Physicochemical Properties of Seed Oil Blends and Their Potential for the Creation of Synthetic Oleosomes with Modulated Polarities.

There is an increasing demand within the pharmaceutical and cosmetic industries for biofriendly lipid-based active ingredient delivery systems. Micelles, liposomes, and lipid nanoparticles are currently the most used systems despite their limitations. Oleosomes, also known as lipid droplets, are promising alternatives to the existing strategies.

CHARMM-GUI Membrane Builder for Lipid Droplet Modeling and Simulation.

Lipid droplets (LDs) are organelles that are necessary for eukaryotic and prokaryotic metabolism and energy storage. They have a unique structure consisting of a spherical phospholipid monolayer encasing neutral lipids such as triacylglycerol (TAG). LDs have garnered increased interest for their implications in disease and for drug delivery applications.

One-step site-specific S-alkylation of full-length caveolin-1: Lipidation modulates the topology of its C-terminal domain.

Caveolin-1 is an integral membrane protein that is known to acquire a number of posttranslational modifications upon trafficking to the plasma membrane. In particular, caveolin-1 is palmitoylated at three cysteine residues (C133, C143, and C156) located within the C-terminal domain of the protein which could have structural and topological implications. Herein, a reliable preparation of full-length S-alkylated caveolin-1, which closely mimics the palmitoylation observed in vivo, is described.

Facile production of tagless membrane scaffold protein for nanodiscs.

The initial step in the preparation of nanodiscs is to express and purify the membrane scaffold protein (MSP) to homogeneity. Current methods used for the isolation and purification of MSP utilize nickel affinity chromatography. However, the presence of a polyhistidine tag on the MSP often interferes with downstream steps where nanodiscs reconstituted with protein need to be isolated from empty ones.

Rapid preparation of nanodiscs for biophysical studies.

Nanodiscs, which are disc-shaped entities that contain a central lipid bilayer encased by an annulus of amphipathic helices, have emerged as a leading native-like membrane mimic. The current approach for the formation of nanodiscs involves the creation of a mixed-micellar solution containing membrane scaffold protein, lipid, and detergent followed by a time consuming process (3-12 h) of dialysis and/or incubation with sorptive beads to remove the detergent molecules from the sample. In contrast, the methodology described herein provides a facile and rapid procedure for the preparation of nanodiscs in a matter of minutes (<15 min) using Sephadex® G-25 resin to remove the detergent from the sample.

Biophysical characterization of full-length oleosin in dodecylphosphocholine micelles.

Oleosin is a hydrophobic protein that punctuates the surface of plant seed lipid droplets, which are 20 nm-100 μm entities that serve as reservoirs for high-energy metabolites. Oleosin is purported to stabilize lipid droplets, but its exact mechanism of stabilization has not been established. Probing the structure of oleosin directly in lipid droplets is challenging due to the size of lipid droplets and their high degree of light scattering.

Preparation and characterization of neutrally-buoyant oleosin-rich synthetic lipid droplets.

Lipid droplets also known as oil bodies are found in a variety of organisms and function as stores of high-energy metabolites. Recently, there has been interest in using lipid droplets for protein production and drug delivery. Artificial lipid droplets have been previously prepared, but their short lifetime in solution and inhomogeneity has severely limited their applicability.

Preparation of Caveolin-1 for NMR Spectroscopy Experiments.

Caveolin-1 is a 20.5 kDa integral membrane protein that is involved in a myriad of cellular processes including signal transduction, relieving mechano-stresses on the cell, endocytosis, and most importantly caveolae formation. As a consequence, there is intense interest in characterizing caveolin-1 structurally.

Reconstitution of full-length human caveolin-1 into phospholipid bicelles: Validation by analytical ultracentrifugation.

A significant hurdle in obtaining biophysical information on membrane proteins is developing a successful strategy for their reconstitution into a suitable membrane mimic. In particular, utilization of the more 'native-like' membrane mimics such as bicelles is generally more challenging than simple micellar solubilization. Caveolin-1, an integral membrane protein involved in membrane curvature, endocytosis, mechano-protection, and signal transduction, has been shown to be particularly recalcitrant to standard reconstitution protocols due to its highly hydrophobic characteristics.

