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. Aggregations of GUVs into multiclusters were also achieved by introduction of bispyridyl-ligand substituted peptides into GUV-membranes succeeded by rhodium diacetate mediated vesicle clustering and, furthermore, by coinstalling a biotin moiety streptavidin addition attenuating the clustering effect visualized by formation of compact superaggregated GUV-multiclusters. Contacting between two different GUV-populations, i.e., GUV-heteroconnection, was achieved by trans-GUV phenyl ester-hydrazine ligations producing GUV-heteroclusters. Indirectly, GUV-clustering was achieved by strain-promoted azide-alkyne cycloaddition (SPAAC) reacting bicyclononyne (BCN)-GUVs with azido-GlcNAc succeeded by biotinylated wheat germ agglutinin (WGA)-lectin/streptavidin incubation arousing cross-binding of GUVs.

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 = 1.3 × 10-8 M) and no cross-reactivity to either sialyl-Tn epitope or blood group A antigens, scFv G2-D11 is an excellent candidate for a well-defined anti-Tn-antigen reagent. Detailed immunohistochemical evaluation of tissue sections from a cohort of 80 patients with gastric carcinoma showed in all cases positive tumor cells. The observed staining was localized to the cytoplasm and in some cases to the membrane, whereas the surrounding tissue was completely negative demonstrating the usefulness of the novel Tn-antigen binding antibody.

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. The development of a facile general synthetic procedure for the synthesis of BODIPY-labeled cholesterylated glycopeptides, which can coat cell-size giant unilamellar vesicles (GUVs), is described. These peptide constructs were synthesized by: 1) solid-phase peptide synthesis (SPPS) using cholesterylated Fmoc-amino acids (Fmoc=9-fluorenylmethoxycarbonyl) followed by tandem click reactions, 2) attachment of a BODIPY-bicyclononyne (BCN) (prepared by Mitsunobu chemistry via novel aryl BCN-ethers) in the absence of a catalyst, and 3) glycosylation by means of copper(I)-catalyzed click reaction of an azidoglycan. Seven different GUV-glycoforms were prepared and four of these were evaluated with their corresponding four specific anti-glycan binding lectins.

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. We succeeded in integrating the glyco modules into giant unilamellar vesicles by electroformation or infusion in buffer solution. The glyco-decorated liposomes were recognized by a lectin and had unique topological membrane features. In conclusion, this work is a proof of principle for the functionalization of artificial membranes with a primitive synthetic glycocalyx useful for studying carbohydrate-protein interactions on a simplified cell-like membrane surface.

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. However, the binding orientation of fluoxetine is reversed in our experimentally supported model compared with the LeuBAT structures, emphasizing the need for careful experimental verification when extrapolating findings from crystal structures of bacterial transporters to human relatives. We find that the selectivity of fluoxetine and nisoxetine, a NET selective structural congener of fluoxetine, is controlled by residues in different regions of the transporters, indicating a complex mechanism for selective recognition of structurally similar compounds in SERT and NET. Our findings add important new information on the molecular basis for SERT/NET selectivity of antidepressants, and provide the first assessment of the potential of LeuBAT as a model system for antidepressant binding in human transporters, which is essential for future structure-based drug development of antidepressant drugs with fine-tuned transporter selectivity.

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. We reacted the ethylthioester of hippuric acid with cyclohexylamine in the absence or presence of various quantities of thiophenol (PhSH) in a slurry of disodium hydrogen phosphate in dry DMF. Quantitative conversions into the resulting amide were observed within a few hours in the presence of equimolar amounts of thiophenol. Ab initio calculations showed that the reaction mechanism in DMF is similar to the well-known aqueous reaction mechanism. The energy barrier of the catalyzed amidation reaction is approximately 40 kJ mol(-1) lower than the non-catalyzed amidation reaction. At least partially this can be explained by a hydrogen bond from the amine to the π-electrons of the thiophenol, stabilizing the transition state in the aromatic thioester amidation reaction. Under similar conditions, cysteine-free ligation was achieved by coupling a fully side-chain protected 15 amino acid phosphopeptide thioester to the free N-terminal of a side-chain protected 9 amino acid peptide producing the corresponding 24 amino acid phosphopeptide.

