regions of the ribbons, respectively, suggesting a correlation between microscopic morphologies and nanoscopic packing arrangements. The tricarboxylate functionalities of the bolaamphiphilic lipid provide a handle for the manipulation of the bisfunctional PDAs' morphology. Increasing solution pH caused the fraying of helical ribbons into nanofibers accompanied by a sharp blue-to-red chromatic transition. A dramatic change in circular dichroism spectra was observed during this process, suggesting the loss of chirality in packing. A model is proposed to account for the pH-induced morphological change and chromatic transition. The color-coded transition between two distinct microstructures would be useful in the design of sensors and other "smart" nanomaterials requiring defined molecular templates.  相似文献   

10.
Physicochemical properties of the plasma membrane have been shown to play an important role in cellular functionality. Among those properties, the molecular order of the lipids, or the lipid packing, is of high importance. Changes in lipid packing are believed to compartmentalize cellular signaling by initiating coalescence and conformational changes of proteins. A common way to infer membrane lipid packing is by using membrane‐embedded polarity‐sensitive dyes, whose emission spectrum is dependent on the molecular order of the immediate membrane environment. Here, we report on an improved determination of such spectral shifts in the emission spectrum of the polarity‐sensitive dyes. This improvement is based on the use of spectral imaging on a scanning confocal fluorescence microscope in combination with an improved analysis, which considers the whole emission spectrum instead of just single wavelength ranges. Using this approach and the polarity‐sensitive dyes C‐Laurdan or Di‐4‐ANEPPDHQ, we were able to image—with high accuracy—minute differences in the lipid packing of model and cellular membranes.  相似文献   

11.
As a small tetrameric helical membrane protein, the M2 proton channel structure is highly sensitive to its environment. As a result, structural data from a lipid bilayer environment have proven to be essential for describing the conductance mechanism. While oriented sample solid-state NMR has provided a high-resolution backbone structure in lipid bilayers, quaternary packing of the helices and many of the side-chain conformations have been poorly restrained. Furthermore, the quaternary structural stability has remained a mystery. Here, the isotropic chemical shift data and interhelical cross peaks from magic angle spinning solid-state NMR of a liposomal preparation strongly support the quaternary structure of the transmembrane helical bundle as a dimer-of-dimers structure. The data also explain how the tetrameric stability is enhanced once two charges are absorbed by the His37 tetrad prior to activation of this proton channel. The combination of these two solid-state NMR techniques appears to be a powerful approach for characterizing helical membrane protein structure.  相似文献   

12.
According to their intramolecular arrangement and position in a cell, membrane proteins are generally classified into the following six types: (1) type I transmembrane, (2) type II transmembrane, (3) multipass transmembrane, (4) lipid chain-anchored membrane, (5) GPI-anchored membrane, and (6) peripheral membrane. Situated in a heteropolar environment, these six types of membrane proteins must have quite different amphiphilic sequence-order patterns in order to stabilize their respective frameworks. To incorporate such a feature into the predictor, the amphiphilic pseudo amino acid composition has been formulated that contains a series of hydrophobic and hydrophilic correlation factors. The success rates thus obtained have been remarkably enhanced in identifying the types of membrane proteins, as demonstrated by the jackknife test and independent data set test, respectively.  相似文献   

13.
An artificial cell membrane that is composed of bilayer lipid membranes (BLMs) with transmembrane proteins incorporated within them represents a well‐defined system for the analysis of membrane proteins, especially ion channel proteins that are major targets for drug design. Because the BLM system has a high compatibility with recently developed cell‐free expression systems, it has attracted attention as a next‐generation drug screening system. However, three issues associated with BLM systems, i. e., their instability, the need for non‐volatile organic solvents and a low efficiency of ion channel incorporation, have limited their use as a drug screening platform. In this personal account, we discuss our recent approaches to address these issues based on microfabrication. We also discuss the potential for using the BLM system combined with cell‐free expression systems as a drug screening system for future personalized medicine.  相似文献   

14.
Bezrukov SM  Rand RP  Vodyanoy I  Parsegian VA 《Faraday discussions》1998,(111):173-83; discussion 225-46
Lipid membranes are not passive, neutral scaffolds to hold membrane proteins. In order to examine the influence of lipid packing energetics on ion channel expression, we study the relative probabilities of alamethicin channel formation in dioleoylphosphatidylserine (DOPS) bilayers as a function of pH. The rationale for this strategy is our earlier finding that the higher-conductance states, corresponding to larger polypeptide aggregates, are more likely to occur in the presence of lipids prone to hexagonal HII-phase formation (specifically DOPE), than in the presence of lamellar L alpha-forming lipids (DOPC). In low ionic strength NaCl solutions at neutral pH, the open channel in DOPS membranes spends most of its time in states of lower conductance and resembles alamethicin channels in DOPC; at lower pH, where the lipid polar groups are neutralized, the channel probability distribution resembles that in DOPE. X-Ray diffraction studies on DOPS show a progressive decrease in the intrinsic curvature of the constituent monolayers as well as a decreased probability of HII-phase formation when the charged lipid fraction is increased. We explore how proton titration of DOPS affects lipid packing energetics, and how these energetics couple titration to channel formation.  相似文献   

