lipidic mesophases

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Membrane protein crystallization in lipidic mesophases with tailored bilayers

Membrane protein crystallization in lipidic mesophases with tailored bilayers

lipidic mesophases, expectations ran high. In the in- There is also the issue of the hosting lipid that is tervening period, the method has been used success- integral to the in meso method and that will surely impact fully for high-resolution structure determination studies on its success. The original approach was developed by on several photocycle intermediates of bR (Lanyi and using monoolein (9.9 MAG; the monoacylglycerol [MAG] Schobert, 2004), halorhodopsin (Kolbe et al., 2000), sen- chain shorthand notation used here is based on the N.T sory rhodopsin II (SRII) (Luecke et al., 2001), an SRII/ system introduced previously (Misquitta and Caffrey,
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Crystallizing membrane proteins for structure determination using lipidic mesophases

Crystallizing membrane proteins for structure determination using lipidic mesophases

Clearly, there are many steps involved in structure determination using macromolecular crystallography. This is illustrated in Figure 3. Typically, these include identifying a membrane protein target, and then producing, purifying and crystallizing it. Diffraction measurements are performed on the crystal using a home or a synchrotron X-ray source. The diffraction data are processed yielding an electron density map that is then fitted with a molecular model. The model, when refined, can be used to explore the mechanism of action of the protein and for structure-based drug design. The focus of this article is to show how we produce diffraction quality crystals of membrane proteins using lipidic mesophases, by the so-called in meso method. A recent review of the method and its scope is available in Reference 1 (Caffrey, 2009). The step-by-step protocol we will follow here is described in Reference 2 (Caffrey and Cherezov, 2009).
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Membrane protein structure determination using crystallography and lipidic mesophases: recent advances and successes

Membrane protein structure determination using crystallography and lipidic mesophases: recent advances and successes

Improvements are needed of course if the method is to become routine. Critically, the specialized materials and supplies upon which the method relies must be made more generally available and the method itself must be made user-friendly. New and improved in meso robots available on the market are tackling the user-friendliness issue. Workshops that involve hands-on demonstrations contribute to making the method more accessible. The author has been active in this area for several years now with recent workshops in Ireland, 147 Mexico 148 , Hawaii, 149 Australia 150 , and China. 151 In the past year alone, over 200 students were trained in the practicalities and finer elements of in meso crystallogenesis and related topics at various locations worldwide. Online video demonstrations covering practical aspects of the method are available and in preparation (Li, D., Boland, C., Aragao, D., Walsh, K., and Caffrey, M. Harvesting and cryo-cooling crystals of membrane proteins grown in lipidic mesophases for structure determination by macromolecular crystallography. In review; Li, D., Boland, C., Walsh, K., and Caffrey, M. Use of a robot for high-throughput crystallization of membrane proteins in lipidic mesophases. In press). 46
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Membrane Protein Crystallization in Lipidic Mesophases   A Mechanism Study Using X ray Microdiffraction

Membrane Protein Crystallization in Lipidic Mesophases A Mechanism Study Using X ray Microdiffraction

The membrane structural biologist seeks to understand how membrane proteins function at a molecular level. One of the most direct ways of accomplishing this requires knowing the structure of the protein, ideally at atomic resolution. To date, this can only be done by the method of macromolecular crystallography. Integral to the method is the need for three-dimensional crystals of diffraction quality and their production represents a major rate-limiting step in the overall process of structure determination. The in meso method is a novel approach for crystallizing membrane proteins. It makes use of lipidic mesophases, the cubic phase in particular. A mechanism for how the method works has been proposed. In this study, we set out to test one aspect of the hypothesis which posits that the protein migrates from the bulk mesophase reservoir to the face of the crystal by way of a lamellar conduit. Using a sub-micrometer-sized X-ray beam the interface between a growing membrane protein crystal and the bulk cubic phase was interrogated with micrometer spatial resolution. Characteristic diffraction from the lamellar phase was observed at the interface as expected. This result supports the proposal that the protein uses a lamellar portal on its way from the bulk mesophase up and into the face of the crystal.
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Use of a Robot for High throughput Crystallization of Membrane Proteins in Lipidic Mesophases

Use of a Robot for High throughput Crystallization of Membrane Proteins in Lipidic Mesophases

