Clustering of Giant Unilamellar Vesicles Promoted by Covalent and Noncovalent Bonding of Functional Groups at Membrane-Embedded Peptides.

Access to clusters of cell-sized globular objects such as giant unilamellar vesicles (GUVs) is of increasing interest due to their potential applications in prototissue and cell-cell adhesion studies. Aggregations of GUVs by four different approaches were observed via covalent as well as noncovalent bond participations of functional groups at membrane embedded cholesterylpeptides using optical microscopy. Passive air oxidation of GUV-surface thiols into trans-GUV disulfide bonds promoted multivesicle aggregation.

ABO Blood Group Antigen Decorated Giant Unilamellar Vesicles Exhibit Distinct Interactions with Plasmodium falciparum Infected Red Blood Cells.

Severe malaria is considered to be the deadliest disease of this century, primarily among children in sub-Saharan Africa. It stems from infection by the virulent parasite Plasmodium falciparum. The pathogenesis of the disease is based on the rosetting phenomenon, which occurs during the life cycle of the parasite in red blood cells (RBCs) and promotes the binding of parasitized RBCs to healthy ones.

Synthesis of Oligo-(alkyne-triplet)peptide Constructs.

Copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) click synthesis of an Fmoc-(trispropargyl)amino acid building block for solid phase peptide synthesis (SPPS) of oligo-(trialkyne)peptide constructs is reported. These can carry potentially indefinite numbers of inherent alkyne-triplets, which are click derivatized with GlcNAc-azide into the corresponding glycopeptides.

Synthesis of BODIPY-Labeled Cholesterylated Glycopeptides by Tandem Click Chemistry for Glycocalyxification of Giant Unilamellar Vesicles (GUVs).

The glycocalyx cover membrane surfaces of all living cells. These complex architectures render their interaction mechanisms on the membrane surface difficult to study. Artificial cell-sized membranes with selected and defined glycosylation patterns may serve as a minimalistic approach to systematically study cell surface glycan interactions.

Epitope mapping of a new anti-Tn antibody detecting gastric cancer cells.

Here, we introduce a novel scFv antibody, G2-D11, specific for two adjacent Tn-antigens (GalNAc-Ser/Thr) binding equally to three dimeric forms of the epitope, Ser-Thr, Thr-Thr and Thr-Ser. Compared to other anti-Tn reagents, the binding of G2-D11 is minimally influenced by the peptide structure, which indicates a high degree of carbohydrate epitope dominance and a low influence from the protein backbone. With a high affinity (KDapp = 1.

A Combinatory Antibody-Antigen Microarray Assay for High-Content Screening of Single-Chain Fragment Variable Clones from Recombinant Libraries.

We have developed a combinatory antibody-antigen microarray for direct screening of multiple single-chain fragment variable (scFv) clones with no need for pre-purification or enrichment before screening. The straightforward workflow allows for early selection of binders to predefined peptide and glycopeptide targets. A capture antibody is contact printed on microarray slides, side by side with the antigens of interest.

Interrogating the Molecular Basis for Substrate Recognition in Serotonin and Dopamine Transporters with High-Affinity Substrate-Based Bivalent Ligands.

The transporters for the neurotransmitters serotonin and dopamine (SERT and DAT, respectively) are targets for drugs used in the treatment of mental disorders and widely used drugs of abuse. Studies of prokaryotic homologues have advanced our structural understanding of SERT and DAT, but it still remains enigmatic whether the human transporters contain one or two high-affinity substrate binding sites. We have designed and employed 24 bivalent ligands possessing a highly systematic combination of substrate moieties (serotonin and/or dopamine) and aliphatic or poly(ethylene glycol) spacers to reveal insight into substrate recognition in SERT and DAT.

Synthesis of Cholesterol-Substituted Glycopeptides for Tailor-Made Glycocalyxification of Artificial Membrane Systems.

Synthetic minimal membrane systems are extremely useful for better understanding of complex cellular structures and cell surface processes. We have developed a facile method for synthesis of cholesterylated peptides, each bearing a carbohydrate moiety and a fluorescent tag. The position of the cholesterol moiety on the peptide can be controlled by using a new Fmoc-protected cholesterol-triazole-lysine group, which we constructed by means of solid-phase peptide synthesis.

Evaluation of the immediate vascular stability of lipoprotein lipase-generated 2-monoacylglycerol in mice.

