High-pressure, high-temperature molecular doping of nanodiamond.

The development of color centers in diamond as the basis for emerging quantum technologies has been limited by the need for ion implantation to create the appropriate defects. We present a versatile method to dope diamond without ion implantation by synthesis of a doped amorphous carbon precursor and transformation at high temperatures and high pressures. To explore this bottom-up method for color center generation, we rationally create silicon vacancy defects in nanodiamond and investigate them for optical pressure metrology. In addition, we show that this process can generate noble gas defects within diamond from the typically inactive argon pressure medium, which may explain the hysteresis effects observed in other high-pressure experiments and the presence of noble gases in some meteoritic nanodiamonds. Our results illustrate a general method to produce color centers in diamond and may enable the controlled generation of designer defects.

From amorphous to nanocrystalline: the effect of nanograins in an amorphous matrix on the thermal conductivity of hot-wire chemical-vapor deposited silicon films.

We have measured the thermal conductivity of amorphous and nanocrystalline silicon films with varying crystalline content from 85 K to room temperature. The films were prepared by the hot-wire chemical-vapor deposition, where the crystalline volume fraction is determined by the hydrogen (H2) dilution ratio to the processing silane gas (SiH4), R = H2/SiH4. We varied R from 1 to 10, where the films transform from amorphous for R < 3 to mostly nanocrystalline for larger R. Structural analyses show that the nanograins, averaging from 2 to 9 nm in sizes with increasing R, are dispersed in the amorphous matrix. The crystalline volume fraction increases from 0 to 65% as R increases from 1 to 10. The thermal conductivities of the two amorphous silicon films are similar and consistent with the most previous reports with thicknesses no larger than a few µm deposited by a variety of techniques. The thermal conductivities of the three nanocrystalline silicon films are also similar, but are about 50-70% higher than those of their amorphous counterparts. The heat conduction in nanocrystalline silicon films can be understood as the combined contribution in both amorphous and nanocrystalline phases, where increased conduction through improved nanocrystalline percolation path outweighs increased interface scattering between silicon nanocrystals and the amorphous matrix.

Chemically exfoliating large sheets of phosphorene via choline chloride urea viscosity-tuning.

Exfoliation of two-dimensional phosphorene from bulk black phosphorous through chemical means is demonstrated where the solvent system of choice (choline chloride urea diluted with ethanol) has the ability to successfully exfoliate large-area multi-layer phosphorene sheets and further protect the flakes from ambient degradation. The intercalant solvent molecules, aided by low-powered sonication, diffuse between the layers of the bulk black phosphorus, allowing for the exfoliation of the multi-layer phosphorene through breaking of the interlayer van der Waals bonds. Through viscosity tuning, the optimal parameters (1:1 ratio between the intercalant and the diluting solvent) at which the exfoliation takes place is determined. Our exfoliation technique is shown to produce multi-layer phosphorene flakes with surface areas greater than 3 µm2 (a factor of three larger than what has previously been reported for a similar exfoliation method) while limiting exposure to the ambient environment, thereby protecting the flakes from degradation. Characterization techniques such as optical microscopy, Raman spectroscopy, ultraviolet-visible spectroscopy, and (scanning) transmission electron microscopy are used to investigate the quality, quantity, and thickness of the exfoliated flakes.

The nature, origin and modification of insoluble organic matter in chondrites, the possibly interstellar source of Earth's C and N.

All chondrites accreted ~3.5 wt.% C in their matrices, the bulk of which was in a macromolecular solvent and acid insoluble organic material (IOM). Similar material to IOM is found in interplanetary dust particles (IDPs) and comets. The IOM accounts for almost all of the C and N in chondrites, and a significant fraction of the H. Chondrites and, to a lesser extent, comets were probably the major sources of volatiles for the Earth and the other terrestrial planets. Hence, IOM was both the major source of Earth's volatiles and a potential source of complex prebiotic molecules. Large enrichments in D and 15N, relative to the bulk solar isotopic compositions, suggest that IOM or its precursors formed in very cold, radiation-rich environments. Whether these environments were in the interstellar medium (ISM) or the outer Solar System is unresolved. Nevertheless, the elemental and isotopic compositions and functional group chemistry of IOM provide important clues to the origin(s) of organic matter in protoplanetary disks. IOM is modified relatively easily by thermal and aqueous processes, so that it can also be used to constrain the conditions in the solar nebula prior to chondrite accretion and the conditions in the chondrite parent bodies after accretion. Here we review what is known about the abundances, compositions and physical nature of IOM in the most primitive chondrites. We also discuss how the IOM has been modified by thermal metamorphism and aqueous alteration in the chondrite parent bodies, and how these changes may be used both as petrologic indicators of the intensity of parent body processing and as tools for classification. Finally, we critically assess the various proposed mechanisms for the formation of IOM in the ISM or Solar System.