An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at atomic resolution. Presently, there is only one way to capture such information as applied to integral membrane proteins (Figure 1), and the complexes they form, and that method is macromolecular X-ray crystallography (MX). To do MX diffraction quality crystals are needed which, in the case of membrane proteins, do not form readily. A method for crystallizing membrane proteins that involves the use of lipidic mesophases, specifically the cubic and sponge phases 1-5 , has gained considerable attention of late due to the successes it has had in the G protein-coupled receptor field 6-21 (www.mpdb.tcd.ie). However, the method, henceforth referred to as the in meso or lipidic cubic phase method, comes with its own technical challenges. These arise, in part, due to the generally viscous and sticky nature of the lipidic mesophase in which the crystals, which are often micro-crystals, grow. Manipulating crystals becomes difficult as a result and particularly so during harvesting 22,23 . Problems arise
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Crystallizing membrane proteins using lipidic mesophases

Crystallizing membrane proteins using lipidic mesophases

In meso crystallization was performed with monoolein as the host lipid. The lipidic mesophase laden with protein was dispensed into home-made 96-well glass sandwich crystallization plates using a home-built in meso crystallization robot. Trials were performed with 50 nL protein/ lipid dispersion and 1 μL precipitant solution. Plates were incubated at 20 oC for up to 30 days. Crystallization was monitored using an automatic imager RockImager RI24 and manually by light microscope. Initial screening was performed using six 96-well blocks filled with precipitant solution from commercial crystallization kits (Crystal Screen HT, Index HT, SaltRx and MembFac from Hampton Research; Wizard I&II from Emerald BioSystems; MemStart from Molecular Dimensions and JBScreen Membrane from Jena Bioscience). Small crystals of the type shown in Figure 18 were found to form in the presence of MPD, pentaerythritol propoxylate (5/4 PO/OH), PEG 550 MME, jeffamine M-600, 1,4-butanediol and potassium thiocyanate. Conditions containing MPD were optimized using coarse concentration-pH grid screens ([MPD] range 6 – 14 %(v/v) with 2 %(v/v) steps, pH range 4 – 8 with 1 pH unit steps), followed by screening with 48 different salts as additives (two concentrations for each salt in the 0.1 – 0.5 M range) and finishing with fine concentration-pH grid screens ([MPD] range 8 – 12 %(v/v) in steps of 1 %(v/v), salt concentration range 0.05 – 0.3 M in 0.05 M steps, pH range 6 – 7 in pH unit steps of 0.5). About five subsequent rounds of optimization (using visual, not diffraction quality cues) were needed to obtain ~100 μm-sized BtuB crystals. The best crystals were grown in 10–12 %(v/v) MPD, 0.1–0.2 M ammonium formate, and 0.1 M MES, pH 6.5. Crystals of BtuB for crystallographic measurement were grown in a 72-well Nunc microbatch plate using a fine concentration-pH grid screen around the best conditions found with sandwich plates. 200 nL lipid/protein dispersion was used in each well in conjunction with 3 μL precipitant solution. Wells were sealed with transparent tape. Crystals grew to their full size (~100 × 60 × 25 μm 3 ) in 10–14 days (Figure 9D). Harvesting was done after 14 days
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Membrane Protein Crystallization in Lipidic Mesophases  Hosting lipid affects on the crystallization and structure of a transmembrane peptide

Membrane Protein Crystallization in Lipidic Mesophases Hosting lipid affects on the crystallization and structure of a transmembrane peptide

Gramicidin is an apolar pentadecapeptide antibiotic consisting of alternating D-and L-amino acids. It functions, in part, by creating pores in membranes of susceptible cells rendering them leaky to monovalent cations. The peptide should be able to traverse the host membrane either as a double stranded, intertwined double helix (DSDH) or as a head-to-head single stranded helix (HHSH). Current structure models are based on macromolecular X-ray crystallography (MX) and nuclear magnetic resonance (NMR). However, the HHSH form has only been observed by NMR. The shape and size of the different gramicidin conformations differ. We speculated therefore that reconstituting it into a lipidic mesophase with bilayers of different microstructures would preferentially stabilize one form over the other. By using such mesophases for in meso
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Why GPCRs behave differently in cubic and lamellar lipidic mesophases

