大肠杆菌MinC/FtsZ蛋白复合物的纯化和结晶

大肠杆菌在细胞分裂时,FtsZ(Filamentous temperature-sensitive protein Z)蛋白会在细胞中部潜在位点聚合形成Z环,而MinC蛋白会抑制Z环形成,从而控制细胞分裂。本研究将min C与fts Z目的基因克隆到合适的载体中,并导入到大肠杆菌中进行表达,采用亲和层析和分子筛纯化的方法得到MinC/FtsZ复合物蛋白进行晶体筛选。通过FtsZ、MinC分别单独转化、表达纯化后混合和FtsZ、MinC共转化法两种方法得到FtsZ/MinC蛋白复合物,并分别对其进行晶体筛选。实验结果表明,在适宜的表达条件下,利用分别转化、纯化再混合的方法得到的FtsZ和MinC蛋白复合比例约为1∶1;混合时加入GTP和Mg Cl2可以促进复合物聚集态更单一,通过晶体筛选初步得到形状为针状的FtsZ/MinC复合蛋白晶体,为MinC/FtsZ复合物的结构解析提供实验基础。     

Protein Patterns and Oscillations on Lipid Monolayers and in Microdroplets

The Min proteins from E.coli position the bacterial cell‐division machinery through pole‐to‐pole oscillations. In vitro, Min protein self‐organization can be reconstituted in the presence of a lipid membrane as a catalytic surface. However, Min dynamics have so far not been reconstituted in fully membrane‐enclosed volumes. Microdroplets interfaced by lipid monolayers were employed as a simple 3D mimic of cellular compartments to reconstitute Min protein oscillations. We demonstrate that lipid monolayers are sufficient to fulfil the catalytic role of the membrane and thus represent a facile platform to investigate Min protein regulated dynamics of the cell‐division protein FtsZ‐mts. In particular, we show that droplet containers reveal distinct Min oscillation modes, and reveal a dependence of FtsZ‐mts structures on compartment size. Finally, co‐reconstitution of Min proteins and FtsZ‐mts in droplets yields antagonistic localization, thus demonstrating that droplets indeed support the analysis of complex bacterial self‐organization in confined volumes.     

Targeting the bacterial Z-ring

FtsZ is a prokaryotic homolog of eukaryotic tubulin and forms the essential bacterial cell division ring (Z-ring). A new study in this issue of Chemistry & Biology, L?ppchen et al., provides further evidence that differences in nucleotide-binding properties of FtsZ and tubulin can be exploited to specifically target the bacterial Z-ring.     

Interaction of FtsZ protein with a DPPE Langmuir film

FtsZ is the key protein in cell division in bacteria. We have proposed that lipid domains in the cytoplasmic membrane play a role in the localisation of FtsZ. In order to test this hypothesis, we used a model system based on Langmuir films to simulate the bacterial membrane. In this simple system we used a single phospholipid, dipalmytoylphosphatidylethanolamine, which is the major constituent of the inner membrane in Escherichia coli. The first results show clearly the importance of the GTP-controlled assembly process in the appearance of circular or fibrillar structures.     

Synthesis of 7‐Oxo‐1,2,3,4,6,7,12,12b‐octahydroindolo[2,3‐a]quinolizine

A short synthesis of 7‐oxo‐1,2,3,4,6,7,12,12b‐octahydroindolo[2,3‐a]quinolizine from 2‐acetylpyridine and phenylhydrazine is described. Ring C is forged using 2‐chloro‐N,N‐dimethylacetamide. This derivative of the natural alkaloid 1,2,3,4,6,7,12,12b‐octahydroindolo[2,3‐a]quinolizine is an inhibitor of the ZipA–FtsZ protein–protein complex, which is a novel antibacterial target.     