Reduction of spin injection efficiency by interface defect spin scattering in ZnMnSe/AlGaAs-GaAs spin-polarized light-emitting diodes.

We report the first experimental demonstration that interface microstructure limits diffusive electrical spin-injection efficiency across heteroepitaxial interfaces. An inverse correlation be-tween spin-polarized electron injection efficiency and interface defect density is demonstrated for ZnMnSe/AlGaAs-GaAs spin-polarized light-emitting diodes that exhibit quantum well spin polarizations up to 85%. A theoretical treatment shows that the suppression of spin injection due to interface defects results from the contribution of the defect potential to the spin-orbit interaction, which increases the spin-flip scattering.

Crystal structure of the macrocycle-forming thioesterase domain of the erythromycin polyketide synthase: versatility from a unique substrate channel.

As the first structural elucidation of a modular polyketide synthase (PKS) domain, the crystal structure of the macrocycle-forming thioesterase (TE) domain from the 6-deoxyerythronolide B synthase (DEBS) was solved by a combination of multiple isomorphous replacement and multiwavelength anomalous dispersion and refined to an R factor of 24.1% to 2.8-A resolution. Its overall tertiary architecture belongs to the alpha/beta-hydrolase family, with two unusual features unprecedented in this family: a hydrophobic leucine-rich dimer interface and a substrate channel that passes through the entire protein. The active site triad, comprised of Asp-169, His-259, and Ser-142, is located in the middle of the substrate channel, suggesting the passage of the substrate through the protein. Modeling indicates that the active site can accommodate and orient the 6-deoxyerythronolide B precursor uniquely, while at the same time shielding the active site from external water and catalyzing cyclization by macrolactone formation. The geometry and organization of functional groups explain the observed substrate specificity of this TE and offer strategies for engineering macrocycle biosynthesis. Docking of a homology model of the upstream acyl carrier protein (ACP6) against the TE suggests that the 2-fold axis of the TE dimer may also be the axis of symmetry that determines the arrangement of domains in the entire DEBS. Sequence conservation suggests that all TEs from modular polyketide synthases have a similar fold, dimer 2-fold axis, and substrate channel geometry.

Multi-targeted antifolates aimed at avoiding drug resistance form covalent closed inhibitory complexes with human and Escherichia coli thymidylate synthases.

Crystal structures of four pyrrolo(2,3-d)pyrimidine-based antifolate compounds, developed as inhibitors of thymidylate synthase (TS) in a strategy to circumvent drug-resistance, have been determined in complexes with their in vivo target, human thymidylate synthase, and with the structurally best-characterized Escherichia coli enzyme, to resolutions of 2.2-3.0 A. The 2.9 A crystal structure of a complex of human TS with one of the inhibitors, the multi-targeted antifolate LY231514, demonstrates that this compound induces a "closed" enzyme conformation and leads to formation of a covalent bond between enzyme and substrate. This structure is one of the first liganded human TS structures, and its solution was aided by mutation to facilitate crystallization. Structures of three other pyrrolo(2,3-d)pyrimidine-based antifolates in complex with Escherichia coli TS confirm the orientation of this class of inhibitors in the active site. Specific interactions between the polyglutamyl moiety and a positively charged groove on the enzyme surface explain the marked increase in affinity of the pyrrolo(2,3-d)pyrimidine inhibitors once they are polyglutamylated, as mediated in vivo by the cellular enzyme folyl polyglutamate synthetase.

Predicting and harnessing protein flexibility in the design of species-specific inhibitors of thymidylate synthase.