2-Monoacylglycerols are gaining increasing interest as signaling lipids, beyond endocannabinoids, for example, as ligands for the receptor GPR119 and as mediators of insulin secretion. In the vascular system, they are formed by the action of lipoprotein lipase (LPL); however, their further disposition is unclear. Assuming similar affinity for uptake and incorporation into tissues of 2-oleoylglycerol and 2-oleylglyceryl ether, we have synthesized a (3)H-labeled 2-ether analog of triolein (labeled in alkyl group) and compared its disposition with (14)C-labeled triolein (labeled in glycerol) 20 min after intravenous coadministration in a ratio of 1:1 to mice.

Mechanistic insight into benzenethiol catalyzed amide bond formations from thioesters and primary amines.

The influence of arylthiols on cysteine-free ligation, i.e. the reaction between an alkyl thioester and a primary amine forming an amide bond, was studied in a polar aprotic solvent.

Molecular basis for selective serotonin reuptake inhibition by the antidepressant agent fluoxetine (Prozac).

Inhibitors of the serotonin transporter (SERT) are widely used antidepressant agents, but the structural mechanism for inhibitory activity and selectivity over the closely related norepinephrine transporter (NET) is not well understood. Here we use a combination of chemical, biological, and computational methods to decipher the molecular basis for high-affinity recognition in SERT and selectivity over NET for the prototypical antidepressant drug fluoxetine (Prozac; Eli Lilly, Indianapolis, IN). We show that fluoxetine binds within the central substrate site of human SERT, in agreement with recent X-ray crystal structures of LeuBAT, an engineered monoamine-like version of the bacterial amino acid transporter LeuT.

Deterministic assembly of linear gold nanorod chains as a platform for nanoscale applications.

We demonstrate a method to assemble gold nanorods highly deterministically into a chain formation by means of directed capillary assembly. This way we achieved straight chains consisting of end-to-end aligned gold nanorods assembled in one specific direction with well-controlled gaps of ∼6 nm between the individual constituents. We determined the conditions for optimum quality and yield of nanorod chain assembly by investigating the influence of template dimensions and assembly temperature.

Probing the role of backbone hydrogen bonds in protein-peptide interactions by amide-to-ester mutations.

One of the most frequent protein-protein interaction modules in mammalian cells is the postsynaptic density 95/discs large/zonula occludens 1 (PDZ) domain, involved in scaffolding and signaling and emerging as an important drug target for several diseases. Like many other protein-protein interactions, those of the PDZ domain family involve formation of intermolecular hydrogen bonds: C-termini or internal linear motifs of proteins bind as β-strands to form an extended antiparallel β-sheet with the PDZ domain. Whereas extensive work has focused on the importance of the amino acid side chains of the protein ligand, the role of the backbone hydrogen bonds in the binding reaction is not known.

Synthetic and mechanistic insight into nosylation of glycine residues.

The Fukuyama-Mitsunobu alkylation procedure is widely used to introduce alkyl substituents to amino groups in general and N-alkylation of peptides in particular. Here we have investigated the procedure in detail for N-alkylation of peptides with N-terminal glycine residues, based on the observation that standard conditions lead to substantial bis-nosylation of the glycine amino group. A systematic evaluation of this observation was carried out and it was demonstrated that for peptides with alanine, β-alanine or γ-aminobutyric acid (GABA) as N-terminal residues mono-nosylation was observed under the same conditions.

Molecular determinants for selective recognition of antidepressants in the human serotonin and norepinephrine transporters.

Inhibitors of the serotonin transporter (SERT) and norepinephrine transporter (NET) are widely used in the treatment of major depressive disorder. Although SERT/NET selectivity is a key determinant for the therapeutic properties of these drugs, the molecular determinants defining SERT/NET selectivity are poorly understood. In this study, the structural basis for selectivity of the SERT selective inhibitor citalopram and the structurally closely related NET selective inhibitor talopram is delineated.

Cell-permeable and plasma-stable peptidomimetic inhibitors of the postsynaptic density-95/N-methyl-D-aspartate receptor interaction.

The protein--protein interaction between the NMDA receptor and its intracellular scaffolding protein, PSD-95, is a potential target for treating ischemic brain diseases, neuropathic pain, and Alzheimer's disease. We have previously demonstrated that N-alkylated tetrapeptides are potent inhibitors of this interaction, and here, this template is exploited for the development of blood plasma-stable and cell-permeable inhibitors. Initially, we explored both the amino acid sequence of the tetrapeptide and the nature of the N-alkyl groups, which consolidated N-cyclohexylethyl-ETAV (1) as the most potent and selective compound.

