Supported Planar Bilayers for Study of the Immunological Synapse

Journal of Lipid Research

as well as carbohydrate side chains. Proteins are singled out for GPI anchoring due to the presence of a GPI signaling sequence (GSS). The GSS contains the later site of GPI attachment (the amino acid in the  position) and a series of hydrophobic amino acids, essentially forming a membraneassociating domain linking the pre-GPI protein to the luminal side of the endoplasmic reticulum. Biosynthesis of the anchor occurs separately and consists of a complex series of enzymatic reactions involving more than 11 enzymes (3). Synthesis starts at the cytosolic side of the endoplasmic reticulum with phosphoinositol, flips to the lumenal side, and sequentially adds the carbohydrate core elements. The transamidase enzyme complex replaces the GSS with the preformed GPI anchor by amide bond formation to the amino acid in the  position. The GPI-APs are then transported to their final destination via the Golgi system. During transport, further modification of the anchor lipids occurs in a process termed lipid remodeling (4). GPI-APs may be lost from the membrane either with their anchors intact, in a process termed shedding, or upon enzymatic cleavage, i.e., by phosphoinositol-specific phospholipases B and C (5) (see Fig. 1). Biosynthesis, biochemistry and cell biology, trafficking, organization, and dynamics at the cell surface and the release of GPI-APs have all been reviewed recently in greater detail (4, 6-11). To these detailed insights into the topic, we would like to add information about the applications of GPI-APs in biotechnology, and more specifically, in biomedicine (12-14). These applications are mainly based on the membranetargeting properties of GPI-APs and directed at modifying or Abstract Glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) use a unique posttranslational modification to link proteins to lipid bilayer membranes. The anchoring structure consists of both a lipid and carbohydrate portion and is highly conserved in eukaryotic organisms regarding its basic characteristics, yet highly variable in its molecular details. The strong membrane targeting property has made the anchors an interesting tool for biotechnological modification of lipid membrane-covered entities from cells through extracellular vesicles to enveloped virus particles. In this review, we will take a closer look at the mechanisms and fields of application for GPI-APs in lipid bilayer membrane engineering and discuss their advantages and disadvantages for biomedicine.-Heider, S., J. A. Dangerfield, and C. Metzner. Biomedical applications of glycosylphosphatidylinositol-anchored proteins.

Biosensors and Bioelectronics, 1996

Phospholipid bilayers deposited on sensor surfaces are excellent substrates for immobilizing proteins via a molecular anchor. An integrated optics sensing device was used to accurately measure the binding kinetics of proteins thus anchored. By comparing the results with measurements using proteins from which the anchor had been enzymatically removed, it was shown that the anchor accounts for essentially all the irreversible binding. The insertion of the anchor into the lipid bilayer is a spontaneous process. This method of immobilization should be widely applicable to many soluble protein molecules, to which an anchor can be attached by routine methods of molecular engineering.

Biophysical Journal, 2003

M13 major coat protein was derivatized with BODIPY (n-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-sindacene-3-yl)methyl iodoacetamide), and its aggregation was studied in 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and DOPC/1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DOPG) or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE)/DOPG (model systems of membranes with hydrophobic thickness matching that of the protein) using photophysical methodologies (time-resolved and steady-state self-quenching, absorption, and emission spectra). It was concluded that the protein is essentially monomeric, even in the absence of anionic phospholipids. The protein was also incorporated in pure bilayers of lipids with a strong mismatch with the protein transmembrane length, 1,2-dierucoyl-sn-glycero-3-phosphocholine (DEuPC, longer lipid) and 1,2-dimyristoleoyl-sn-glycero-3-phosphocholine (DMoPC, shorter lipid), and in lipidic mixtures containing DOPC and one of these lipids. The protein was aggregated in the pure vesicles of mismatching lipid but remained essentially monomeric in the mixtures as detected from BODIPY fluorescence emission self-quenching. From fluorescence resonance energy transfer (FRET) measurements (donor-n-(iodoacetyl)aminoethyl-1-sulfonaphthylamine (IAEDANS)-labeled protein; acceptor-BODIPY labeled protein), it was concluded that in the DEuPC/DOPC and DMoPC/DOPC lipid mixtures, domains enriched in the protein and the matching lipid (DOPC) are formed.