BACKGROUND: Protein plasticity in response to ligand binding abrogates the notion of a rigid receptor site. Thus, computational docking alone misses important prospective drug design leads. Bacterial-specific inhibitors of an essential enzyme, thymidylate synthase (TS), were developed using a combination of computer-based screening followed by in-parallel synthetic elaboration and enzyme assay [Tondi et al. (1999) Chem. Biol. 6, 319-331]. Specificity was achieved through protein plasticity and despite the very high sequence conservation of the enzyme between species. RESULTS: The most potent of the inhibitors synthesized, N,O-didansyl-L-tyrosine (DDT), binds to Lactobacillus casei TS (LcTS) with 35-fold higher affinity and to Escherichia coli TS (EcTS) with 24-fold higher affinity than to human TS (hTS). To reveal the molecular basis for this specificity, we have determined the crystal structure of EcTS complexed with DDT and 2'-deoxyuridine-5'-monophosphate (dUMP). The 2.0 A structure shows that DDT binds to EcTS in a conformation not predicted by molecular docking studies and substantially differently than other TS inhibitors. Binding of DDT is accompanied by large rearrangements of the protein both near and distal to the enzyme's active site with movement of C alpha carbons up to 6 A relative to other ternary complexes. This protein plasticity results in novel interactions with DDT including the formation of hydrogen bonds and van der Waals interactions to residues conserved in bacterial TS but not hTS and which are hypothesized to account for DDT's specificity. The conformation DDT adopts when bound to EcTS explains the activity of several other LcTS inhibitors synthesized in-parallel with DDT suggesting that DDT binds to the two enzymes in similar orientations. CONCLUSIONS: Dramatic protein rearrangements involving both main and side chain atoms play an important role in the recognition of DDT by EcTS and highlight the importance of incorporating protein plasticity in drug design. The crystal structure of the EcTS/dUMP/DDT complex is a model system to develop more selective TS inhibitors aimed at pathogenic bacterial species. The crystal structure also suggests a general formula for identifying regions of TS and other enzymes that may be treated as flexible to aid in computational methods of drug discovery.

Atomic structure of a glycerol channel and implications for substrate permeation in aqua(glycero)porins.

The structure of a glycerol channel from Escherichia coli at 2.2 A resolution serves as a basis for the understanding of selective transmembrane substrate permeation. In the course of permeation, glycerol molecules diffuse through a tripathic channel with their alkyl backbone wedged against a hydrophobic corner, such that OH groups become acceptors and donors of hydrogen bonds at the same time. The structure of the channel explains the preferential permeability for linear carbohydrates and absolute exclusion of ions and charged solutes. Its gene-duplicated sequence has a structural counterpart in a pseudo two-fold symmetry within the monomeric channel protein.

The signal recognition particle.

The signal recognition particle (SRP) and its membrane-associated receptor (SR) catalyze targeting of nascent secretory and membrane proteins to the protein translocation apparatus of the cell. Components of the SRP pathway and salient features of the molecular mechanism of SRP-dependent protein targeting are conserved in all three kingdoms of life. Recent advances in the structure determination of a number of key components in the eukaryotic and prokaryotic SRP pathway provide new insight into the molecular basis of SRP function, and they set the stage for future work toward an integrated picture that takes into account the dynamic and contextual properties of this remarkable cellular machine.

Approaches to solving the rigid receptor problem by identifying a minimal set of flexible residues during ligand docking.

BACKGROUND: Using fixed receptor sites derived from high-resolution crystal structures in structure-based drug design does not properly account for ligand-induced enzyme conformational change and imparts a bias into the discovery and design of novel ligands. We sought to facilitate the design of improved drug leads by defining residues most likely to change conformation, and then defining a minimal manifold of possible conformations of a target site for drug design based on a small number of identified flexible residues. RESULTS: The crystal structure of thymidylate synthase from an important pathogenic target Pneumocystis carinii (PcTS) bound to its substrate and the inhibitor, BW1843U89, is reported here and reveals a new conformation with respect to the structure of PcTS bound to substrate and the more conventional antifolate inhibitor, CB3717. We developed an algorithm for determining which residues provide 'soft spots' in the protein, regions where conformational adaptation suggests possible modifications for a drug lead that may yield higher affinity. Remodeling the active site of thymidylate synthase with new conformations for only three residues that were identified with this algorithm yields scores for ligands that are compatible with experimental kinetic data. CONCLUSIONS: Based on the examination of many protein/ligand complexes, we develop an algorithm (SOFTSPOTS) for identifying regions of a protein target that are more likely to accommodate plastically to regions of a drug molecule. Using these indicators we develop a second algorithm (PLASTIC) that provides a minimal manifold of possible conformations of a protein target for drug design, reducing the bias in structure-based drug design imparted by structures of enzymes co-crystallized with inhibitors.

Structure of Cry2Aa suggests an unexpected receptor binding epitope.

