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1.
Biosensors that make use of transport processes across lipid membranes are very rare even though a stimulus, the binding of a single analyte molecule, can enhance the sensor response manifold if the analyte leads to the transport of more than one ion or molecule across the membrane. Prerequisite for a proper function of such membrane based biosensors is the formation of lipid bilayers attached to a support that allow for the insertion of membrane peptides and proteins in a functional manner. In this review, the current state of the art technologies to obtain lipid membranes on various supports are described. Solid supported membranes on transparent and electrically conducting surfaces, lipid bilayers on micromachined apertures and on porous materials are discussed. The focus lies on the applicability of such membranes for the investigation of transport phenomena across lipid bilayers facilitated by membrane embedded peptides, channel proteins and transporters. Carriers and channel forming peptides, which are easy to handle and rather robust, are used frequently to build up membrane based biosensors. However, channel forming proteins and transporters are more difficult to insert functionally and thus, there are yet only few examples that demonstrate the applicability of such systems as biosensor devices.
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Oligomers of (R)‐3‐hydroxybutanoate (OHB) have previously been shown to transport cations through lipid bilayers. The ion‐transport activity has been attributed to the formation of hydrophobic aggregates or pores, which have been identified by fluorescence‐microscopy measurements of membrane‐incorporated fluorescence‐labeled OHBs. To obtain more information about these aggregates, we describe here the synthesis of the specifically F‐labeled HB oligomers II – IV for structural investigation by means of solid‐state 19F‐NMR spectroscopic techniques. 相似文献
4.
Corticosteroid hormone-induced factor (CHIF) is a major regulatory subunit of the Na,K-ATPase, and a member of an evolutionarily conserved family of membrane proteins that regulate the function of the enzyme complex in a tissue-specific and physiological-state-specific manner. Here we present the structure of CHIF oriented in the membrane, determined by solid-state NMR orientation-dependent restraints. Because CHIF adopts a similar structure in lipid micelles and bilayers, it is possible to assign the solid-state NMR spectrum measured for (15)N-labeled CHIF in oriented bilayers from the structure determined in micelles, to obtain the global orientation of the protein in the membrane. 相似文献
5.
Hall AC Suarez C Hom-Choudhury A Manu AN Hall CD Kirkovits GJ Ghiriviga I 《Organic & biomolecular chemistry》2003,1(16):2973-2982
The synthesis, cation binding and transmembrane conductive properties of a novel group of synthetic ion channels containing a redox-active centre are described. Experiments using a black lipid membrane preparation revealed that these compounds function effectively as ion channels. Subsequent 23Na NMR spectroscopy studies focused on a synthesized ion channel with a ferrocene centre. When incorporated in vesicular bilayers, this channel was demonstrated to support a Na+ flux that was at least six times faster than ion transport by monensin. Since oxidation of the ferrocene moiety completely inhibited the Na+ transport, the redox-active centre provides a potential mechanism for controlling ion flux. 相似文献
6.
Structural information about the interactions between membrane proteins and their ligands provides insights into the membrane protein functions. A variety of surfactants have been used for structural analyses of membrane proteins, and in some cases, they yielded successful results. However, the use of surfactants frequently increases the conformational instability of membrane proteins and distorts their normal function. Here, we propose a new strategy of membrane protein reconstitution into lipid bilayers on affinity beads, which maintains the native conformation and function of the protein for NMR studies. The reconstituted membrane proteins are suitable for NMR analyses of interactions, by using the transferred cross-saturation method. The strategy was successfully applied to the interaction between a potassium ion channel, KcsA, and a pore-blocker, agitoxin2 (AgTx2). This strategy would be useful for analyzing the interactions between various membrane proteins and their ligands. 相似文献
7.
This study, for the first time, demonstrates that it is possible to mechanically align lipid bilayers at a very low temperature (as low as the gel-to-liquid crystalline phase transition temperature). Performing NMR experiments on mechanically aligned lipid bilayers at a low temperature increases the signal-to-noise ratio, the resolution, and the span of NMR parameters. The increased lifetime of the alignment and the nature of the bilayer sample would enhance the application of solid-state NMR techniques to study membrane proteins. 相似文献
8.