Secondary structure of caveolins: a mini review.

Caveolae are 50-100 nm invaginations found within the plasma membrane of cells. Caveolae are involved in many processes that are essential for homeostasis, most notably endocytosis, mechano-protection, and signal transduction. Within these invaginations, the most important proteins are caveolins, which in addition to participating in the aforementioned processes are structural proteins responsible for caveolae biogenesis.

Low- q Bicelles Are Mixed Micelles.

Bicelles are used in many membrane protein studies because they are thought to be more bilayer-like than micelles. We investigated the properties of "isotropic" bicelles by small-angle neutron scattering, small-angle X-ray scattering, fluorescence anisotropy, and molecular dynamics. All data suggest that bicelles with a q value below 1 deviate from the classic bicelle that contains lipids in the core and detergent in the rim.

Efficient solubilization and purification of highly insoluble membrane proteins expressed as inclusion bodies using perfluorooctanoic acid.

The purification of membrane proteins can be challenging due to their low solubility in conventional detergents and/or chaotropic solutions. The introduction of fusion systems that promote the formation of inclusion bodies has facilitated the over-expression of membrane proteins. In this protocol, we describe the use of perfluorooctanoic acid (PFOA) as an aid in the purification of highly hydrophobic membrane proteins expressed as inclusion bodies.

Evidence for Surface Recognition by a Cholesterol-Recognition Peptide.

Two cholesterol recognition/interaction amino-acid consensus peptides, N-acetyl-LWYIKC-amide, and N-acetyl-CLWYIK-amide, have been coupled to exchangeable mimics of Chol (cholesterol) and Phos (1,2-dipalmitoyl-sn-glycerol-3-phospho-(1'rac-glycerol)) via disulfide bond formation. Equilibration between Chol and Phos via thiolate-disulfide interchange reactions has revealed that both peptides favor Chol as a nearest-neighbor in liquid-disordered (ld) bilayers to the same extent. In contrast, no Chol- or Phos-recognition could be detected by these peptides in analogous liquid-ordered (lo) bilayers.

Reconstitution and spectroscopic analysis of caveolin-1 residues 62-178 reveals that proline 110 governs its structure and solvent exposure.

Caveolin-1 is a membrane protein that possesses an unusual topology where both N- and C-termini are cytoplasmic as a result of a membrane-embedded turn. In particular, proline 110 has been postulated to be the linchpin of this unusual motif. Using a caveolin-1 construct (residues 62-178) reconstituted into dodecylphosphocholine micelles with and without a cholesterol mimic, the changes that occurred upon P110A mutation were probed.

Secondary Structure Analysis of a Functional Construct of Caveolin-1 Reveals a Long C-Terminal Helix.

Caveolin-1 is an integral membrane protein that is the primary component of cell membrane invaginations called caveolae. While caveolin-1 is known to participate in a myriad of vital cellular processes, structural data on caveolin-1 of any kind is severely limited. In order to rectify this dearth, secondary structure analysis of a functional construct of caveolin-1, containing the intact C-terminal domain, was performed using NMR spectroscopy in lyso-myristoylphosphatidylglycerol micelles.

Recent progress in the topology, structure, and oligomerization of caveolin: a building block of caveolae.

Caveolae are cholesterol-rich plasma membrane invaginations that are found in a plethora of cell types. They play many roles including signal transduction, endocytosis, and mechanoprotection. The most critical protein in caveolae is the integral membrane protein, caveolin, which has been shown to be necessary for caveolae formation, and governs the major functions attributed to caveolae.

Probing the U-shaped conformation of caveolin-1 in a bilayer.

Caveolin induces membrane curvature and drives the formation of caveolae that participate in many crucial cell functions such as endocytosis. The central portion of caveolin-1 contains two helices (H1 and H2) connected by a three-residue break with both N- and C-termini exposed to the cytoplasm. Although a U-shaped configuration is assumed based on its inaccessibility by extracellular matrix probes, caveolin structure in a bilayer remains elusive.