Immobilization of acetylcholinesterase in lipid membranes deposited on self-assembled monolayers

Human red blood cell acetylcholinesterase was incorporated into planar lipid membranes deposited on alkanethiol self-assembled monolayers (SAMs) on gold substrates. Activity of the protein in the membrane was detected with a standard photometric assay and was determined to be similar to the protein in detergent solution or incorporated in lipid vesicles. Monolayer and bilayer lipid membranes were generated by fusing liposomes to hydrophobic and hydrophilic SAMs, respectively. Liposomes were formed by the injection method using the lipid dimyristoylphosphatidylcholine (DMPC). The formation of alkanethiol SAMs and lipid monolayers on SAMs was confirmed by sessile drop goniometry, ellipsometry, and electrochemical impedance spectroscopy. In this work, we report acetylcholinesterase immobilization in lipid membranes deposited on SAMs formed on the gold surface and compare its activity to enzyme in solution.     

Phospholipid morphologies on photochemically patterned silane monolayers

We have studied the spreading of phospholipid vesicles on photochemically patterned n-octadecylsiloxane monolayers using epifluorescence and imaging ellipsometry measurements. Self-assembled monolayers of n-octadecylsiloxanes were patterned using short-wavelength ultraviolet radiation and a photomask to produce periodic arrays of patterned hydrophilic domains separated from hydrophobic surroundings. Exposing these patterned surfaces to a solution of small unilamellar vesicles of phospholipids and their mixtures resulted in a complex lipid layer morphology epitaxially reflecting the underlying pattern of hydrophilicity. The hydrophilic square regions of the photopatterned OTS monolayer reflected lipid bilayer formation, and the hydrophobic OTS residues supported lipid monolayers. We further observed the existence of a boundary region composed of a nonfluid lipid phase and a lipid-free moat at the interface between the lipid monolayer and bilayer morphologies spontaneously corralling the fluid bilayers. The outer-edge of the boundary region was found to be accessible for subsequent adsorption by proteins (e.g., streptavidin and BSA), but the inner-edge closer to the bilayer remained resistant to adsorption by protein or vesicles. Mechanistic implications of our results in terms of the effects of substrate topochemical character are discussed. Furthermore, our results provide a basis for the construction of complex biomembrane models, which exhibit fluidity barriers and differentiate membrane properties based on correspondence between lipid leaflets. We also envisage the use of this construct where two-dimensionally fluid, low-defect lipid layers serve as sacrificial resists for the deposition of protein and other material patterns.     

Characterization and in Vitro Inhibition Studies of Bacillus anthracis FtsZ: A Potential Antibacterial Target

FtsZ is an essential bacterial cell division protein that is an attractive target for the development of antibacterial agents. FtsZ is a homologue of eukaryotic tubulin, has GTPase activity, and forms a ring-type structure to initiate cell division. In this study, the FtsZ of Bacillus anthracis was cloned into a bacterial expression vector and overexpressed into Escherichia coli BL21 (DE3) cells. The overexpressed B. anthracis FtsZ was soluble and purified to homogeneity using Ni-His-tag affinity chromatography. Like other known FtsZs, the recombinant B. anthracis FtsZ also showed GTP-dependent polymerization, which was analyzed using both spectrophotometric and Transmission Electronic Microscopic (TEM) analysis. Using the purified FtsZ, we screened a naturally extracted chemical library to identify potent and novel inhibitors. The screening yielded three chemicals, SA-011, SA-059, and SA-069, that inhibited the in vitro polymerization activity of FtsZ in the micromolar range (IC₅₀ of 55–168 μM). The inhibition potency was significantly comparable with that of berberine, a known potential inhibitor of FtsZ. Understanding the biochemical basis of the effect of these inhibitors on B. anthracis growth would provide a promising path for the development of new antianthracis drugs.     

Phospholipids as an alternative to direct covalent coupling: surface functionalization of nanoporous alumina for protein recognition and purification