BACKGROUND: Genetically modified (GM) crops that express insecticidal protein toxins are an integral part of modern agriculture. Proteins produced by Bacillus thuringiensis (Bt) during sporulation mediate the pathogenicity of Bt toward a spectrum of insect larvae whose breadth depends upon the Bt strain. These transmembrane channel-forming toxins are stored in Bt as crystalline inclusions called Cry proteins. These proteins are the active agents used in the majority of biorational pesticides and insect-resistant transgenic crops. Though Bt toxins are promising as a crop protection alternative and are ecologically friendlier than synthetic organic pesticides, resistance to Bt toxins by insects is recognized as a potential limitation to their application. RESULTS: We have determined the 2.2 A crystal structure of the Cry2Aa protoxin by multiple isomorphous replacement. This is the first crystal structure of a Cry toxin specific to Diptera (mosquitoes and flies) and the first structure of a Cry toxin with high activity against larvae from two insect orders, Lepidoptera (moths and butterflies) and Diptera. Cry2Aa also provides the first structure of the proregion of a Cry toxin that is cleaved to generate the membrane-active toxin in the larval gut. CONCLUSIONS: The crystal structure of Cry2Aa reported here, together with chimeric-scanning and domain-swapping mutagenesis, defines the putative receptor binding epitope on the toxin and so may allow for alteration of specificity to combat resistance or to minimize collateral effects on nontarget species. The putative receptor binding epitope of Cry2Aa identified in this study differs from that inferred from previous structural studies of other Cry toxins.

Enzyme-catalyzed therapeutic agent (ECTA) design: activation of the antitumor ECTA compound NB1011 by thymidylate synthase.

The in vivo administration of enzyme-inhibiting drugs for cancer and infectious disease often results in overexpression of the targeted enzyme. We have developed an enzyme-catalyzed therapeutic agent (ECTA) approach in which an enzyme overexpressed within the resistant cells is recruited as an intracellular catalyst for converting a relatively non-toxic substrate to a toxic product. We have investigated the potential of the ECTA approach to circumvent the thymidylate synthase (TS) overexpression-based resistance of tumor cells to conventional fluoropyrimidine [i.e. 5-fluorouracil (5-FU)] cancer chemotherapy. (E)-5-(2-Bromovinyl)-2'-deoxy-5'-uridyl phenyl L-methoxyalaninylphosphoramidate (NB1011) is a pronucleotide analogue of (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVdU), an antiviral agent known to be a substrate for TS when in the 5'-monophosphorylated form. NB1011 was synthesized and found to be at least 10-fold more cytotoxic to 5-FU-resistant, TS-overexpressing colorectal tumor cells than to normal cells. This finding demonstrates that the ECTA approach to the design of novel chemotherapeutics results in compounds that are selectively cytotoxic to tumor cell lines that overexpress the target enzyme, TS, and therefore may be useful in the treatment of fluoropyrimidine-resistant cancer.

Functional consequences of the Kaposi's sarcoma-associated herpesvirus protease structure: regulation of activity and dimerization by conserved structural elements.

The structure of Kaposi's sarcoma-associated herpesvirus protease (KSHV Pr), at 2.2 A resolution, reveals the active-site geometry and defines multiple possible target sites for drug design against a human cancer-producing virus. The catalytic triad of KSHV Pr, (Ser114, His46, and His157) and transition-state stabilization site are arranged as in other structurally characterized herpesviral proteases. The distal histidine-histidine hydrogen bond is solvent accessible, unlike the situation in other classes of serine proteases. As in all herpesviral proteases, the enzyme is active only as a weakly associated dimer (K(d) approximately 2 microM), and inactive as a monomer. Therefore, both the active site and dimer interface are potential targets for antiviral drug design. The dimer interface in KSHV Pr is compared with the interface of other herpesviral proteases. Two conserved arginines (Arg209), one from each monomer, are buried within the same region of the dimer interface. We propose that this conserved arginine may provide a destabilizing element contributing to the tuned micromolar dissociation of herpesviral protease dimers.

Structure of a glycerol-conducting channel and the basis for its selectivity.

Membrane channel proteins of the aquaporin family are highly selective for permeation of specific small molecules, with absolute exclusion of ions and charged solutes and without dissipation of the electrochemical potential across the cell membrane. We report the crystal structure of the Escherichia coli glycerol facilitator (GlpF) with its primary permeant substrate glycerol at 2.2 angstrom resolution. Glycerol molecules line up in an amphipathic channel in single file. In the narrow selectivity filter of the channel the glycerol alkyl backbone is wedged against a hydrophobic corner, and successive hydroxyl groups form hydrogen bonds with a pair of acceptor, and donor atoms. Two conserved aspartic acid-proline-alanine motifs form a key interface between two gene-duplicated segments that each encode three-and-one-half membrane-spanning helices around the channel. This structure elucidates the mechanism of selective permeability for linear carbohydrates and suggests how ions and water are excluded.