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. A systematic structure-activity relationship study allowed identification of the substituents that control activity and selectivity toward SERT and NET and revealed a common pattern showing that SERT and NET have opposite preference for the stereochemical configuration of these inhibitors. Mutational analysis of nonconserved SERT/NET residues within the central substrate binding site was performed to determine the molecular basis for inhibitor selectivity. Changing only five residues in NET to the complementary residues in SERT transferred a SERT-like affinity profile for R- and S-citalopram into NET, showing that the selectivity of these compounds is determined by amino acid differences in the central binding site of the transporters. In contrast, the activity of R- and S-talopram was largely unaffected by any mutations within the central substrate binding site of SERT and NET and in the outer vestibule of NET, suggesting that citalopram and talopram bind to distinct sites on SERT and NET. Together, these findings provide important insight into the molecular basis for SERT/NET selectivity of antidepressants, which can be used to guide rational development of unique transporter inhibitors with fine-tuned transporter selectivity.

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 ß-strands to form an extended antiparallel ß-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. Using amide-to-ester substitutions to perturb the backbone hydrogen-bonding pattern, we have systematically probed putative backbone hydrogen bonds between four different PDZ domains and peptides corresponding to natural protein ligands. Amide-to-ester mutations of the three C-terminal amides of the peptide ligand severely affected the affinity with the PDZ domain, demonstrating that hydrogen bonds contribute significantly to ligand binding (apparent changes in binding energy, ΔΔG = 1.3 to >3.8 kcal mol(-1)). This decrease in affinity was mainly due to an increase in the dissociation rate constant, but a significant decrease in the association rate constant was found for some amide-to-ester mutations suggesting that native hydrogen bonds have begun to form in the transition state of the binding reaction. This study provides a general framework for studying the role of backbone hydrogen bonds in protein-peptide interactions and for the first time specifically addresses these for PDZ domain-peptide interactions.

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, ß-alanine or γ-aminobutyric acid (GABA) as N-terminal residues mono-nosylation was observed under the same conditions. Moreover, bis-nosylation was independent of the type of resin, neighboring amino acid and nature of the peptide. Calculations suggest that the reason for the bis-nosylation is the fact that the deprotonated mono-nosyl species is particularly stable in the case of the terminal Gly residue because the N(-) residue can become closer to the SO(2) unit. Finally, the mono-nosylated N-terminal glycine could be obtained by careful optimization of the procedure, adding only one equivalent of 2-nitrobenzenesulfonyl chloride.

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 10.1 microM). Mutational analysis, together with computational docking of the compound in simulations starting from the PDZ domain structure, identified the binding mode of FSC231. The specificity of FSC231 for the PICK1 PDZ domain was supported by the lack of binding to PDZ domains of postsynaptic density protein 95 (PSD-95) and glutamate receptor interacting protein 1 (GRIP1). Pretreatment of cultured hippocampal neurons with FSC231 inhibited coimmunopreciptation of the AMPA receptor GluR2 subunit with PICK1. In agreement with inhibiting the role of PICK1 in GluR2 trafficking, FSC231 accelerated recycling of pHluorin-tagged GluR2 in hippocampal neurons after internalization in response to NMDA receptor activation. FSC231 blocked the expression of both long-term depression and long-term potentiation in hippocampal CA1 neurons from acute slices, consistent with inhibition of the bidirectional function of PICK1 in synaptic plasticity. Given the proposed role of the PICK1/AMPA receptor interaction in neuropathic pain, excitotoxicity, and cocaine addiction, FSC231 might serve as a lead in the future development of new therapeutics against these conditions.

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. Thereby, docking studies were used to correlate biological affinities with structural considerations for ligand-protein interactions. The most potent analogue obtained in this study showed an improvement in affinity compared to 1 and is currently a lead in further studies of PICK1 inhibition.

Single-electron transistor of a single organic molecule with access to several redox states.