The BH3 mimetics targeting the interaction between the BH3-only proteins and their prosurvival Bcl-2 family proteins have shown enormous potential as cancer therapeutics. Herein, seven analogues targeting anti-apoptotic Bcl-2 proteins derived from the Bim BH3 domain via sequence simplification and/or modification are described. The in vitro binding affinity on anti-apoptotic Bcl-2 proteins and cell killing activity were evaluated. The results showed that analogues could significantly bind to target proteins and exhibited anti-cancer effect against three cancer cell lines. Of particular interest were the analogue SM-5 (KD=9.48 nmol/L for Bcl-2) and SM-6 (KD=0.08 nmol/L for Bcl-xL), which exhibited improved binding affinity compared with the lead Bim (KD=16.90 nmol/L for Bcl-2 and 22.2 nmol/L for Bcl-xL, respectively). These results indicated that the peptide sequence containing the four hydrophobic side chains occupying pockets within the BH3-recognition cleft of anti-apoptotic Bcl-2 proteins might be the minimum sequence required for the bioactivity and the active core region of Bim. Promising inhibitors of anti-apoptotic Bcl-2 proteins with high bioactivity might be designed based on the active core. 相似文献
9.
Freezing point depression of water in phospholipid membranes: a solid-state NMR study 总被引:1,自引:0,他引:1
Lee DK Kwon BS Ramamoorthy A 《Langmuir : the ACS journal of surfaces and colloids》2008,24(23):13598-13604
Lipid-water interaction plays an important role in the properties of lipid bilayers, cryoprotectants, and membrane-associated peptides and proteins. The temperature at which water bound to lipid bilayers freezes is lower than that of free water. Here, we report a solid-state NMR investigation on the freezing point depression of water in phospholipid bilayers in the presence and absence of cholesterol. Deuterium NMR spectra at different temperatures ranging from -75 to + 10 degrees C were obtained from fully (2)H2O-hydrated POPC (1-palmitoyl-2-oleoylphosphatidylcholine) multilamellar vesicles (MLVs), prepared with and without cholesterol, to determine the freezing temperature of water and the effect of cholesterol on the freezing temperature of water in POPC bilayers. Our 2H NMR experiments reveal the motional behavior of unfrozen water molecules in POPC bilayers even at temperatures significantly below 0 degrees C and show that the presence of cholesterol further lowered the freezing temperature of water in POPC bilayers. These results suggest that in the presence of cholesterol the fluidity and dynamics of lipid bilayers can be retained even at very low temperatures as exist in the liquid crystalline phase of the lipid. Therefore, bilayer samples prepared with a cryoprotectant like cholesterol should enable the performance of multidimensional solid-state NMR experiments to investigate the structure, dynamics, and topology of membrane proteins at a very low temperature with enhanced sample stability and possibly a better sensitivity. Phosphorus-31 NMR data suggest that lipid bilayers can be aligned at low temperatures, while 15N NMR experiments demonstrate that such aligned samples can be used to enhance the signal-to-noise ratio of is 15N chemical shift spectra of a 37-residue human antimicrobial peptide, LL-37. 相似文献
10.
Manipulating recognition and transport at the nanoscale holds great promise for technological breakthroughs in energy conversion, catalysis, and information processing. Living systems evolve specialized membrane proteins (MPs) embedded in lipid bilayers to exquisitely control communications across the insulating membrane boundaries. Harnessing MP functions directly in synthetic systems opens up enormous opportunities for nanotechnology, but there exist fundamental challenges of how to address the labile nature of lipid bilayers that renders them of inadequate value under a broad range of harsh non-biological conditions, and how to reconstitute MPs coherently in two or three dimensions into non-lipid-based artificial membranes. Here we show that amphiphilic block copolymers can be designed to direct proteorhodopsin reconstitution and formation of hierarchically ordered proteopolymer membrane arrays spontaneously, even when the membrane-forming polymer blocks are in entangled states. These findings unfold a viable approach for the development of robust and chemically versatile nanomembranes with MP-regulated recognition and transport performance. 相似文献
11.