Replacement set mutagenesis of the four phosphate-binding arginine residues of thymidylate synthase.

Arginines R23, R178, R179 and R218 in thymidylate synthase (TS, EC 2. 1.1.45) are hydrogen bond donors to the phosphate moiety of the substrate, dUMP. In order to investigate how these arginines contribute to enzyme function, we prepared complete replacement sets of mutants at each of the four sites in Lactobacillus casei TS. Mutations of R23 increase K:(m) for dUMP 2-20-fold, increase K:(m) for cofactor 8-40-fold and decrease k(cat) 9-20-fold, reflecting the direct role of the R23 side chain in binding and orienting the cofactor in ternary complexes of the enzyme. Mutations of R178 increase K:(m) for dUMP 40-2000-fold, increase K:(m) for cofactor 3-20-fold and do not significantly affect k(cat). These results are consistent with the fact that this residue is an integral part of the dUMP-binding wall and contributes to the orientation and ordering of several other dUMP binding residues. Kinetic parameters for all R179 mutations except R179P were not significantly different from wild-type values, reflecting the fact that this external arginine does not directly contact the cofactor or other ligand-binding residues. R218 is essential for the structure of the catalytic site and all mutations of this arginine except R218K were inactive.

Mesoscopic surfactant organization and membrane protein crystallization.

The paucity of detailed X-ray crystallographic structures of integral membrane proteins arises from substantive technical obstacles in the overexpression of multimilligram quantities of protein, and in the crystallization of purified protein-detergent complexes (PDCs). With rare exception, crystal contacts within the lattice are mediated by protein-protein interaction, and the detergent surrounding the protein behaves as a disordered solvent. The addition and use of surfactants that display mesoscopic self-assembly behavior in membrane protein crystallization experiments presents a novel alternative strategy. Well-ordered crystals of the water channel human aquaporin-1 (hAQP1) that diffract to 4 A resolution have been obtained with this approach.

Site-directed ligand discovery.

We report a strategy (called "tethering") to discover low molecular weight ligands ( approximately 250 Da) that bind weakly to targeted sites on proteins through an intermediary disulfide tether. A native or engineered cysteine in a protein is allowed to react reversibly with a small library of disulfide-containing molecules ( approximately 1,200 compounds) at concentrations typically used in drug screening (10 to 200 microM). The cysteine-captured ligands, which are readily identified by MS, are among the most stable complexes, even though in the absence of the covalent tether the ligands may bind very weakly. This method was applied to generate a potent inhibitor for thymidylate synthase, an essential enzyme in pyrimidine metabolism with therapeutic applications in cancer and infectious diseases. The affinity of the untethered ligand (K(i) approximately 1 mM) was improved 3,000-fold by synthesis of a small set of analogs with the aid of crystallographic structures of the tethered complex. Such site-directed ligand discovery allows one to nucleate drug design from a spatially targeted lead fragment.

Crystal structure of the HIV-1 integrase catalytic core and C-terminal domains: a model for viral DNA binding.

Insolubility of full-length HIV-1 integrase (IN) limited previous structure analyses to individual domains. By introducing five point mutations, we engineered a more soluble IN that allowed us to generate multidomain HIV-1 IN crystals. The first multidomain HIV-1 IN structure is reported. It incorporates the catalytic core and C-terminal domains (residues 52-288). The structure resolved to 2.8 A is a Y-shaped dimer. Within the dimer, the catalytic core domains form the only dimer interface, and the C-terminal domains are located 55 A apart. A 26-aa alpha-helix, alpha6, links the C-terminal domain to the catalytic core. A kink in one of the two alpha6 helices occurs near a known proteolytic site, suggesting that it may act as a flexible elbow to reorient the domains during the integration process. Two proteins that bind DNA in a sequence-independent manner are structurally homologous to the HIV-1 IN C-terminal domain, suggesting a similar protein-DNA interaction in which the IN C-terminal domain may serve to bind, bend, and orient viral DNA during integration. A strip of positively charged amino acids contributed by both monomers emerges from each active site of the dimer, suggesting a minimally dimeric platform for binding each viral DNA end. The crystal structure of the isolated catalytic core domain (residues 52-210), independently determined at 1.6-A resolution, is identical to the core domain within the two-domain 52-288 structure.