Anodic aluminum oxide (AAO) substrates with aligned, cylindrical, non-intersecting pores with diameters of 75 nm and depths of 3.5 or 10 μm were functionalized with lipid monolayers harboring different receptor lipids. AAO was first functionalized with dodecyl-trichlorosilane, followed by fusion of small unilamellar vesicles (SUVs) forming a lipid monolayer. The SUVs' lipid composition was transferred onto the AAO surface, allowing us to control the surface receptor density. Owing to the optical transparency of the AAO, the overall vesicle spreading process and subsequent protein binding to the receptor-doped lipid monolayers could be investigated in situ by optical waveguide spectroscopy (OWS). SUV spreading occurred at the pore-rim interface, followed by lateral diffusion of lipids within the pore-interior surface until homogeneous coverage was achieved with a lipid monolayer. The functionality of the system was demonstrated through streptavidin binding onto a biotin-DOPE containing POPC membrane, showing maximum protein coverage at 10 mol% of biotin-DOPE. The system enabled us to monitor in real-time the selective extraction of two histidine-tagged proteins, PIGEA14 (14 kDa) and ezrin (70 kDa), directly from cell lysate solutions using a DOGS-NTA(Ni)/DOPC (1:9) membrane. The purification process including protein binding and elution was monitored by OWS and confirmed by SDS-PAGE.     

Interaction of antimicrobial arginine-based cationic surfactants with liposomes and lipid monolayers

The present work examines the relationship between the antimicrobial activity of novel arginine-based cationic surfactants and the physicochemical process involved in the perturbation of the cell membrane. To this end, the interaction of these surfactants with two biomembrane models, namely, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) multilamellar lipid vesicles (MLVs) and monolayers of DPPC, 1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] sodium salt (DPPG), and Escherichia coli total lipid extract, was investigated. For the sake of comparison, this study included two commercial antimicrobial agents, hexadecyltrimethylammonium bromide and chlorhexidine dihydrochloride. Changes in the thermotropic phase transition parameters of DPPC MLVs in the presence of the compounds were studied by differential scanning calorimetry analysis. The results show that variations in both the transition temperature (Tm) and the transition width at half-height of the heat absorption peak (deltaT1/2) were consistent with the antimicrobial activity of the compounds. Penetration kinetics and compression isotherm studies performed with DPPC, DPPG, and E. coli total lipid extract monolayers indicated that both steric hindrance effects and electrostatic forces explained the antimicrobial agent-lipid interaction. Overall, in DPPC monolayers single-chain surfactants had the highest penetration capacity, whereas gemini surfactants were the most active in DPPG systems. The compression isotherms showed an expansion of the monolayers compared with that of pure lipids, indicating an insertion of the compounds into the lipid molecules. Owing to their cationic character, they are incorporated better into the negatively charged DPPG than into zwitterionic DPPC lipid monolayers.     

Binding of a truncated form of lecithin:retinol acyltransferase and its N- and C-terminal peptides to lipid monolayers

Lecithin:retinol acyltransferase (LRAT) is a 230 amino acid membrane-associated protein which catalyzes the esterification of all-trans-retinol into all-trans-retinyl ester. A truncated form of LRAT (tLRAT), which contains the residues required for catalysis but which is lacking the N- and C-terminal hydrophobic segments, was produced to study its membrane binding properties. Measurements of the maximum insertion pressure of tLRAT, which is higher than the estimated lateral pressure of membranes, and the positive synergy factor a argue in favor of a strong binding of tLRAT to phospholipid monolayers. Moreover, the binding, secondary structure and orientation of the peptides corresponding to its N- and C-terminal hydrophobic segments of LRAT have been studied by circular dichroism and polarization-modulation infrared reflection absorption spectroscopy in monolayers. The results show that these peptides spontaneously bind to lipid monolayers and adopt an α-helical secondary structure. On the basis of these data, a new membrane topology model of LRAT is proposed where its N- and C-terminal segments allow to anchor this protein to the lipid bilayer.     

Laminar order within Langmuir-Blodgett multilayers from phospholipid and myelin basic protein: a neutron reflectivity study