ChemistryOpen, 2016

Biochemistry, 2007

In the prokaryote Acholeplasma laidlawii, membrane bilayer properties are sensed and regulated by two interface glycosyltransferases (GTs), synthesizing major nonbilayer-(alMGS GT) and bilayerprone glucolipids. These enzymes are of similar structure, as many soluble GTs, but are sensitive to lipid charge and curvature stress properties. Multivariate and bioinformatic sequence analyses show that such interface enzymes, in relation to soluble ones of similar fold, are characterized by high cationic charge, certain distances between small and cationic amino acids, and by amphipathic helices. Varying surface contents of Lys/Arg pairs and Trp indicate different membrane-binding subclasses. A predicted potential (cationic) binding helix from alMGS was structurally verified by solution NMR and CD. The helix conformation was induced by a zwitterionic as well as anionic lipid environment, and the peptide was confined to the bilayer interface. Bilayer affinity of the peptide, analyzed by surface plasmon resonance, was higher than that for soluble membrane-seeking proteins/peptides and rose with anionic lipid content. Interface intercalation was supported by phase equilibria in membrane lipid mixtures, analyzed by 31 P NMR and DSC. An analogous, potentially binding helix has a similar location in the structurally determined Escherichia coli cell wall precursor GT MurG. These two helices have little sequence conservation in alMGS and MurG homologues but maintain their amphipathic character. The evolutionary modification of the alMGS binding helix and its location close to the acceptor substrate site imply a functional importance in enzyme catalysis, potentially providing a mechanism by which glycolipid synthesis will be sensitive to membrane surface charge and intrinsic curvature strain. † This work was supported by the Swedish Science Research Council, and the Natural Sciences and Engineering Council of Canada. ‡ The chemical shifts for 65S-L87 in DHPC (zwitterionic bicelles) and 10% negatively charged bicelles have been deposited with the BMRB (www.bmrb.wisc.edu) under accession code 15171. The coordinates for the solution structure of 65S-L87 in DHPC have been deposited with the PDB under accession code 1Z2T (www.rcsb.org).

Current Biology, 2004

Private Bag 92019 to the putative transmembrane boundary and retaining a C-terminal histidine tag, were expressed and purified. Auckland New Zealand A nickel-chelating lipid then captured the histidine tag to reattach the molecule to a lipid bilayer [7] and mimic 2 University of Strasbourg F67412 Illkirch, Cedex the normal in vivo display. The membrane bound molecules were fully functional and accessible to their natural France 3 Department of Cell Biology ligand and to antibody binding [7]. Two-dimensional crystals of lipid-tethered H-2K b were previously gener-4 Department of Molecular Biology 5 The Skaggs Institute for Chemical Biology and ated by using this technique and preserved in uranyl acetate to produce a 3-dimensional structure of the 6 Department of Immunology The Scripps Research Institute membrane-anchored molecule by electron microscopy. The resolution of this map, as deduced by us now, using La Jolla, California 92037 the point-spread-function criterion [8] is nominally ‫41ف‬Å in plane and ‫42ف‬Å normal to the membrane plane. Notwithstanding the low resolution of this map, it was shown Summary by Celia et al. [7] to agree qualitatively with the commonly

Colloids and Surfaces B: Biointerfaces, 2007

Many different processes take place at the cell membrane interface. Indeed, for instance, ligands bind membrane proteins which in turn activate peripheral membrane proteins, some of which are enzymes whose action is also located at the membrane interface. Native cell membranes are difficult to use to gain information on the activity of individual proteins at the membrane interface because of the large number of different proteins involved in membranous processes. Model membrane systems, such as monolayers at the air-water interface, have thus been extensively used during the last 50 years to reconstitute proteins and to gain information on their organization, structure and activity in membranes. In the present paper, we review the recent work we have performed with membrane and peripheral proteins as well as enzymes in monolayers at the air-water interface. We show that the structure and orientation of gramicidin has been determined by combining different methods. Furthermore, we demonstrate that the secondary structure of rhodopsin and bacteriorhodopsin is indistinguishable from that in native membranes when appropriate conditions are used. We also show that the kinetics and extent of monolayer binding of myristoylated recoverin is much faster than that of the nonmyristoylated form and that this binding is highly favored by the presence polyunsaturated phospholipids. Moreover, we show that the use of fragments of RPE65 allow determine which region of this protein is most likely involved in membrane binding. Monomolecular films were also used to further understand the hydrolysis of organized phospholipids by phospholipases A2 and C.

