Biochemistry

Pore-forming toxins (PFTs) have been discovered in a wide range of organisms. Their functions are essential to the survival or virulence of many species. PFTs often interact with lipid membranes. Large unilamellar vesicles (LUV), also known as liposomes, have been commonly used as reliable membrane models for testing PFTs activity. Liposomes have great adaptability in size, lipid composition, and loading cargo. Incorporating the fluorescent dye/quencher pair, 8-Aminonaphthalene-1,3,6-Trisulfonic Acid (ANTS) and p-Xylene-Bis-Pyridinium Bromide (DPX), in liposomes is an effective approach for measuring membrane leakage. When ANTS and DPX are encapsulated in a liposome, the fluorescence of ANTS is quenched by DPX. However, disruption of liposome integrity and subsequent leakage result in measurable fluorescence emitted by ANTS. Here, we report our protocol for optimal liposome preparation for measuring liposome leakage by fluorescence dequenching.

Modern microscopy methods are powerful tools for studying live cell signaling and biochemical reactions, enabling us to observe when and where these reactions take place from the level of a cell down to single molecules. With microscopy, each cell or molecule can be observed both before and after a given perturbation, facilitating better inference of cause and effect than is possible with destructive modes of signaling quantitation. As many inputs to cell signaling and biochemical systems originate as protein-protein interactions near the cell membrane, an outstanding challenge lies in controlling the timing, location and the magnitude of protein-protein interactions in these unique environments. Here, we detail our procedure for manipulating such spatial and temporal protein-protein interactions in a closed microscopy system using a LOVTRAP-based light-responsive protein-protein interaction system on a supported lipid bilayer. The system responds in seconds and can pattern details down to the one micron level. We used this technique to unlock fundamental aspects of T cell signaling, and this approach is generalizable to many other cell signaling and biochemical contexts.

The emergence and rapid spread of multidrug resistance in bacteria have led to the urgent need for novel antibacterial agents. Membrane permeabilization is the mechanism for many antibacterial molecules that are being developed against gram-negative bacteria. Thus, to determine the efficacy of a potential antibacterial molecule, it is important to assess the change in bacterial membrane permeability after treatment. This study describes the protocol for the assays of outer and inner membrane permeability using the fluorescent probes N-phenyl-1-naphthylamine and propidium iodide. Compared with other experiments, such as electron microscopy and the assay of minimal bactericidal concentration, this methodology provides a simpler, faster, and cost-effective way of estimating the membrane-permeabilizing effect and bactericidal efficacy of antibacterial molecules. This study presents an optimized protocol with respect to the classical protocols by incubating bacteria with antibacterial molecules in the culture condition identical to that of antibacterial assays and then detecting the signal of the fluorescent probe in the buffer without broth and antibacterial molecules. This protocol avoids the effect of nutrient deficiency on the physiological status of bacteria and the interference of antibacterial molecules towards the fluorescent probe. Thus, this method can effectively and precisely evaluate the membrane permeability and match the results obtained from other antibacterial assays, such as minimum inhibitory concentration and time–kill curve assays.

Non-covalent binding of cholesterol to the transmembrane region of proteins affect their functionalities, but methods to prove such an interaction are rare. We describe our protocol to label the hemagglutinin (HA) of Influenza virus with a cholesterol derivative in living cells or with immunoprecipitated protein. We synthesized a “clickable” photocholesterol compound, which closely mimics authentic cholesterol. It contains a reactive diazirine group that can be activated by UV-illumination to form a covalent bond with amino acids in its vicinity. Incorporation of photocholesterol into HA is then visualized by “clicking” it to a fluorophore, which can be detected in an SDS-gel by fluorescence scanning. This method provides a convenient and practical way to demonstrate cholesterol-binding to other proteins and probably to identify the binding site.

Interactions of lipids with proteins are essential events in the framework of biological membranes. Assessment of the affinity and specificity of protein-lipid binding can give useful information to elucidate cell membrane functions. Surface Plasmon Resonance (SPR) is a powerful technology to study macromolecular interactions, allowing direct and rapid determination of association and dissociation rates using small amounts of samples. An extensive range of binding analyses can be performed by SPR such as protein–protein, protein–membrane (lipids), protein–carbohydrate, protein–nucleic acid and even protein-small molecules. This protocol describes the binding of an antimicrobial protein (used as ligand) to a lipopolysaccharide (LPS) (used as analyte) after immobilization onto a CM sensor chip by amine coupling.