The B-cell lymphoma 2 (Bcl-2) family of proteins regulates the intrinsic pathway of apoptosis. Interactions between specific
anti- and pro-apoptotic Bcl-2 proteins determine the fate of a cell. Anti-apoptotic Bcl-2 proteins have been shown to be over-expressed
in certain cancers and they are attractive targets for developing anti-cancer drugs. Peptides from the BH3 region of pro-apoptotic
proteins have been shown to interact with anti-apoptotic Bcl-2 proteins and induce biological activity similar to that observed
in parent proteins. However, the specificity of BH3 peptides derived from different pro-apoptotic proteins differ for different
anti-apoptotic Bcl-2 proteins. In this study, we have investigated the relationship between the stable helical nature of BH3
peptides and their affinities to Bcl-XL, an anti-apoptotic Bcl-2 protein. We have carried out molecular dynamics simulations of six BH3 peptides derived from Bak,
Bad and Bim pro-apoptotic proteins for a period of 50 ns each in aqueous medium. Due to the amphipathic nature of BH3 peptides,
the hydrophobic residues on the hydrophobic face tend to cluster together in all BH3 peptides. While this process resulted
in a complete loss of helical structure in 16-mer Bak and 16-mer Bad wild type peptides, stabilizing interactions in the hydrophilic
face of the BH3 peptides and capping interactions helped to maintain partial helical character in 16-mer Bad mutant and 16-mer
Bim peptides. The latter two 16-mer peptides exhibit higher affinity for Bcl-XL. Similarly the longer BH3 peptides, 25-mer Bad and 33-mer Bim, also resulted in smaller and stable helical fragments and
their helical conformation is stabilized by interactions between residues in the solvent-exposed hydrophilic half of the peptide.
The stable nature of helical segment in a BH3 peptide can be directly correlated to its binding affinity and the helical region
encompassed the highly conserved Leu residue. We propose that upon approaching the hydrophobic groove of anti-apoptotic proteins,
a longer helix will be induced in high affinity BH3 peptides by extending the smaller stable helical segments around the conserved
Leu residue in both N- and C-terminal regions. The results reported in this study will have implications in developing peptide-based
inhibitors for anti-apoptotic Bcl-2 proteins. 相似文献
12.
A novel solid-state NMR technique for identifying the asymmetric insertion depths of membrane proteins in lipid bilayers is introduced. By applying Mn (2+) ions on the outer but not the inner leaflet of lipid bilayers, the sidedness of protein residues in the lipid bilayer can be determined through paramagnetic relaxation enhancement (PRE) effects. Protein-free lipid membranes with one-side Mn (2+)-bound surfaces exhibit significant residual (31)P and lipid headgroup (13)C intensities, in contrast to two-side Mn (2+)-bound membranes, where lipid headgroup signals are mostly suppressed. Applying this method to a cell-penetrating peptide, penetratin, we found that at low peptide concentrations, penetratin is distributed in both leaflets of the bilayer, in contrast to the prediction of the electroporation model, which predicts that penetratin binds to only the outer lipid leaflet at low peptide concentrations to cause an electric field that drives subsequent peptide translocation. The invalidation of the electroporation model suggests an alternative mechanism for intracellular import of penetratin, which may involve guanidinium-phosphate complexation between the peptide and the lipids. 相似文献
13.
Zhi Jia Hui Hui Yang Yun-Jun Liu Xiu-Zhen Wang 《Applied biochemistry and biotechnology》2018,186(1):145-160
A new series of dibenzoxanthenes 4a–4f were synthesized through the nucleophilic substitution and characterized by NMR and MS spectra. Their antitumor activity was screened by MTT assay. Compounds (except 4b and 4c) displayed strong growth inhibitory effects against chosen five tumor cells under light irradiation. The molecular mechanism of compound-induced cell apoptosis was investigated by AO/EB staining, comet assay, DCFH-DA, JC-1 fluorescent probe, and western blotting. Compounds induced the apoptosis of HepG2 cells and DNA damage. Location assay showed that compounds entered the nucleus of tumor cells. Furthermore, it was found that compounds induced loss of mitochondrial membrane potential, acceleration of ROS production, and activation of caspse-3, caspase-7, and caspase-9 proteins. Compounds upregulated the expression of pro-apoptotic Bim and Bax and downregulated the expression of anti-apoptotic Bcl-xl and Bcl-2. These results indicated that compounds induced the apoptosis of HepG2 cells through ROS-mediated mitochondrial pathway. The induction of apoptosis by dibenzoxanthenes may provide an important mechanism for their cancer chemopreventive function. 相似文献
14.