Why GPCRs behave differently in cubic and lamellar lipidic mesophases

the protein and the bilayers of the two lipidic phases in the nucleation process. We note that the nucleation is triggered by the addition of the precipitant which is known to cause transient water depletion from the LCP interior, 19 resulting in structural changes in the cubic phase bilayer. Our current study has not attempted to quantify the e ff ects of the precipitant, but it is reasonable to speculate that dehydration of the cubic phase will only increase the unfavorable residual interactions between the GPCR and the LCP bilayer. 19 This is likely to increase the drive for protein oligomerization inside the LCP upon precipitant addition, which could lead to the formation of locally fl attened lamellar bilayers as a prelude to protein crystallization. In order to address quantitatively the structural perturbations due to precipitant, such as changes in curvature of the LCP bilayer, it is critical to calculate the corresponding deformation energies in the presence of the protein. However, the representation of the complex geometry of the LCP around the insertion (Figures 6 − 7) in the numerical approach developed in the current work for quantifying the LCP shape (i.e., fi tting the MD data to the analytical solution) is not su ffi ciently re fi ned to serve in the calculation of reliable energies. We will be addressing these numerical challenges in future work.
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Crystallizing Membrane Proteins in Lipidic Mesophases  A Host Lipid Screen

Crystallizing Membrane Proteins in Lipidic Mesophases A Host Lipid Screen

Default Hosting Lipid, Monoolein 9.9 MAG—To date, the structures of two outer membrane proteins crystallized by the in meso method, have been reported.14, 15 In both cases, monoolein was[r]

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Crystallizing membrane proteins for structure function studies using lipidic mesophases

Crystallizing membrane proteins for structure function studies using lipidic mesophases

The lipidic cubic phase method for crystallizing membrane proteins has posted some high-profile successes recently. This is especially true in the area of G-protein-coupled receptors, with six new crystallographic structures emerging in the last 3 1/2 years. Slowly, it is becoming an accepted method with a proven record and convincing generality. However, it is not a method that is used in every membrane structural biology laboratory and that is unfortunate. The reluctance in adopting it is attributable, in part, to the anticipated difficulties associated with handling the sticky viscous cubic mesophase in which crystals grow. Harvesting and collecting diffraction data with the mesophase-grown crystals is also viewed with some trepidation. It is acknowledged that there are challenges associated with the method. However, over the years, we have worked to make the method user-friendly. To this end, tools for handling the mesophase in the pico- to nano-litre volume range have been developed for efficient crystallization screening in manual and robotic modes. Glass crystallization plates have been built that provide unparalleled optical quality and sensitivity to nascent crystals. Lipid and precipitant screens have been implemented for a more rational approach to crystallogenesis, such that the method can now be applied to a wide variety of membrane protein types and sizes. In the present article, these assorted advances are outlined, along with a summary of the membrane proteins that have yielded to the method. The challenges that must be overcome to develop the method further are described.
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Novel oligomeric and polymeric materials based upon the dibenzotetraaza[14]annulenes

Novel oligomeric and polymeric materials based upon the dibenzotetraaza[14]annulenes

This example demonstrates that polymers that are rod-like with a symmetrical structure are unsuitable for obtaining thermotropic mesophases since decomposition occurs prior to the event. As the number of repeat units, n, in the polymer increases so the difference between the melting point and the decomposition temperature increases. The problem posed by the high temperatures that are required to transform the polymer from the crystalline to the mesomorphic phase can be approached in two ways, both involving modifications to the main chain of the polymer. Firstly, the linearity of the polymer backbone may be reduced by the introduction of non-linear yet rigid components, and this has the effect of deforming the cylindrical shape of the polymer. As the amount of the non-linear comonomer unit increases, the melting point of the polymer decreases due to the loss of order caused by the reduction in stacking efficiency of the molecules.
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Aqueous Viscosity Is the Primary Source of Friction in Lipidic Pore Dynamics