15.
Proteins imbedded in solid-supported lipid bilayers can serve as model systems for investigations of cellular membranes and protein behavior on surfaces. We have investigated the self-assembly of streptavidin on mica-supported bilayer membranes. Using fluorescence microscopy and atomic force microscopy, our studies reveal that the concentration of surface ligand influences the molecular packing of the resulting protein arrays, which in turn affects overall crystal morphology. Two-dimensional streptavidin crystals are obtained when the biotinylated lipid density on the substrate reaches 1.5% mole fraction, yielding high-aspect morphologies that comprise primarily of crystals with P1 symmetry. At 3% and above, crystals with C222 symmetry are formed and result in H-shaped and confluent structures. In intermediate densities between 2 and 3%, a coexistence of P1 and C222 crystal forms is observed. The relationship between macroscopic morphology and molecular configuration is similar to previously reported data obtained at the air/water interface. This suggests that, under our experimental conditions, protein interactions with the supporting substrate are less significant for defining self-assembly behavior than interactions between protein molecules. Ligand-inhibition and fluorescence recovery after photobleaching were used to elucidate the concentration-dependent mechanism for the divergent crystal forms. We have measured the diffusion coefficient of molecules in P1-forming conditions to be approximately twice that of molecules in C222-forming concentrations, which is consistent with proteins bound to the surface through one and two ligands, respectively. The differential flexibility associated with the binding state is therefore likely to alter the subtle protein interactions involved in crystallization.  相似文献   

16.
Phase-contrast transmission electron microscopy (PC-TEM) and quick freezing method have been combined to study the initial growing process of a self-assembled lipid nanotube in water. The PC-TEM enabled us to detect thin lamellar edge structure and the very fast growth of the newborn edge to a thin tube with high contrast. The thin tube acts as a core structure for further growth into thick complete lipid nanotube. The initially formed nanotube structure is denoted as a "core tube". The core tube has uniform wall structure that consists of five lamellar layers and the inner and outer diameters of the core tube are 130 and 180 nm, respectively. The evaluated lamellar spacing of 4.6 nm is well compatible with that measured by X-ray diffraction. We also discussed the molecular packing of the nanotube from the pitch angle determined by the PC-TEM images, X-ray diffraction pattern in wide-angle region, and IR spectroscopy. The subcell structure of the nanotube is assigned to an orthorhombic type. The twisting angle between the neighboring lipid molecules is determined as ca. 0.26 degrees for the first time; it is a crucial parameter for the formation of a lipid nanotube in chiral packing but has not been elucidated before.  相似文献   

17.
Intrinsically disordered proteins (IDPs) are proteins that possess large unstructured regions. Their importance is increasingly recognized in biology but their characterization remains a challenging task. We employed field swept Electron Spin Echoes in pulsed EPR to investigate low-temperature stochastic molecular librations in a spin-labeled IDP, casein (the main protein of milk). For comparison, a spin-labeled globular protein, hen egg white lysozyme, is also investigated. For casein these motions were found to start at 100 K while for lysozyme only above 130 K, which was ascribed to a denser and more ordered molecular packing in lysozyme. However, above 120 K, the motions in casein were found to depend on temperature much slower than those in lysozyme. This abrupt change in casein was assigned to an ordering transition in which peptide residues rearrange making the molecular packing more rigid and/or more cohesive. The found features of molecular motions in these two proteins turned out to be very similar to those known for gel-phase lipid bilayers composed of conformationally ordered and conformationally disordered lipids. This analogy with a simpler molecular system may appear helpful for elucidation properties of molecular packing in IDPs.  相似文献   

18.
We utilize supported lipid/protein bilayers to probe the dimerization of transmembrane (TM) helices in a membrane environment. The bilayers are formed by incubating substrates with liposomes containing the proteins, and are characterized using fluorescence recovery after photobleaching and imaging Forster resonance energy transfer (FRET). We show that the FRET signal, as a measure of TM helix dimerization, is the same in suspended liposomes and in surface-supported bilayers. This work is the first step toward the development of a new tool for probing the association of TM helices in lipid bilayers.  相似文献   

19.
The impact of in-line coagulation pre-treatment of secondary effluent on the operation of an immersed hollow-fibre ultrafiltration membrane pilot was evaluated as part of a larger study on optimising phosphorus removal. The efficacy of alum and ferric chloride was investigated, with an emphasis on alum use. Both coagulants were found to shift the particle-size distribution of organic matter in the feed towards larger fractions, with a notable reduction in colloidal matter. This was reflected in a reduction of both average daily transmembrane pressure increases, as well as a reduction of transmembrane pressure increases within backpulse intervals. Fouling reduction was observed with both lower and higher membrane packing density modules (membrane surface areas of 55.7 and 62.7 m2/module). The results of one-way analysis of variance (ANOVA) testing indicate that for this pilot system, chemical pre-treatment and solids concentrations in the feed water played a statistically significant role in determining transmembrane pressure variations. Membrane packing density and membrane production method did not exhibit a statistically significant effect on transmembrane pressure under the conditions of this study.  相似文献   

20.
Although lipid phases are routinely studied by X-ray diffraction, construction of their unit cell structures from the diffraction data is difficult except for the lamellar phases. This is due to the well-known phase problem of X-ray diffraction. Here we successfully applied the multiwavelength anomalous dispersion (MAD) method to solve the phase problem for an inverted hexagonal phase of a phospholipid with brominated chains. Although the principle of the MAD method for all systems is the same, we found that for lipid structures it is necessary to use a procedure of analysis significantly different from that used for protein crystals. The inverted hexagonal phase has been used to study the chain packing in a hydrophobic interstice where three monolayers meet. Hydrophobic interstices are of great interest, because they occur in the intermediate states of membrane fusion. It is generally believed that chain packing in such a region is energy costly. Consequently, it has been speculated that the inverted lipid tube is likely to deviate from a circular shape, and the chain density distribution might be nonuniform. The bromine distribution obtained from the MAD analysis provides the information for the chain packing in the hexagonal unit cell. The intensity of the bromine distribution is undulated around the unit cell. The analysis shows that the lipid chains pack the hexagonal unit cell at constant volume per chain, with no detectable effect from a high-energy interstitial region.  相似文献   

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