Diacylglycerol (DAG) is a bioactive lipid with diverse biological roles. DAG transiently accumulates in a membrane upon receipt of an appropriate stimulus that activates phospholipase C to cleave phospholipids. The resulting hydrolysis product DAG binds to proteins such as protein kinase C to initiate a variety of downstream cellular processes. DAG kinases attenuate such responses by converting DAG to phosphatidic acid.

This protocol describes an assay designed to quantify cellular DAG levels. The assay exploits the enzymatic conversion of DAG (sn-1,2-diacylglycerol) to phosphatidic acid (1,2-diacyl- sn-glycerol-3-phosphate) in conjunction with the incorporation of a radiolabeled phosphate group by DAG kinase (Figure 1). This assay was described in (Strijbis et al., 2013).

Dye release experiments are a widely used method to assess the interactions between membrane-active molecules and lipid membranes. Of particular interest is the ability to assess the degree of the lipid bilayer perturbation by simultaneously encapsulating dye of different sizes, such as dextrans grafted with a chromophore. In this assay, dextran linked to rhodamine or fluorescein are both encapsulated in lipid vesicles to allow quantifying the leakage of each dextran individually from a single sample. For instance, the size evaluation of the lipid pore formed by an antimicrobial peptide has been recently achieved using this protocol (Sani et al., 2013).

The PIs coated beads assay or “PIP-Beads” developed by Echelon Biosciences (Salt Lake City, USA) is a quick assay to recognize which PIs are able to bind to a given protein domain, in a quantitative way. It is much faster and cheaper than liposomes and more reproducible than PIP-strip assays. The “PIP-Beads” assay is a biochemical assay that basically involves an incubation of a purified protein or protein domain with the appropriate PI-coated set of beads. After washing, drying and resuspending the samples, they can be easily analyzed by SDS-PAGE separation. Phosphoinositides (PIs) have been characterized as important determinants of cell membrane domains, such as the apical and basolateral domains in epithelial polarized cells (Martin-Belmonte and Mostov, 2007), controlling membrane trafficking (Szentpetery et al., 2010) or determining the presynaptic or postsynaptic terminal in neurons, among other functions (Di Paolo and De Camilli, 2006). These phosphoinositides enriched membranes bring the proteomic machinery together, confers to these membrane their different identities and functions. This protein-PIs interaction in many cases involves direct binding of specific protein membrane domains with certain PIs. Some of these domains are characterized such as PH domains from phospholipase-C- or synaptotagmin-like C2 domains (Galvez-Santisteban et al., 2012), while some of them are not. To determine which PI is binding to a given protein domain, it is important to have a quick and efficient assay. The liposome binding assays are very good to establish the kinetic properties of binding, but they are expensive and permit only to test a few PIs per experiment. On the other hand, PIP-strip (phosphatidil-inositol-phosphate) based analysis is easy and fast, however the PIs are presented in a flat surface and the reproducibility is sometimes limited.

The lipid and protein interactions are an integral and important part of many cellular signaling pathways. The understanding of the selective and specific interaction of the given lipid molecule with the target protein is required for studying cellular signaling. In this assay, different lipids are spotted onto a nitrocellulose membrane to which they attach. Then the membrane is incubated with a lipid binding protein possessing an epitope tag. The protein binds to the lipid which is detected by immunoblotting with an antibody recognizing the epitope tag (see Figure 1). PTEN is an important tumor suppressor which functions as both protein and lipid phosphatase. The primary physiological substrate of PTEN is signaling lipid PtdIns (3, 4, 5) P3, by dephosphrylating PtdIns (3, 4, 5) P3 to PtdIns (4, 5) P2 PTEN negatively regulates PI3K signaling and mediates its tumor-suppressor function by inactivating downstream oncogenic AKT-mediated signaling. The PTEN lipid binding assay is conducted to study the specific binding of PTEN to different lipid molecules.