Aqueous Viscosity Is the Primary Source of Friction in Lipidic Pore Dynamics

ABSTRACT A new theory, to our knowledge, is developed that describes the dynamics of a lipidic pore in a liposome. The equa- tions of the theory capture the experimentally observed three-stage functional form of pore radius over time—stage 1, rapid pore enlargement; stage 2, slow pore shrinkage; and stage 3, rapid pore closure. They also show that lipid flow is kinetically limited by the values of both membrane and aqueous viscosity; therefore, pore evolution is affected by both viscosities. The theory predicts that for a giant liposome, tens of microns in radius, water viscosity dominates over the effects of membrane viscosity. The edge tension of a lipidic pore is calculated by using the theory to quantitatively account for pore kinetics in stage 3, rapid pore closing. This value of edge tension agrees with the value as standardly calculated from the stage of slow pore closure, stage 2. For small, submicron liposomes, membrane viscosity affects pore kinetics, but only if the viscosity of the aqueous solution is comparable to that of distilled water. A first-principle fluid-mechanics calculation of the friction due to aqueous viscosity is in excellent agreement with the friction obtained by applying the new theory to data of previously published experimental results.
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Novel phytoandrogens and lipidic augmenters from Eucommia ulmoides

Novel phytoandrogens and lipidic augmenters from Eucommia ulmoides

Animal studies To establish the dose response relationship between intramuscular IM testosterone Sustanon 5000 injections and ventral prostate weight as a measure of prostate development[r]

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A lipidic α-amino acid based adjuvant/carrier system for peptide delivery and enhancing peptide immunogenicity

A lipidic α-amino acid based adjuvant/carrier system for peptide delivery and enhancing peptide immunogenicity

Ever since the commercial introduction of insulin and thyroid hormone, peptides and proteins have been investigated as potential drug candidates. For a new drug to be clinically effective it should be a highly active compound, exerting its pharmacological properties with minimal side effects and should reach the required site of action in therapeutic concentrations. In recent years, the rapid progress made in molecular biology has given us the opportunity to identify target molecules and to design drugs of a highly specific nature to tackle poorly controlled diseases. However, even though numerous peptides with great therapeutic promise are known, an intense effort is still required if these potential drug molecules are to be delivered to their active site. In particular, peptides and proteins must negotiate a multitude of barriers if they are to be administered by the oral route. With the goal of oral peptide delivery in mind, this thesis exploits the use of a novel group of compounds, the lipidic amino acids, not only for their potential in peptide delivery, but also their possible use as immunoadjuvants for synthetic peptide vaccines.
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Novel lipidic-amino acid based drug and peptide delivery system

Novel lipidic-amino acid based drug and peptide delivery system

The lipidic amino acids are synthetic a-amino acids with long alkyl side chains, they and their homo-oligomers represent a class of compounds which combine the structural features of lipids with those of amino acids and peptides. The lipidic amino acids can be condensed in the same way as natural amino acids to give oligomers and polymers. Interest has been shown in the amino acids and their derivatives as lubricants', cosmetics^ polishes^ and surface improvers for ceramics^ In addition, the lipidic amino acids and oligomers can be used as detergents, water resistant (waterproof) and biocompatible films and coatings^ Our particular interest is to use them as a drug delivery system.
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Preparation, Characterization and Applications of Nanoemulsions: An Insight

Preparation, Characterization and Applications of Nanoemulsions: An Insight

Nanoemulsions are defined as isotropic, thermodynamically stable, transparent or translucent; dispersions of oil and water stabilized by surfactant molecules (forms an interfacial film) having the droplet size of 20-500nm. Ease of preparation and scale-up, stability and increased bioavailability are features of these formulations which have attracted the attention of researchers. Its basic principle lies in its ability to spontaneously generate fine o/w microemulsion under mild agitation following dilution with aqueous phases. These conditions mimic the digestive motility in the GIT necessary to provide the agitation required for In vivo self emulsification. Unlike emulsions, self- nanoemulsified drug delivery systems (SNEDDS) generates microemulsion with a narrow droplet size distribution of less than 50 nm due to which these systems have also been addressed as nanoemulsions. Nanoemulsions (NE) are lipidic nanoformulations with droplet diameter in nanometer range have established tremendous attention as drug delivery formulations for lipophilic drugs due to their capability to increase solubility, permeation across biological membranes as well as their therapeutic efficiency of lipid soluble drugs due to predictable size- distribution, high drug loading and stability under biological environment. However there is still relatively narrow insight regarding preparation, characterization and applications of nanoemulsions. This limitation unfolds the premise for current review article. In this review, we attempt to explore varying intricacies, methods of preparation, characteristics, and drug delivery applications of nanoemulsions to spike interest of those contemplating a foray in this field.
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“RECENT CHARACTERIZATION
                                AND APPLICABILITY OF POLYMORPHISM: LIQUID CRYSTAL” by *Dr. Gopal
                            Rai, Deepak Jain, India.