Journal of Cellular Biochemistry, 2001

Isolated glycosylphosphatidylinositol (GPI)-anchored proteins, when added to cells in vitro, incorporate into their surface membranes and, once incorporated, exert their native functions. Virtually any protein of interest, if expressed as a GPI-reanchored derivative, can be modi®ed to acquire this capacity. Such transfer of proteins directly to cells, termed``protein engineering'' or``painting'' constitutes an alternative to conventional gene transfer for manipulating cell surface composition that has many potential applications. Previous studies with incorporated GPIanchored proteins have focused almost entirely on their extracellular functions. In this study, biotinylated human erythrocyte (E hu ) decay accelerating factor, E hu acetylcholinesterase, and GPI-reanchored murine B7-1 and B7-2 were used as GPI-anchored reporters to characterize their plasma membrane organization and cell signalling properties following addition to Hela or Chinese hamster ovary cells. For each reporter, three types of cell-association were documented; (1) nonphysiological attachment and/or incomplete insertion, (2) uncomplexed membrane integration, and (3) organization into TX-100-resistant microdomains. Transit from the ®rst two compartments into the third, i.e., microdomains, progressed slowly, continuing even after 24 to 36 h and was associated with the acquisition of cell signalling capacity. All four reporters, incorporated in two different detergents, behaved similarly. When organized in microdomains, caveolin and other GPI proteins co-isolated with the incorporated reporter. These results have implications for protein engineering of cells in general, and in particular, for cells such as modi®ed tumor cell immunogens administered to patients for therapeutic purposes.

Colloids and Surfaces B: Biointerfaces, 2007

Two decapeptide fragments of the non-structural hepatitis G NS3 protein (GBV-C/HGV), 513-522 (RGRTGRGRSG) and 505-514 (SAELSMQRRG), as well as their palmitoylated derivatives were synthesized. The physico-chemical properties of the peptides were analyzed in both the absence and presence of the zwitterionic 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), the negative 1,2-dipalmitoyl-snglycero-3-[phospho-rac-(1-glycerol)] (DPPG) and the positive 1,2-dioeloyl-3-trimethylammonium-propane (DOTAP) lipid monolayers. Based on their high hydrophilic properties, neither parent peptide presented surface activity and their incorporation into lipid monolayers was low. In contrast, their palmitoylated derivatives showed concentration-dependent surface activity and could be inserted into lipid monolayers to varying degrees depending on their sequence. Compression isotherms showed that the presence of palmitoylated peptides in the subphase resulted in a molecular arrangement less condensed than that corresponding to the pure phospholipid. In concordance with the monolayer results, differential scanning calorimetry (DSC) demonstrated that the parent peptides did not have any effect on the thermograms, while the palmitoylated derivatives affected the thermotropic properties of DPPC bilayers.

Biochimica et Biophysica Acta (BBA) - Biomembranes, 2009

Knowledge about the vertical movement of a protein with respect to the lipid bilayer plane is important to understand protein functionality in the biological membrane. In this work, the vertical displacement of bacteriophage M13 major coat protein in a lipid bilayer is used as a model system to study the molecular details of its anchoring mechanism in a homologue series of lipids with the same polar head group but different hydrophobic chain length. The major coat proteins were reconstituted into 14:1PC, 16:1PC, 18:1PC, 20:1PC, and 22:1PC bilayers, and the fluorescence spectra were measured of the intrinsic tryptophan at position 26 and BADAN attached to an introduced cysteine at position 46, located at the opposite ends of the transmembrane helix. The fluorescence maximum of tryptophan shifted for 700 cm-1 on going from 14:1PC to 22:1PC, the corresponding shift of the fluorescence maximum of BADAN at position 46 was approximately 10 times less (∼ 70 cm-1). Quenching of fluorescence with the spin label CAT 1 indicates that the tryptophan is becoming progressively inaccessible for the quencher with increasing bilayer thickness, whereas quenching of BADAN attached to the T46C mutant remained approximately unchanged. This supports the idea that the BADAN probe at position 46 remains at the same depth in the bilayer irrespective of its thickness and clearly indicates an asymmetrical nature of the protein dipping in the lipid bilayer. The anchoring strength at the C-terminal domain of the protein (provided by two phenylalanine residues together with four lysine residues) was estimated to be roughly 5 times larger than the anchoring strength of the N-terminal domain.