Interaction with the Src homology (SH3-SH2) region of the Src-family kinase Hck structures the HIV-1 Nef dimer for kinase activation and effector recruitment.

HIV-1 Nef supports high titer viral replication in vivo and is essential for AIDS progression. Nef function depends on interactions with multiple host cell effectors, including Hck and other Src-family kinases. Here we describe the x-ray crystal structure of Nef in complex with the Hck SH3-SH2 regulatory region to a resolution of 1.

Effector kinase coupling enables high-throughput screens for direct HIV-1 Nef antagonists with antiretroviral activity.

HIV-1 Nef, a critical AIDS progression factor, represents an important target protein for antiretroviral drug discovery. Because Nef lacks intrinsic enzymatic activity, we developed an assay that couples Nef to the activation of Hck, a Src family member and Nef effector protein. Using this assay, we screened a large, diverse chemical library and identified small molecules that block Nef-dependent Hck activity with low micromolar potency.

Damaged DNA induced UV-damaged DNA-binding protein (UV-DDB) dimerization and its roles in chromatinized DNA repair.

UV light-induced photoproducts are recognized and removed by the nucleotide-excision repair (NER) pathway. In humans, the UV-damaged DNA-binding protein (UV-DDB) is part of a ubiquitin E3 ligase complex (DDB1-CUL4A(DDB2)) that initiates NER by recognizing damaged chromatin with concomitant ubiquitination of core histones at the lesion. We report the X-ray crystal structure of the human UV-DDB in a complex with damaged DNA and show that the N-terminal domain of DDB2 makes critical contacts with two molecules of DNA, driving N-terminal-domain folding and promoting UV-DDB dimerization.

First-in-human trial of a STAT3 decoy oligonucleotide in head and neck tumors: implications for cancer therapy.

Unlabelled: Despite evidence implicating transcription factors, including STAT3, in oncogenesis, these proteins have been regarded as "undruggable." We developed a decoy targeting STAT3 and conducted a phase 0 trial. Expression levels of STAT3 target genes were decreased in head and neck cancers following injection with the STAT3 decoy compared with tumors receiving saline control.

Interaction of haptoglobin with hemoglobin octamers based on the mutation Asn78Cys or Gly83Cys.

Octameric hemoglobins have been developed by the introduction of surface cysteines in either the alpha or beta chain. Originally designed as a blood substitute, we report here the structure and ligand binding function; in addition the interaction with haptoglobin was studied. The recombinant Hbs (rHbs) with mutations alpha Asn78Cys or beta Gly83Cys spontaneously form octamers under conditions where the cysteines are oxidized.

HIV-1 Nef interaction influences the ATP-binding site of the Src-family kinase, Hck.

Background: Nef is an HIV-1 accessory protein essential for viral replication and AIDS progression. Nef interacts with a multitude of host cell signaling partners, including members of the Src kinase family. Nef preferentially activates Hck, a Src-family kinase (SFK) strongly expressed in macrophages and other HIV target cells, by binding to its regulatory SH3 domain.

Metal-adeninate vertices for the construction of an exceptionally porous metal-organic framework.

Metal-organic frameworks comprising metal-carboxylate cluster vertices and long, branched organic linkers are the most porous materials known, and therefore have attracted tremendous attention for many applications, including gas storage, separations, catalysis and drug delivery. To increase metal-organic framework porosity, the size and complexity of linkers has increased. Here we present a promising alternative strategy for constructing mesoporous metal-organic frameworks that addresses the size of the vertex rather than the length of the organic linker.

Microfluidic chip-based nanoelectrode array as miniaturized biochemical sensing platform for prostate-specific antigen detection.

A microfluidic biosensor chip with an embedded three-electrode configuration is developed for the study of the voltammetric response of a nanoelectrode array with controlled inter-electrode distance in a nanoliter-scale sample volume. The on-chip three-electrode cell consists of a 5 × 5 array of Au working nanoelectrodes with radii between 60 and 120 nm, a Cl(2)-plasma-treated Ag/AgCl reference electrode, and a Au counter electrode. The nanoelectrode array is fabricated by creating high-aspect-ratio pores through an alumina insulating layer using an I(2) gas-assisted focused-ion-beam (FIB) milling, ion beam sculpting, and electrodeposition of Au.

