Site-controlled Ag nanocrystals grown by molecular beam epitaxy-Towards plasmonic integration technology

We demonstrate site-controlled growth of epitaxial Ag nanocrystals on patterned GaAs substrates by molecular beam epitaxy with high degree of long-range uniformity. The alignment is based on lithographically defined holes in which position controlled InAs quantum dots are grown. The Ag nanocrystals self-align preferentially on top of the InAs quantum dots. No such ordering is observed in the absence of InAs quantum dots, proving that the ordering is strain-driven. The presented technique facilitates the placement of active plasmonic nanostructures at arbitrarily defined positions enabling their integration into complex devices and plasmonic circuits.

From ab initio quantum mechanics to molecular neurobiology: A cation–π binding site in the nicotinic receptor

The nicotinic acetylcholine receptor is the prototype ligand-gated ion channel. A number of aromatic amino acids have been identified as contributing to the agonist binding site, suggesting that cation–π interactions may be involved in binding the quaternary ammonium group of the agonist, acetylcholine. Here we show a compelling correlation between: (i) ab initio quantum mechanical predictions of cation–π binding abilities and (ii) EC50 values for acetylcholine at the receptor for a series of tryptophan derivatives that were incorporated into the receptor by using the in vivo nonsense-suppression method for unnatural amino acid incorporation. Such a correlation is seen at one, and only one, of the aromatic residues—tryptophan-149 of the α subunit. This finding indicates that, on binding, the cationic, quaternary ammonium group of acetylcholine makes van der Waals contact with the indole side chain of α tryptophan-149, providing the most precise structural information to date on this receptor. Consistent with this model, a tethered quaternary ammonium group emanating from position α149 produces a constitutively active receptor.

Identification of the binding site for Gqα on its effector Bruton’s tyrosine kinase

Heterotrimeric G proteins and tyrosine kinases are two major cellular signal transducers. Although G proteins are known to activate tyrosine kinases, the activation mechanism is not clear. Here, we demonstrate that G protein Gqα binds directly to the nonreceptor Bruton’s tyrosine kinase (Btk) to a region composed of a Tec-homology (TH) domain and a sarcoma virus tyrosine kinase (Src)-homology 3 (SH3) domain both in vitro and in vivo. Only active GTP-bound Gqα, not inactive GDP-bound Gqα, can bind to Btk. Mutations of Btk that disrupt its ability to bind Gqα also eliminate Btk stimulation by Gqα, suggesting that this interaction is important for Btk activation. Remarkably, the structure of this TH (including a proline-rich sequence) -SH3 fragment of the Btk family of tyrosine kinases shows an intramolecular interaction. Furthermore, the crystal structure of the Src family of tyrosine kinases reveals that the intramolecular interaction of SH3 and its ligand is the major determining factor keeping the kinase inactive. Thus, we propose an activation model that entails binding of Gqα to the TH-SH3 region, thereby disrupting the TH-SH3 intramolecular interaction and activating Btk.

Conversion of agonist site to metal-ion chelator site in the β2-adrenergic receptor

Previously metal-ion sites have been used as structural and functional probes in seven transmembrane receptors (7TM), but as yet all the engineered sites have been inactivating. Based on presumed agonist interaction points in transmembrane III (TM-III) and -VII of the β2-adrenergic receptor, in this paper we construct an activating metal-ion site between the amine-binding Asp-113 in TM-III—or a His residue introduced at this position—and a Cys residue substituted for Asn-312 in TM-VII. No increase in constitutive activity was observed in the mutant receptors. Signal transduction was activated in the mutant receptors not by normal catecholamine ligands but instead either by free zinc ions or by zinc or copper ions in complex with small hydrophobic metal-ion chelators. Chelation of the metal ions by small hydrophobic chelators such as phenanthroline or bipyridine protected the cells from the toxic effect of, for example Cu2+, and in several cases increased the affinity of the ions for the agonistic site. Wash-out experiments and structure–activity analysis indicated, that the high-affinity chelators and the metal ions bind and activate the mutant receptor as metal ion guided ligand complexes. Because of the well-understood binding geometry of the small metal ions, an important distance constraint has here been imposed between TM-III and -VII in the active, signaling conformation of 7TM receptors. It is suggested that atoxic metal-ion chelator complexes could possibly in the future be used as generic, pharmacologic tools to switch 7TM receptors with engineered metal-ion sites on or off at will.