Multilayers consisting of negatively charged phospholipid DMPA and myelin basic protein (MBP) were assembled by Langmuir-Blodgett deposition of floating Langmuir monolayers from the air/water interface to solid substrates. Protein/lipid samples were obtained by binding MBP from the aqueous subphase to the phospholipid monolayers before deposition. The vertical organization of these model membranes (i.e., with organization perpendicular to the substrate surface) was investigated in detail by neutron reflectivity measurements, and the internal distribution of water molecules was determined from the change of contrast after in-situ H2O/D2O exchange. The multilayers were well ordered, with repeating lipid bilayers as fundamental structural unit. MBP was inserted in between adjacent lipid headgroups, such as in the natural myelin membrane. Water molecules in the multilayers were present mainly in the lipid headgroup and protein slab. On exposition of the pure lipid multilayers to a dry atmosphere, a reduction of the bilayer spacing was determined, whereas the global lamellar order was not affected. In contrast, drying of the protein/lipid multilayers induced degradation of the laminar order. The data demonstrate that ordered Langmuir-Blodgett multilayers are versatile model systems for studying how competing interactions between lipid, protein, water, and ions affect the global organization of such multilamellar lipid/protein assemblies. Here, the water molecules were found to be a necessary mediator to maintain the laminar order in a multilayer from DMPA and myelin basic protein.     

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.     

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.     

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.     

Probing FtsZ and tubulin with C8-substituted GTP analogs reveals differences in their nucleotide binding sites

The cytoskeletal proteins, FtsZ and tubulin, play a pivotal role in prokaryotic cell division and eukaryotic chromosome segregation, respectively. Selective inhibitors of the GTP-dependent polymerization of FtsZ could constitute a new class of antibiotics, while several inhibitors of tubulin are widely used in antiproliferative therapy. In this work, we set out to identify selective inhibitors of FtsZ based on the structure of its natural ligand, GTP. We found that GTP analogs with small hydrophobic substituents at C8 of the nucleobase efficiently inhibit FtsZ polymerization, whereas they have an opposite effect on the polymerization of tubulin. The inhibitory activity of the GTP analogs on FtsZ polymerization allowed us to crystallize FtsZ in complex with C8-morpholino-GTP, revealing the binding mode of a GTP derivative containing a nonmodified triphosphate chain.     

Formation of artificial lipid bilayers using droplet dielectrophoresis

We describe the formation of artificial bilayer lipid membranes (BLMs) by the controlled, electrical manipulation of aqueous droplets immersed in a lipid-alkane solution. Droplet movement was generated using dielectrophoresis on planar microelectrodes covered in a thin insulator. Droplets, surrounded by lipid monolayers, were brought into contact and spontaneously formed a BLM. The method produced BLMs suitable for single-channel recording of membrane protein activity and the technique can be extended to create programmable BLM arrays and networks.     

Controlled immobilization of membrane proteins to surfaces for fourier transform infrared investigations

We show that it is possible to immobilize membrane proteins uniformly and reversibly as self-assembled (sub)monolayers on nitrilotriacetic acid-covered sensor surfaces via hexahistidine sequences present either in the protein or in lipid membranes. Fourier transform infrared spectra of such self-assembled (sub)monolayers deliver important structural information of the membrane proteins and are suited to screen the function of cellular receptors.     

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.     

A combined CoMFA and CoMSIA 3D-QSAR study of benzamide type antibacterial inhibitors of the FtsZ protein in drug-resistant Staphylococcus aureus

A major problem today is bacterial resistance to antibiotics and the small number of new therapeutic agents approved in recent years. The development of new antibiotics capable of acting on new targets is urgently required. The filamenting temperature-sensitive Z (FtsZ) bacterial protein is a key biomolecule for bacterial division and survival. This makes FtsZ an attractive new pharmacological target for the development of antibacterial agents. There have been several attempts to develop ligands able to inhibit FtsZ. Despite the large number of synthesized compounds that inhibit the FtsZ protein, there are no quantitative structure–activity relationships (QSAR) that allow for the rational design and synthesis of promising new molecules. We present the first 3D-QSAR study of a large and diverse set of molecules that are able to inhibit the FtsZ bacterial protein. We summarize a set of chemical changes that can be made in the steric, electrostatic, hydrophobic and donor/acceptor hydrogen-bonding properties of the pharmacophore, to generate new bioactive molecules against FtsZ. These results provide a rational guide for the design and synthesis of promising new antibacterial agents, supported by the strong statistical parameters obtained from CoMFA (r²_pred = 0.974) and CoMSIA (r²_pred = 0.980) analyses.