“RECENT CHARACTERIZATION AND APPLICABILITY OF POLYMORPHISM: LIQUID CRYSTAL” by *Dr. Gopal Rai, Deepak Jain, India.

In contrast to thermotropic mesophases, lyotropic liquid crystal transitions occur with the influence of solvents, not by a change in temperature. Lyotropic mesophases occur as a result of solvent-induced aggregation of the constituent mesogens into micellar structures. Lyotropic mesogens are typically amphiphilic, meaning that they are composed of both lyophilic (solvent-attracting) and lyophobic (solvent-repelling) parts. This causes them to form into micellar structures in the presence of a solvent, since the lyophobic ends will stay together as the lyophilic ends extend outward toward the solution. As the concentration of the solution is increased and the solution is cooled, the micelles increase in size and eventually coalesce. This separates the newly formed liquid crystalline state from the solvent.
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Hybrid delivery systems for methotrexate-lipidic liquid crystalline cubic phases and cubosomes with magnetic nanoparticles

Hybrid delivery systems for methotrexate-lipidic liquid crystalline cubic phases and cubosomes with magnetic nanoparticles

The effect magnetic nanoparticles have on drug release, as well as other molecules from various lipidic structures, under the influence of the external magnetic field has been well documented. Vallooran et al. [27] showed that under a constant magnetic stimuli, the hexagonal phase with MNPs, aligns the orientation of the hexagonal domains in the direction of the external (constant) magnetic field, facilitating the transport of a hydrophilic drug across the liquid crystalline phase in this direction. Two cases were considered: when the magnetically induced orientation of the hexagonal domains in the membrane was in the direction of diffusion, effects the rate of transport of a hydrophilic drug, and when the forced position of the hexagonal domains was perpendicular to the diffusion direction, affects inhibition of drug diffusion. Mendozza et al. showed that the monoolein cubic phase with MNPs subjected to AMF for 10 minutes indicate Pn3m to H II phase transition. [40]
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Antiprotozoal activities of quassinoids

Antiprotozoal activities of quassinoids

either a lipidic a-amino acid or acetic acid produced a compound of equivalent antimalarial activity, when compared with the parent quassinoid, but with reduced KB cell t[r]

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Targeted Macromolecules Delivery by Large Lipidic Nanovesicles Electrofusion with Mammalian Cells

Targeted Macromolecules Delivery by Large Lipidic Nanovesicles Electrofusion with Mammalian Cells

Lipidic nanovesicles (so called liposomes) were one the earliest forms of nanovectors. One of their limits was our lack of knowledge on the delivery pathway of their content to the target cell cytoplasm. The present communication de- scribes an efficient way to enhance the delivery. Pulsed electric fields (PEF) are known since the early 80’s to mediate a fusogenic state of plasma membranes when applied to a cell suspension or a tissue. Polykaryons are detected when PEF are applied on cells in contact during or after the pulses. Heterofusion can be obtained when a cell mixture is pulsed. When lipidic nanovesicles, either small unilamellar vesicles (SUVs) or large unilamellar vesicles (LUVs), are electrostatically brought in contact with electropermeabilized cells by a salt bridge, their content is delivered into the cytoplasm in electropermeabilized cells. The PEF parameters are selected to affect specifically the cells leaving the vesicles unaffected. It is the electropermeabilized state of the cell membrane that is the trigger of the merging between the plasma membrane and the lipid bilayer. The present investigation shows that the transfer of macromolecules can be obtained; i.e. 20 kD dextrans can be easily transferred while a direct transfer does not take place under the same elec- trical parameters. Cell viability was not affected by the treatment. As delivery is present only on electropermeabilized cells, a targeting of the effect is obtained in the volume where the PEF parameters are over the critical value for elec- tropermeabilization. A homogeneous cytoplasm labeling is observed under digitised videomicroscopy. The process is a content and “membrane” mixing, following neither a kiss and run or an endocytotic pathway.
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