Active membrane viscoelasticity by the bacterial FtsZ-division protein

At the early stages of the division process in Escherichia coli, the protein FtsZ forms a septal ring at the midcell. This Z-ring causes membrane constriction during bacterial division. The Z-ring associates to the lipid membrane through several membrane proteins, ZipA among them. Here, a simplified FtsZ-ZipA model was reconstituted onto Langmuir monolayers based in E. coli polar lipid extract. Brewster angle and atomic force microscopy have revealed membrane FtsZ-polymerization upon GTP hydrolysis. The compression viscoelasticity of these monolayers has been also investigated. The presence of protein induced softening and fluidization with respect to the bare lipid membrane. An active mechanism, based on the internal forces stressed by FtsZ filaments and transduced to the lipid membrane by ZipA, was suggested to underlie the observed behavior.     

Proton‐Detected Solid‐State NMR Spectroscopy of a Zinc Diffusion Facilitator Protein in Native Nanodiscs

The structure, dynamics, and function of membrane proteins are intimately linked to the properties of the membrane environment in which the proteins are embedded. For structural and biophysical characterization, membrane proteins generally need to be extracted from the membrane and reconstituted in a suitable membrane‐mimicking environment. Ensuring functional and structural integrity in these environments is often a major concern. The styrene/maleic acid co‐polymer has recently been shown to be able to extract lipid/membrane protein patches directly from native membranes to form nanosize discoidal proteolipid particles, also referred to as native nanodiscs. In this work, we show that high‐resolution solid‐state NMR spectra can be obtained from an integral membrane protein in native nanodiscs, as exemplified by the 2×34 kDa bacterial cation diffusion facilitator CzcD.     

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.     

Probing Deviation of Adhered Membrane Dynamics between Reconstituted Liposome and Cellular System

The dynamics of cell‐cell adhesion are complicated due to complexities in cellular interactions and intra‐membrane interactions. In the present work, we have reconstituted a liposome‐based model system to mimic the cell‐cell adhesion process. Our model liposome system consists of one fluorescein‐tagged and one TRITC (tetramethyl‐rhodamine isothiocyanate)‐tagged liposome, adhered through biotin‐neutravidin interaction. We monitored the adhesion process in liposomes using Förster Resonance Energy Transfer (FRET) between fluorescein (donor) and TRITC (acceptor). Occurrence of FRET is confirmed by the decrease in donor lifetime as well as distinct rise time of the acceptor fluorescence. Interestingly, the acceptor's emission exhibits fluctuations in the range of ≈3±1 s. This may be attributed to structural oscillations associated in two adhered liposomes arising from the flexible nature of biotin‐neutravidin interaction. We have compared the dynamics in a cell‐mimicking liposome system with that in an in vitro live cell system. In the adhered live cell system, we used CPM (7‐diethylamino‐3‐(4‐maleimido‐phenyl)‐4‐methylcoumarin, donor) and nile red (acceptor), which are known to stain the membrane of CHO (Chinese Hamster Ovary) cells. The dynamics of the adhered membranes of two live CHO cells were observed through FRET between CPM and nile red. The acceptor fluorescence intensity exhibits an oscillation in the time‐scale of ≈1±0.75 s, which is faster compared to the reconstituted liposome system, indicating the contributions and involvement of multiple dynamic protein complexes around the cell membrane. This study offers simple reconstituted model systems to understand the complex membrane dynamics using a FRET‐based physical chemistry approach.     

Activity monitoring of functional OprM using a biomimetic microfluidic device

This paper describes the fabrication and use of a biomimetic microfluidic device for the monitoring of a functional porin reconstituted within a miniaturized suspended artificial bilayer lipid membrane (BLM). Such a microfluidic device allows for (1) fluidic and electrical access to both sides of the BLM and (2) reproducible membrane protein insertion and long-term electrical monitoring of its conductance (G(i)), thanks to the miniaturization of the BLM. We demonstrate here for the first time the feasibility to insert a large trans-membrane protein through its β-barrel, and monitor its functional activity for more than 1 hour (limited by buffer evaporation). In this paper, we specifically used our device for the monitoring of OprM, a bacterial efflux channel involved in the multidrug resistance of the bacteria Pseudomonas aeruginosa. Sub-steps of the OprM channel conductance were detected during the electrical recordings within our device, which might be due to oscillations between several structural conformations (sub-states) adopted by the protein, as part of its opening mechanism. This work is a first step towards the establishment of a genuine platform dedicated to the investigation of bacterial proteins under reconstituted conditions, a very promising tool for the screening of new inhibitors against bacterial channels involved in drug resistance.     

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.     

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.     