Structure of the HIV-1 full-length capsid protein in a conformationally trapped unassembled state induced by small-molecule binding.

The capsid (CA) protein plays crucial roles in HIV infection and replication, essential to viral maturation. The absence of high-resolution structural data on unassembled CA hinders the development of antivirals effective in inhibiting assembly. Unlike enzymes that have targetable, functional substrate-binding sites, the CA does not have a known site that affects catalytic or other innate activity, which can be more readily targeted in drug development efforts.

The crystal structure of non-modified and bipyridine-modified PNA duplexes.

Peptide nucleic acid (PNA) is a synthetic analogue of DNA that commonly has an N-aminoethyl glycine backbone. The crystal structures of two PNA duplexes, one containing eight standard nucleobase pairs (GGCATGCC)(2), and the other containing the same nucleobase pairs and a central pair of bipyridine ligands, have been solved with a resolution of 1.22 and 1.

Crystal structure of the Src family kinase Hck SH3-SH2 linker regulatory region supports an SH3-dominant activation mechanism.

Most mammalian cell types depend on multiple Src family kinases (SFKs) to regulate diverse signaling pathways. Strict control of SFK activity is essential for normal cellular function, and loss of kinase regulation contributes to several forms of cancer and other diseases. Previous x-ray crystal structures of the SFKs c-Src and Hck revealed that intramolecular association of their Src homology (SH) 3 domains and SH2 kinase linker regions has a key role in down-regulation of kinase activity.

Crystal structure of chiral gammaPNA with complementary DNA strand: insights into the stability and specificity of recognition and conformational preorganization.

We have determined the structure of a PNA-DNA duplex to 1.7 A resolution by multiple-wavelength anomalous diffraction phasing method on a zinc derivative. This structure represents the first high-resolution 3D view of a hybrid duplex containing a contiguous chiral PNA strand with complete gamma-backbone modification ("gammaPNA").

Nanoelectrodes for biological measurements.

Nanoelectrodes are electrodes with a critical dimension in the range of one to hundreds of nanometers and include individual electrodes, nanoelectrode ensembles, and arrays. Metallic nanowires, carbon nanotubes, magnetic nanoparticles, and metal oxide nanowires have been employed to fabricate nanoelectrodes and platforms. In this review, applications of single electrodes, nanoelectrode arrays, and ensembles are briefly evaluated, with emphasis on biological analysis.

Comparison of the mechanism of toxicity of zinc oxide and cerium oxide nanoparticles based on dissolution and oxidative stress properties.

Nanomaterials (NM) exhibit novel physicochemical properties that determine their interaction with biological substrates and processes. Three metal oxide nanoparticles that are currently being produced in high tonnage, TiO(2), ZnO, and CeO(2), were synthesized by flame spray pyrolysis process and compared in a mechanistic study to elucidate the physicochemical characteristics that determine cellular uptake, subcellular localization, and toxic effects based on a test paradigm that was originally developed for oxidative stress and cytotoxicity in RAW 264.7 and BEAS-2B cell lines.

Structural characterizations of glycerol kinase: unraveling phosphorylation-induced long-range activation.

Glycerol metabolism provides a central link between sugar and fatty acid catabolism. In most bacteria, glycerol kinase plays a crucial role in regulating channel/facilitator-dependent uptake of glycerol into the cell. In the firmicute Enterococcus casseliflavus, this enzyme's activity is enhanced by phosphorylation of the histidine residue (His232) located in its activation loop, approximately 25 A from its catalytic cleft.

Structure of glycerol-3-phosphate dehydrogenase, an essential monotopic membrane enzyme involved in respiration and metabolism.

Sn-glycerol-3-phosphate dehydrogenase (GlpD) is an essential membrane enzyme, functioning at the central junction of respiration, glycolysis, and phospholipid biosynthesis. Its critical role is indicated by the multitiered regulatory mechanisms that stringently controls its expression and function. Once expressed, GlpD activity is regulated through lipid-enzyme interactions in Escherichia coli.

Part II: coordinated biosensors--development of enhanced nanobiosensors for biological and medical applications.