Wysoczanski P Mart RJ Loveridge EJ Williams C Whittaker SB Crump MP Allemann RK 《Journal of the American Chemical Society》2012,134(18):7644-7647
The Bcl-2 family of proteins includes the major regulators and effectors of the intrinsic apoptosis pathway. Cancers are frequently formed when activation of the apoptosis mechanism is compromised either by misregulated expression of prosurvival family members or, more frequently, by damage to the regulatory pathways that trigger intrinsic apoptosis. Short peptides derived from the pro-apoptotic members of the Bcl-2 family can activate mechanisms that ultimately lead to cell death. The recent development of photocontrolled peptides that are able to change their conformation and activity upon irradiation with an external light source has provided new tools to target cells for apoptosis induction with temporal and spatial control. Here, we report the first NMR solution structure of a photoswitchable peptide derived from the proapoptotic protein Bak in complex with the antiapoptotic protein Bcl-x(L). This structure provides insight into the molecular mechanism, by which the increased affinity of such photopeptides compared to their native forms is achieved, and offers a rationale for the large differences in the binding affinities between the helical and nonhelical states. 相似文献
15.
The characterization of interactions between membrane proteins as they take place within the lipid bilayer poses a technical challenge, which is currently very difficult and, in many cases, impossible to overcome. The recent development of a method based in the combination two-color fluorescence cross-correlation spectroscopy with scanning of the focal volume allows the detection and quantification of interactions between biomolecules inserted in biological membranes. This powerful strategy has allowed the quantitative analysis of diverse systems, such as the association between proteins of the Bcl-2 family involved in apoptosis regulation or the binding between a growth factor and its receptor during signaling. Here, we review the last developments to quantify protein/protein interactions in lipid membranes and focus on the use of fluorescence-correlation-spectroscopy approaches for that purpose. 相似文献
16.
Dr. Julian de Mos Dr. Andreas Jakob Dr. Johanna Becker-Baldus Prof. Dr. Alexander Heckel Prof. Dr. Clemens Glaubitz 《Chemistry (Weinheim an der Bergstrasse, Germany)》2020,26(30):6789-6792
Light-induced activation of biomolecules by uncaging of photolabile protection groups has found many applications for triggering biochemical reactions with minimal perturbations directly within cells. Such an approach might also offer unique advantages for solid-state NMR experiments on membrane proteins for initiating reactions within or at the membrane directly within the closed MAS rotor. Herein, we demonstrate that the integral membrane protein E. coli diacylglycerol kinase (DgkA), which catalyzes the phosphorylation of diacylglycerol, can be controlled by light under MAS-NMR conditions. Uncaging of NPE-ATP or of lipid substrate NPE-DOG by in situ illumination triggers its enzymatic activity, which can be monitored by real-time 31P-MAS NMR. This proof-of-concept illustrates that combining MAS-NMR with uncaging strategies and illumination methods offers new possibilities for controlling biochemical reactions at or within lipid bilayers. 相似文献
17.