Probing the properties of lipopolysaccharide monolayers and their interaction with the antimicrobial peptide polymyxin B by atomic force microscopy

In contrast to the majority of all known cell types, Gram-negative bacteria have a second membrane, the outer membrane, which is an asymmetric bilayer composed of a phospholipid inner leaflet and a glycolipid outer leaflet. The glycolipid layer, in most cases being composed of a lipopolysaccharide (LPS), is the first target for antimicrobial agents. To get a basic understanding of the membrane-forming properties of LPS, we reconstituted monolayers of deep rough mutant LPS from Salmonella enterica serova Minnesota (R595 LPS), its lipid A moiety, and of the synthetic tetraacyl compound 406 (resembling the biosynthetic lipid A precursor IVa) at the air-water interface of a film balance. The liquid-expanded (LE) and liquid-condensed (LC) domains in the coexisting region were investigated with epifluorescence and, after transferring the monolayer onto mica, as a Langmuir-Blodgett film, with atomic force microscopy (AFM). The fluorescence and the AFM images showed identical domain structure. The higher resolution of the AFM images, however, contained more topographic details. Different heights and adhesion forces between the LE and LC domains could be observed. Differences in the adhesion forces between the AFM tip and the sample were determined in the repulsive and the attractive dynamic scanning modes, demonstrating the importance of a careful interpretation of height images. We propose that an increase in the lateral pressure causing the LE-LC transition of the monolayers leads to a reorientation of the molecules due to a tilt angle between the alkyl chains and the diglucosamine backbone. LPS monolayers have been utilized as a simplified reconstitution model of the outer membrane to study the interaction with antimicrobial agents. We investigated the action of the polycationic peptide polymyxin B (PMB) and found dramatic influences on the domain structures.     

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.     

Modulation of Higher‐order Behaviour in Model Protocell Communities by Artificial Phagocytosis

Collective behaviour in mixed populations of synthetic protocells is an unexplored area of bottom‐up synthetic biology. The dynamics of a model protocell community is exploited to modulate the function and higher‐order behaviour of mixed populations of bioinorganic protocells in response to a process of artificial phagocytosis. Enzyme‐loaded silica colloidosomes are spontaneously engulfed by magnetic Pickering emulsion (MPE) droplets containing complementary enzyme substrates to initiate a range of processes within the host/guest protocells. Specifically, catalase, lipase, or alkaline phosphatase‐filled colloidosomes are used to trigger phagocytosis‐induced buoyancy, membrane reconstruction, or hydrogelation, respectively, within the MPE droplets. The results highlight the potential for exploiting surface‐contact interactions between different membrane‐bounded droplets to transfer and co‐locate discrete chemical packages (artificial organelles) in communities of synthetic protocells.     

CHARMM‐GUI Membrane Builder toward realistic biological membrane simulations

CHARMM‐GUI Membrane Builder, http://www.charmm‐gui.org/input/membrane , is a web‐based user interface designed to interactively build all‐atom protein/membrane or membrane‐only systems for molecular dynamics simulations through an automated optimized process. In this work, we describe the new features and major improvements in Membrane Builder that allow users to robustly build realistic biological membrane systems, including (1) addition of new lipid types, such as phosphoinositides, cardiolipin (CL), sphingolipids, bacterial lipids, and ergosterol, yielding more than 180 lipid types, (2) enhanced building procedure for lipid packing around protein, (3) reliable algorithm to detect lipid tail penetration to ring structures and protein surface, (4) distance‐based algorithm for faster initial ion displacement, (5) CHARMM inputs for P₂₁ image transformation, and (6) NAMD equilibration and production inputs. The robustness of these new features is illustrated by building and simulating a membrane model of the polar and septal regions of E. coli membrane, which contains five lipid types: CL lipids with two types of acyl chains and phosphatidylethanolamine lipids with three types of acyl chains. It is our hope that CHARMM‐GUI Membrane Builder becomes a useful tool for simulation studies to better understand the structure and dynamics of proteins and lipids in realistic biological membrane environments. © 2014 Wiley Periodicals, Inc.     

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.     

Frame‐Guided Assembly of Vesicles with Programmed Geometry and Dimensions

In molecular self‐assembly molecules form organized structures or patterns. The control of the self‐assembly process is an important and challenging topic. Inspired by the cytoskeletal‐membrane protein lipid bilayer system that determines the shape of eukaryotic cells, we developed a frame‐guided assembly process as a general strategy to prepare heterovesicles with programmed geometry and dimensions. This method offers greater control over self‐assembly which may benefit the understanding of the formation mechanism as well as the functions of the cell membrane.     