In this review, we summarize recent developments in nanobiosensors and their applications in biology and potential in medical diagnostics. We first highlight the concept of coordinated nanobiosensors, which integrate desirable properties of the individual components: protein machinery for sensitivity and specificity of binding, peptide or nucleic acid chemistry for aligning the various electron-transducing units and the nanoelectrodes for enhancing sensitivity in electronic detection. The fundamental basis of coordinated nanobiosensing is in applying the precise 3D atomic resolution structural information to rationally design and fabricate biosensors with high specificity and sensitivity.

Part I: recent developments in nanoelectrodes for biological measurements.

Biosensors are a type of analytical device that use biological molecules to monitor biorecognition events and interactions. Coupled with the progress in nanotechnologies over recent years, the development of a nanobiosensor based on individual nanoelectrodes and nanoelectrode arrays or nanoelectrode ensembles offers unprecedented avenues for screening and detection at ultrahigh sensitivities. These capabilities provide the basis for a paradigmatic change in biomedical diagnostics and treatment.

Comparison of the abilities of ambient and manufactured nanoparticles to induce cellular toxicity according to an oxidative stress paradigm.

Nanomaterial properties differ from those bulk materials of the same composition, allowing them to execute novel activities. A possible downside of these capabilities is harmful interactions with biological systems, with the potential to generate toxicity. An approach to assess the safety of nanomaterials is urgently required.

Prediction of membrane proteins in Mycobacterium tuberculosis using a support vector machine algorithm.

We report our finding of linear clustering of signal sequences at the N-terminus of M.tb membrane proteins, directing membrane localization. Although it is widely accepted that membrane proteins have signal peptides at the N-terminus, statistical ensemble analysis of Support Vector Machine prediction results indicate that M.

Peptergents: peptide detergents that improve stability and functionality of a membrane protein, glycerol-3-phosphate dehydrogenase.

Toward enhancing in vitro membrane protein studies, we have utilized small self-assembling peptides with detergent properties ("peptergents") to extract and stabilize the integral membrane flavoenzyme, glycerol-3-phosphate dehydrogenase (GlpD), and the soluble redox flavoenzyme, NADH peroxidase (Npx). GlpD is a six transmembrane spanning redox enzyme that catalyzes the oxidation of glycerol-3-phosphate to dihydroxyacetone phosphate. Although detergents such as n-octyl-beta-D-glucpyranoside can efficiently solubilize the enzyme, GlpD is inactivated within days once reconstituted into detergent micelles.

Nanowiring of a redox enzyme by metallized peptides.

A molecular assembly consisting of a redox enzyme, NADH peroxidase, a metallized double-helical peptide, and a gold nanoparticle immobilized onto a gold wire derivatized with a benzenedithiol compound, initiated and conducted redox signals in the presence of H(2)O(2) and NADH. The current generated by the binding of NADH, the electron donor, was transduced through the molecular assembly with apparently little loss of signal to the solution. The currents measured correlate to an electron transfer rate constant on the order of 3,000 s(-1) within each assembly.

The effects of flash-annealing on glycerol kinase crystals.

Reflection profiles from glycerol kinase crystals were analyzed to determine the effect of flash-cooling on mosaicity (eta) and peak intensity in order to reveal changes in mosaic domain structure and composition. The results showed that repeated flash-annealing causes a significant decrease in the averaged mosaicity along with an increase in the overall peak counts of reflections and an enhanced signal-to-noise ratio. Individual reflection-profile analysis revealed a mostly dual domain structure, showing the minimization of one domain as a result of flash-annealing.

Structures of enterococcal glycerol kinase in the absence and presence of glycerol: correlation of conformation to substrate binding and a mechanism of activation by phosphorylation.

The first structure of a glycerol kinase from a Gram-positive organism, Enterococcus casseliflavus, has been determined to 2.8 A resolution in the presence of glycerol and to 2.5 A resolution in the absence of substrate.

A manual nanoscale method for protein crystallization.

To overcome one of the major hurdles in three-dimensional crystal structure determination - the requirement for large quantities of purified material to grow crystals - crystallization methodologies have been developed that require only a total of 2-5 microl of a concentrated macromolecular solution to screen more than 100 conditions. These procedures employ a circular slide containing an array of 25 wells designed for crystallization setups in the nanolitre volume range. These 'crystallization slides' fit into the wells of standard crystallization trays.