Rapid screening of membrane protein activity: electrophysiological analysis of OmpF reconstituted in proteoliposomes 总被引:1,自引:0,他引:1
Solvent-free planar lipid bilayers were formed in an automatic manner by bursting of giant unilamellar vesicles (GUVs) after gentle suction application through micron-sized apertures in a borosilicate glass substrate. Incubation of GUVs with the purified ion channel protein of interest yielded proteoliposomes. These proteoliposomes allow for immediate recording of channel activity after GUV sealing. This approach reduces the time-consuming, laborious and sometimes difficult protein reconstitution processes normally performed after bilayer formation. Bilayer recordings are attractive for investigations of membrane proteins not accessible to patch clamp analysis, like e.g. proteins from organelles. In the presented work, we show the example of the outer membrane protein OmpF from Escherichia coli. We reconstituted OmpF in proteoliposomes and observed the characteristic trimeric conductance levels and the typical gating induced by pH and transmembrane voltage. Moreover, OmpF is the main entrance for beta-lactam antibiotics and we investigated translocation processes of antibiotics and modulation of OmpF by spermine. We suggest that the rapid formation of porin containing lipid bilayers is of potential for the efficient electrophysiological characterization of the OmpF protein, for studying membrane permeation processes and for the rapid screening of antibiotics. 相似文献
18.
Heron AJ Thompson JR Mason AE Wallace MI 《Journal of the American Chemical Society》2007,129(51):16042-16047
We form planar lipid bilayers between an aqueous droplet and a hydrogel support immersed in a lipid-oil solution. By scanning the bilayer over the surface of an SDS-PAGE gel, we are able to directly detect membrane proteins from gels using single-channel recording. Using this technique, we are able to examine low levels of endogenous protein from cell extracts without the need for over-expression. We also use droplet bilayers to detect small molecules from hydrogels. The bilayers show enhanced stability compared to conventional planar lipid bilayers, and both bilayer size and position can be controlled during an experiment. Hydrogel scanning with droplet bilayers provides a new method for the discovery and characterization of ion channels with the potential for high-throughput screening. 相似文献
19.
Subramaniam V Alves ID Salgado GF Lau PW Wysocki RJ Salamon Z Tollin G Hruby VJ Brown MF Saavedra SS 《Journal of the American Chemical Society》2005,127(15):5320-5321
Transmembrane proteins (TMPs), particularly ion channels and receptors, play key roles in transport and signal transduction. Many of these proteins are pharmacologically important and therefore targets for drug discovery. TMPs can be reconstituted in planar-supported lipid bilayers (PSLBs), which has led to development of TMP-based biosensors and biochips. However, PSLBs composed of natural lipids lack the high stability desired for many technological applications. One strategy is to use synthetic lipid monomers that can be polymerized to form robust bilayers. A key question is how lipid polymerization affects TMP structure and activity. In this study, we have examined the effects of UV polymerization of bis-Sorbylphosphatidylcholine (bis-SorbPC) on the photoactivation of reconstituted bovine rhodopsin (Rho), a model G-protein-coupled receptor. Plasmon-waveguide resonance spectroscopy (PWR) was used to compare the degree of Rho incorporation and activation in fluid and poly(lipid) PSLBs. The results show that reconstitution of Rho into a supported lipid bilayer composed only of bis-SorbPC, followed by photoinduced lipid cross-linking, does not measurably diminish protein function. 相似文献
20.
The role of electrostatic forces in indole-lipid interactions was studied by (1)H and (2)H NMR in ether- and ester-linked phospholipid bilayers with incorporated indole. Indole-ring-current-induced (1)H NMR chemical shifts of lipid resonances in bilayers of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphocholine, 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine, and 1,2-di-O-octadecenyl-sn-glycero-3-phosphomethanol show a bimodal indole distribution, with indole residing at the upper hydrocarbon chain/glycerol region of the lipid and near the choline group, when present. (2)H NMR of indole-d(7)-incorporated lipid bilayers reveals that the former site is occupied by about two-thirds of the indole, which adopts a distinct preferred orientation with respect to the bilayer normal. The results suggest that the upper hydrocarbon chain/glycerol location is dictated by many factors, including interactions with the electric charges and dipoles, van der Waals interactions, entropic contributions, and hydrogen bonding. Indole diffusion rates are higher in lipids with ester bonds and lower in choline-containing lipids, suggesting that interactions between indole and carbonyl groups are of minor importance for lipid-indole association and that cation-pi interactions with choline drive the second indole location. Nuclear Overhauser effect spectroscopy cross-relaxation rates suggest a 30-ns lifetime for indole-lipid associations. These results may have important implications for sidedness and structural transitions in tryptophan-rich membrane proteins. 相似文献