A dimeric crystal structure for the N-terminal two domains of intercellular adhesion molecule-1

The 3.0-Å structure of a 190-residue fragment of intercellular adhesion molecule-1 (ICAM-1, CD54) reveals two tandem Ig-superfamily (IgSF) domains. Each of two independent molecules dimerizes identically with a symmetry-related molecule over a hydrophobic interface on the BED sheet of domain 1, in agreement with dimerization of ICAM-1 on the cell surface. The residues that bind to the integrin LFA-1 are well oriented for bivalent binding in the dimer, with the critical Glu-34 residues pointing away from each other on the periphery. Residues that bind to rhinovirus are in the flexible BC and FG loops at the tip of domain 1, and these and the upper half of domain 1 are well exposed in the dimer for docking to virus. By contrast, a residue important for binding to Plasmodium falciparum-infected erythrocytes is in the dimer interface. The presence of A′ strands in both domains 1 and 2, conserved hydrogen bonds at domain junctions, and elaborate hydrogen bond networks around the key integrin binding residues in domain 1 make these domains suited to resist tensile forces during adhesive interactions. A subdivision of the intermediate (I) set of IgSF domains is proposed in which domain 1 of ICAM-1 and previously described I set domains belong to the I1 set and domain 2 of ICAM-1, ICAM-2, and vascular cell adhesion molecule-1 belong to the I2 set.

A peptide that inhibits hydroxyapatite growth is in an extended conformation on the crystal surface

Proteins play an important role in the biological mechanisms controlling hard tissue development, but the details of molecular recognition at inorganic crystal interfaces remain poorly characterized. We have applied a recently developed homonuclear dipolar recoupling solid-state NMR technique, dipolar recoupling with a windowless sequence (DRAWS), to directly probe the conformation of an acidic peptide adsorbed to hydroxyapatite (HAP) crystals. The phosphorylated hexapeptide, DpSpSEEK (N6, where pS denotes phosphorylated serine), was derived from the N terminus of the salivary protein statherin. Constant-composition kinetic characterization demonstrated that, like the native statherin, this peptide inhibits the growth of HAP seed crystals when preadsorbed to the crystal surface. The DRAWS technique was used to measure the internuclear distance between two 13C labels at the carbonyl positions of the adjacent phosphoserine residues. Dipolar dephasing measured at short mixing times yielded a mean separation distance of 3.2 ± 0.1 Å. Data obtained by using longer mixing times suggest a broad distribution of conformations about this average distance. Using a more complex model with discrete α-helical and extended conformations did not yield a better fit to the data and was not consistent with chemical shift analysis. These results suggest that the peptide is predominantly in an extended conformation rather than an α-helical state on the HAP surface. Solid-state NMR approaches can thus be used to determine directly the conformation of biologically relevant peptides on HAP surfaces. A better understanding of peptide and protein conformation on biomineral surfaces may provide design principles useful for the modification of orthopedic and dental implants with coatings and biological growth factors that are designed to enhance biocompatibility with surrounding tissue.

Crystal structure of Taq DNA polymerase in complex with an inhibitory Fab: The Fab is directed against an intermediate in the helix-coil dynamics of the enzyme

We report the crystal structure of Thermus aquaticus DNA polymerase I in complex with an inhibitory Fab, TP7, directed against the native enzyme. Some of the residues present in a helical conformation in the native enzyme have adopted a γ turn conformation in the complex. Taken together, structural information that describes alteration of helical structure and solution studies that demonstrate the ability of TP7 to inhibit 100% of the polymerase activity of the enzyme suggest that the change in conformation is probably caused by trapping of an intermediate in the helix-coil dynamics of this helix by the Fab. Antibodies directed against modified helices in proteins have long been anticipated. The present structure provides direct crystallographic evidence. The Fab binds within the DNA binding cleft of the polymerase domain, interacting with several residues that are used by the enzyme in binding the primer:template complex. This result unequivocally corroborates inferences drawn from binding experiments and modeling calculations that the inhibitory activity of this Fab is directly attributable to its interference with DNA binding by the polymerase domain of the enzyme. The combination of interactions made by the Fab residues in both the polymerase and the vestigial editing nuclease domain of the enzyme reveal the structural basis of its preference for binding to DNA polymerases of the Thermus species. The orientation of the structure-specific nuclease domain with respect to the polymerase domain is significantly different from that seen in other structures of this polymerase. This reorientation does not appear to be antibody-induced and implies remarkably high relative mobility between these two domains.