古紫质AR4重组的磷脂脂质体中的定向自组装

This paper reports, for the first time, that Archaerhodopsin-4 (AR4) could be reconstituted into phospholipid liposomes by self-assembly. AR4 is a new membrane protein isolated from halobacteria H.sp. xz515 in a salt lake of Tibet, China. This is a bacteriorhodopsin (bR) like protein, function as a light-driven proton pump. Experimental measurements verified that similar to bR, AR not only remains its biological activity in pmteoliposome, but also keeps a preferred orientation in self-assembly.     

Zwitterionic Character and Lipid Composition Determine the Behaviour of Glycosylphosphatidylinositol Fragments in Monolayers

Glycosylphosphatidylinositols (GPIs) are complex glycolipids found in free form or anchoring proteins to the outer leaflet of the cell membrane in eukaryotes. GPIs have been associated with the formation of lipid rafts and protein sorting on membranes. The presence of a conserved glycan core with cell-specific modifications together with lipid remodelling during biosynthesis suggest that the properties of the glycolipids are being fine-tuned. We synthesized a series of GPI fragments and evaluated the interactions and arrangement of these glycolipids in monolayers as a 2-D membrane model. GIXD and IRRAS analyses showed the need of N-acetylglucosamine deacetylation for the formation of hydrogen bonds to obtain highly structured domains in the monolayers and an effect of the unsaturated lipids in formation and localization of the glycolipids within or between membrane microdomains. These results contribute to understand the role of these glycolipids and their modifications in the organization of membranes.     

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.     

Design and application of stimulus-responsive droplets and bubbles stabilized by phospholipid monolayers

Biomimetic colloidal particles are promising agents for biosensing, but current technologies fall far short of Nature's capabilities for sensing, assessing, and responding to stimuli. Phospholipid-containing cell membranes are capable of binding and responding to an enormous variety of biomolecules by virtue of membrane organization and the presence of receptor proteins. By tuning the composition and functionalization of simulated membranes, soft colloids such as droplets and bubbles can be designed to respond to various stimuli. Moreover, because lipid monolayers can surround almost any hydrophobic phase, the interior of the colloid can be selected to provide a sensitive readout, for example in the form of optical microscopy or acoustic detection. In this work, we review some advances made by our group and others in the formulation of lipid-coated particles with different internal phases such as fluorocarbons, hydrocarbons, or liquid crystals. In some cases, binding or displacement of stabilizing lipids gives rise to conformational changes or disruptions in local membrane geometry, which can be amplified by the interior phase. In other cases, multivalent analytes can promote aggregation or even membrane fusion, which can be utilized for an optical or acoustic readout. By highlighting a few recent examples, we hope to show that lipid monolayers represent a versatile biosensing platform that can react to and detect biomolecules by leveraging the unique capabilities of phospholipid membranes.     

Photoswitchable Phase Separation and Oligonucleotide Trafficking in DNA Coacervate Microdroplets

Coacervate microdroplets produced by liquid–liquid phase separation have been used as synthetic protocells that mimic the dynamical organization of membrane‐free organelles in living systems. Achieving spatiotemporal control over droplet condensation and disassembly remains challenging. Herein, we describe the formation and photoswitchable behavior of light‐responsive coacervate droplets prepared from mixtures of double‐stranded DNA and an azobenzene cation. The droplets disassemble and reassemble under UV and blue light, respectively, due to azobenzene trans/cis photoisomerisation. Sequestration and release of captured oligonucleotides follow the dynamics of phase separation such that light‐activated transfer, mixing, hybridization, and trafficking of the oligonucleotides can be controlled in binary populations of the droplets. Our results open perspectives for the spatiotemporal control of DNA coacervates and provide a step towards the dynamic regulation of synthetic protocells.     

Micelles,Bicelles, and Nanodiscs: Comparing the Impact of Membrane Mimetics on Membrane Protein Backbone Dynamics

Detergents are often used to investigate the structure and dynamics of membrane proteins. Whereas the structural integrity seems to be preserved in detergents for many membrane proteins, their functional activity is frequently compromised, but can be restored in a lipid environment. Herein we show with per‐residue resolution that while OmpX forms a stable β‐barrel in DPC detergent micelles, DHPC/DMPC bicelles, and DMPC nanodiscs, the pico‐ to nanosecond and micro‐ to millisecond motions differ substantially between the detergent and lipid environment. In particular for the β‐strands, there is pronounced dynamic variability in the lipid environment, which appears to be suppressed in micelles. This unexpected complex and membrane‐mimetic‐dependent dynamic behavior indicates that the frequent loss of membrane protein activity in detergents might be related to reduced internal dynamics and that membrane protein activity correlates with lipid flexibility.