Structure-activity relationships of a small-molecule inhibitor of the PDZ domain of PICK1.

Recently, we described the first small-molecule inhibitor, (E)-ethyl 2-cyano-3-(3,4-dichlorophenyl)acryloylcarbamate (1), of the PDZ domain of protein interacting with Calpha-kinase 1 (PICK1), a potential drug target against brain ischemia, pain and cocaine addiction. Herein, we explore structure-activity relationships of 1 by introducing subtle modifications of the acryloylcarbamate scaffold and variations of the substituents on this scaffold. The configuration around the double bond of 1 and analogues was settled by a combination of X-ray crystallography, NMR and density functional theory calculations.

Identification of a small-molecule inhibitor of the PICK1 PDZ domain that inhibits hippocampal LTP and LTD.

Proteins containing PSD-95/Discs-large/ZO-1 homology (PDZ) domains play key roles in the assembly and regulation of cellular signaling pathways and represent putative targets for new pharmacotherapeutics. Here we describe the first small-molecule inhibitor (FSC231) of the PDZ domain in protein interacting with C kinase 1 (PICK1) identified by a screening of approximately 44,000 compounds in a fluorescent polarization assay. The inhibitor bound the PICK1 PDZ domain with an affinity similar to that observed for endogenous peptide ligands (K(i) approximately 10.

4-Bromo-seleno-anisole.

The title compound, 1-bromo-4-methyl-seleno-benzene, C(7)H(7)BrSe, was prepared by methyl-ation of 4-bromo-seleno-phenolate with methyl iodide, and crystals suitable for structure determination were obtained by sublimation. The mol-ecule is essentially planar; the Se-Me bond is rotated by only 2.59 (19)° out of the least-squares plane of the benzene ring.

1,4-Bis(4-chloro-phenyl-seleno)-2,5-di-methoxy-benzene.

The title compound, C(20)H(16)Cl(2)O(2)Se(2), utilizes the symmetry of the crystallographic inversion center. Mol-ecular chains are formed through symmetric C-H⋯Cl inter-actions around inversion centers, mimicking the commonly observed symmetric hydrogen-bonded dimer pattern often found in carboxylic acids.

Electronic transport in single molecule junctions: control of the molecule-electrode coupling through intramolecular tunneling barriers.

We report on single molecule electron transport measurements of two oligophenylenevinylene (OPV3) derivatives placed in a nanogap between gold (Au) or lead (Pb) electrodes in a field effect transistor device. Both derivatives contain thiol end groups that allow chemical binding to the electrodes. One derivative has additional methylene groups separating the thiols from the delocalized pi-electron system.

Electronic excitations of a single molecule contacted in a three-terminal configuration.

Low-temperature three-terminal transport measurements through a thiol end-capped pi-conjugated molecule have been carried out. Electronic excitations, including zero and finite-bias Kondo-effects, have been observed and studied as a function of magnetic field. Using a simplified two-orbital model, we have accounted for the spin and the electronic configuration of the first four charge states of the molecule.

Molecular three-terminal devices: fabrication and measurements.

Incorporation of a third, gate electrode in the device geometry of molecular junctions necessary to identify the transport mechanism. At present, the most popular technique fabricate three-terminal molecular devices makes use of electromigration. Although it statistical process, we show that control over the gap resistance can be obtained.

(E,E,E)-1,3,5-Tris[4-(acetylsulfanyl)styryl]benzene toluene hemisolvate.

The first crystal structure of a three-terminal sulfur end-capped oligophenylenevinylene, C36H30O3S3 x 0.5C7H8, has been determined at 122 (1) K. The molecular threefold symmetry is not utilized in the crystal structure.

Probing the effects of conjugation path on the electronic transmission through single molecules using scanning tunneling microscopy.

A systematic study of the relationship between the molecular structure of a series of thiol end-capped oligo-phenylenevinylenes (OPVs) and the coherent electronic transmission at the single molecule level was measured by scanning tunneling microscopy (STM). This reveals a significant change in the electronic transparency of various OPV derivatives due to the insertion of a methylene spacer group or due to nitro group substitution. Apparently, changes in the conjugation path through the central benzene ring from para to meta substitution does not have a profound effect on the electronic transparency of the molecules.