A combination of classical Coulomb charging, electronic level spacings, spin, and vibrational modes determines the single-electron transfer reactions through nanoscale systems connected to external electrodes by tunnelling barriers. Coulomb charging effects have been shown to dominate such transport in semiconductor quantum dots, metallic and semiconducting nanoparticles, carbon nanotubes, and single molecules. Recently, transport has been shown to be also influenced by spin--through the Kondo effect--for both nanotubes and single molecules, as well as by vibrational fine structure. Here we describe a single-electron transistor where the electronic levels of a single pi-conjugated molecule in several distinct charged states control the transport properties. The molecular electronic levels extracted from the single-electron-transistor measurements are strongly perturbed compared to those of the molecule in solution, leading to a very significant reduction of the gap between the highest occupied molecular orbital and the lowest unoccupied molecular orbital. We suggest, and verify by simple model calculations, that this surprising effect could be caused by image charges generated in the source and drain electrodes resulting in a strong localization of the charges on the molecule.

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. The most pronounced intermolecular interactions are two hydrogen bonds of the type C-H⋯π, which determine a herring-bone pattern in the crystal packing.

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.

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. The role of the ABO blood group antigens in relation to the phenomenon has previously only been investigated in clinical isolates obtained from malaria patients. Here, we aim to clarify their role using synthetic ABO-decorated giant unilamellar vesicles (GUVs), which serve as simple biomimetic models of RBC-size cell membranes. Our results suggest clearly and for the first time that the blood group A and O antigens have a direct impact on receptor-specific rosetting phenomena when compared to the B antigen, which only participates in rosetting to an insignificant degree. Thus, glycodecorated GUVs represent a practical tool for studying cell-surface interactions.

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.

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. A large number of scFv clones, in supernatants, are printed on top of the capture antibody and the antigen in a "spot-on-spot" print. The printed scFv clones, which bind to the capture antibody, are detected using biotinylated antigen, while the binding of scFv clones to the printed antigen is detected through a mouse anti-tag antibody. Two different analyses are thus performed on the same slide, generating two kinds of information: one on the ability of an individual scFv clone to bind to the soluble form of the antigen, which may favour selection for higher affinity rather than avidity, while the other allows the identification of large numbers of clones, simultaneously, due to the binding of scFv clones to densely presented antigens, thus providing an overall increased hit rate. The functionality of the new screening approach was illustrated through the generation of antibodies against peptides from the chaperone complex Ku70/Ku80 and the GalNAcα-serine/threonine epitope on the IgA1 alpha chain hinge region. In total, 659 scFv clones were screened with a hit rate of approximately 20%. This approach allowed the identification of functional antibodies in both cases, illustrating the usefulness and capacity of this combinatory microarray screening technique for efficient analysis and validation of antibodies at an early stage of antibody generation.

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. An optimized bivalent ligand comprising two serotonin moieties binds SERT with 3,800-fold increased affinity compared to that of serotonin, suggesting that the human transporters have two distinct substrate binding sites. We show that the bivalent ligands are inhibitors of SERT and an experimentally validated docking model suggests that the bivalent compounds bind with one substrate moiety in the central binding site (the S1 site), whereas the other substrate moiety binds in a distinct binding site (the S2 site). A systematic study of nonconserved SERT/DAT residues surrounding the proposed binding region showed that nonconserved binding site residues do not contribute to selective recognition of substrates in SERT or DAT. This study provides novel insight into the molecular basis for substrate recognition in human transporters and provides an improved foundation for the development of new drugs targeting SERT and DAT.

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. We found that peripheral tissues and the liver in particular are able to take up 2-monoacylglycerols as seen from (3)H uptake. In muscle and adipose tissue, 2-monoacylglycerols are probably further hydrolyzed as seen by an increased (3)H/(14)C ratio, whereas in the liver and the heart, data suggest that they are also subjected to re-esterification to triacylglycerol, as seen by an unchanged (3)H/(14)C ratio in the lipid fraction of the tissues. Our findings suggest that LPL-generated 2-monoacylglycerol is likely to be stable in the vascular system and thus have a potential to circulate or at least exert effects in tissues where it may be locally produced.

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. In addition, we transferred the gold nanorod chains from the assembly template onto a Si/SiO2 target substrate, thus establishing a platform for a variety of nanoscale electronic and optical applications ranging from molecular electronics to optical and plasmonic devices. As a first example, electrical measurements are performed on contacted gold nanorod chains before and after their immersion in a solution of thiol end-capped oligophenylenevinylene molecules showing an increase in the conductance by three orders of magnitude, indicating molecular-mediated transport.