Probing the caveolin-1 P132L mutant: critical insights into its oligomeric behavior and structure.

Caveolin-1 is the most important protein found in caveolae, which are cell surface invaginations of the plasma membrane that act as signaling platforms. A single point mutation in the transmembrane domain of caveolin-1 (proline 132 to leucine) has deleterious effects on caveolae formation in vivo and has been implicated in various disease states, particularly aggressive breast cancers. Using a combination of gel filtration chromatography and analytical ultracentrifugation, we found that a fully functional construct of caveolin-1 (Cav1(62-178)) was a monomer in dodecylphosphocholine micelles.

The transmembrane domain of caveolin-1 exhibits a helix-break-helix structure.

Caveolin is an integral membrane protein that is found in high abundance in caveolae. Both the N- and C- termini lie on the same side of the membrane, and the transmembrane domain has been postulated to form an unusual intra-membrane horseshoe configuration. To probe the structure of the transmembrane domain, we have prepared a construct of caveolin-1 that encompasses residues 96-136 (the entire intact transmembrane domain).

Discoid Bicelles as Efficient Templates for Pillared Lamellar Periodic Mesoporous Silicas at pH 7 and Ultrafast Reaction Times.

We report the first synthesis of periodic mesoporous silicas templated by bicelles. The obtained materials form novel pillared lamellar structures with a high degree of periodic order, narrow pore size distributions, and exceptionally high surface areas.

Lag periods during the self-assembly of {Mo(72)Fe(30)} macroions: connection to the virus capsid formation process.

The kinetic properties of the self-assembly of hydrophilic Keplerate-type polyoxometalate (POM) {Mo(72)Fe(30)} macroanions into single-layer, vesicle-like blackberry structures in solutions were monitored by the static and dynamic laser light scattering techniques. In the presence of additional electrolytes, an obvious lag period at the initial stage of self-assembly was observed, followed by a fast increase of the scattered intensity. The whole kinetic curve is sigmoidal with a lag phase.

Reliable expression and purification of highly insoluble transmembrane domains.

A general procedure for the reliable preparation of insoluble transmembrane domains has been developed. Improved expression schemes were developed by expressing the transmembrane domains of caveolin proteins 1, 2, and 3 as a fusion to the Trp leader protein. This construct readily formed inclusion bodies during overexpression, allowing high levels of protein to be achieved.

From peptide to protein: comparative analysis of the substrate specificity of N-linked glycosylation in C. jejuni.

The gram-negative bacterium Campylobacter jejuni was recently discovered to contain a general N-linked protein glycosylation pathway. Central to this pathway is PglB, a homologue of the Stt3p subunit of the eukaryotic oligosaccharyl transferase (OT), which is involved in the transfer of an oligosaccharide from a polyisoprenyl pyrophosphate carrier to the asparagine side chain of proteins within the conserved glycosylation sites D/E-X1-N-X2-S/T, where X1 and X2 can be any amino acids except proline. Using a library of peptide substrates and a quantitative radioactivity-based in vitro assay, we assessed the amino acids at each position of the consensus glycosylation sequence for their impact on glycosylation efficiency, whereby the sequence DQNAT was found to be the optimal acceptor substrate.

Direct biochemical evidence for the utilization of UDP-bacillosamine by PglC, an essential glycosyl-1-phosphate transferase in the Campylobacter jejuni N-linked glycosylation pathway.

Campylobacter jejuni has a general N-linked glycosylation pathway, encoded by the pgl gene cluster. In C. jejuni, a heptasaccharide is transferred from an undecaprenyl pyrophosphate donor [GalNAc-alpha1,4-GalNAc-alpha1,4-(Glcbeta1,3)-GalNAc-alpha1,4-GalNAc-alpha1,4-GalNAc-alpha1,3-Bac-alpha1-PP-undecaprenyl, where Bac is bacillosamine (2,4-diacetamido-2,4,6-trideoxyglucose)] to the asparagine side chain of target proteins at the Asn-X-Ser/Thr motif.