Preferential interaction of the his pause RNA hairpin with RNA polymerase β subunit residues 904–950 correlates with strong transcriptional pausing

RNA secondary structures (hairpins) that form as the nascent RNA emerges from RNA polymerase are important components of many signals that regulate transcription, including some pause sites, all ρ-independent terminators, and some antiterminators. At the his leader pause site, a 5-bp-stem, 8-nt-loop pause RNA hairpin forms 11 nt from the RNA 3′ end and stabilizes a transcription complex conformation slow to react with NTP substrate. This stabilization appears to depend at least in part on an interaction with RNA polymerase. We tested for RNA hairpin interaction with the paused polymerase by crosslinking 5-iodoUMP positioned specifically in the hairpin loop. In the paused conformation, strong and unusual crosslinking of the pause hairpin to β904–950 replaced crosslinking to β′ and to other parts of β that occurred in nonpaused complexes prior to hairpin formation. These changes in nascent RNA interactions may inhibit reactive alignment of the RNA 3′ end in the paused complex and be related to events at ρ-independent terminators.

Propeptide and glutamate-containing substrates bound to the vitamin K-dependent carboxylase convert its vitamin K epoxidase function from an inactive to an active state

The vitamin K-dependent γ-glutamyl carboxylase catalyzes the posttranslational conversion of glutamic acid to γ-carboxyglutamic acid in precursor proteins containing the γ-carboxylation recognition site (γ-CRS). During this reaction, glutamic acid is converted to γ-carboxyglutamic acid while vitamin KH2 is converted to vitamin K 2,3-epoxide. Recombinant bovine carboxylase was purified free of γ-CRS-containing propeptide and endogenous substrate in a single-step immunoaffinity procedure. We show that in the absence of γ-CRS-containing propeptide and/or glutamate-containing substrate, carboxylase has little or no epoxidase activity. Epoxidase activity is induced by Phe-Leu-Glu-Glu-Leu (FLEEL) (9.2 pmol per min per pmol of enzyme), propeptide, residues −18 to −1 of proFactor IX (3.4 pmol per min per pmol of enzyme), FLEEL and propeptide (100 pmol per min per pmol of enzyme), and proPT28 (HVFLAPQQARSLLQRVRRANTFLEEVRK, residues −18 to +10 of human acarboxy-proprothrombin), (5.3 pmol per min per pmol of enzyme). These results indicate that in the absence of propeptide or glutamate-containing substrate, oxygenation of vitamin K by the carboxylase does not occur. Upon addition of propeptide or glutamate-containing substrate, the enzyme is converted to an active epoxidase. This regulatory mechanism prevents the generation of a highly reactive vitamin K intermediate in the absence of a substrate for carboxylation.

Immunotargeting of liposomes to activated vascular endothelial cells: A strategy for site-selective delivery in the cardiovascular system

Endothelial-selective delivery of therapeutic agents, such as drugs or genes, would provide a useful tool for modifying vascular function in various disease states. A potential molecular target for such delivery is E-selectin, an endothelial-specific cell surface molecule expressed at sites of activation in vivo and inducible in cultured human umbilical vein endothelial cells (HUVEC) by treatment with cytokines such as recombinant human interleukin 1β (IL-1β). Liposomes of various types (classical, sterically stabilized, cationic, pH-sensitive), each conjugated with mAb H18/7, a murine monoclonal antibody that recognizes the extracellular domain of E-selectin, bound selectively and specifically to IL-1β-activated HUVEC at levels up to 275-fold higher than to unactivated HUVEC. E-selectin-targeted immunoliposomes appeared in acidic, perinuclear vesicles 2–4 hr after binding to the cell surface, consistent with internalization via the endosome/lysosome pathway. Activated HUVEC incubated with E-selectin-targeted immunoliposomes, loaded with the cytotoxic agent doxorubicin, exhibited significantly decreased cell survival, whereas unactivated HUVEC were unaffected by such treatment. These results demonstrate the feasibility of exploiting cell surface activation markers for the endothelial-selective delivery of biologically active agents via immunoliposomes. Application of this targeting approach in vivo may lead to novel therapeutic strategies in the treatment of cardiovascular disease.