The roles of the two proton input channels in cytochrome c oxidase from Rhodobacter sphaeroides probed by the effects of site-directed mutations on time-resolved electrogenic intraprotein proton transfer

The crystal structures of cytochrome c oxidase from both bovine and Paracoccus denitrificans reveal two putative proton input channels that connect the heme-copper center, where dioxygen is reduced, to the internal aqueous phase. In this work we have examined the role of these two channels, looking at the effects of site-directed mutations of residues observed in each of the channels of the cytochrome c oxidase from Rhodobacter sphaeroides. A photoelectric technique was used to monitor the time-resolved electrogenic proton transfer steps associated with the photo-induced reduction of the ferryl-oxo form of heme a3 (Fe4+ = O2−) to the oxidized form (Fe3+OH−). This redox step requires the delivery of a “chemical” H+ to protonate the reduced oxygen atom and is also coupled to proton pumping. It is found that mutations in the K channel (K362M and T359A) have virtually no effect on the ferryl-oxo-to-oxidized (F-to-Ox) transition, although steady-state turnover is severely limited. In contrast, electrogenic proton transfer at this step is strongly suppressed by mutations in the D channel. The results strongly suggest that the functional roles of the two channels are not the separate delivery of chemical or pumped protons, as proposed recently [Iwata, S., Ostermeier, C., Ludwig, B. & Michel, H. (1995) Nature (London) 376, 660–669]. The D channel is likely to be involved in the uptake of both “chemical” and “pumped” protons in the F-to-Ox transition, whereas the K channel is probably idle at this partial reaction and is likely to be used for loading the enzyme with protons at some earlier steps of the catalytic cycle. This conclusion agrees with different redox states of heme a3 in the K362M and E286Q mutants under aerobic steady-state turnover conditions.

A technique for detecting matrix proteins in the crystalline spicule of the sea urchin embryo.

The presence of proteins associated with the CaCO3-containing biocrystals found in a wide variety of marine organisms is well established. In these organisms, including the primitive skeleton (spicule) of the sea urchin embryo, the structural and functional role of these proteins either in the biomineralization process or in control of the structural features of the biocrystals is unclear. Recently, one of the matrix proteins of the sea urchin spicule, SM 30, has been shown to contain a carbohydrate chain (the 1223 epitope) that has been implicated in the process whereby Ca2+ is deposited as CaCo3. Because an understanding of the localization of this protein, as well as other proteins found within the spicule, is central to understanding their function, we undertook to develop methods to localize spicule matrix proteins in intact spicules, using immunogold techniques and scanning electron microscopy. Gold particles indicative of this matrix glycoprotein could not be detected on the surface of spicules that had been isolated from embryo homogenates and treated with alkaline hypochlorite to remove any associated membranous material. However, when isolated spicules were etched for 2 min with dilute acetic acid (10 mM) to expose more internal regions of the crystal, SM 30 and perhaps other proteins bearing the 1223 carbohydrate epitope were detected in the calcite matrix. These results, indicating that these two antigens are widely distributed in the spicule, suggest that this technique should be applicable to any matrix protein for which antibodies are available.

The crystal structure of an N-terminal two-domain fragment of vascular cell adhesion molecule 1 (VCAM-1): a cyclic peptide based on the domain 1 C-D loop can inhibit VCAM-1-alpha 4 integrin interaction.

Vascular cell adhesion molecule 1 (VCAM-1) represents a structurally and functionally distinct class of immunoglobulin superfamily molecules that bind leukocyte integrins and are involved in inflammatory and immune functions. X-ray crystallography defines the three-dimensional structure of the N-terminal two-domain fragment that participates in ligand binding. Residues in domain 1 important for ligand binding reside in the C-D loop, which projects markedly from one face of the molecule near the contact between domains 1 and 2. A cyclic peptide that mimics this loop inhibits binding of alpha 4 beta 1 integrin-bearing cells to VCAM-1. These data demonstrate how crystallographic structural information can be used to design a small molecule inhibitor of biological function.

Crystal Structures, Magnetic Structures and Photophysics in Supramolecular Transition-Metal Oxalate Compounds

Polymeric two- and three-dimensional, homo- and heterometallic oxalatebridged coordination compounds offer exciting opportunities, mainly in the fields of molecular magnetism and photophysics. Given that a large variety of magnetic phenomena have been reported so far from these molecular magnets, very limited experience is gained from elastic neutron scattering experiments. Therefore, with two examples, we will address the topic of the elucidation of magnetic structures by means of the neutron scattering technique. In addition, due to the possibility of the variation of different metal ions in varying oxidation states, interesting photophysical processes can be observed within the extended three-dimensional host/guest systems.

Diphenyltin(IV) complexes of the 2-quinolinecarboxaldehyde Schiff bases of S-methyl- and S-benzyldithiocarbazate (Hqaldsme and Hqaldsbz): X-ray crystal structures of Hqaldsme and two conformers of its diphenyltin(IV) complex

New organometallic tin(IV) complexes of the empirical formula Sn(NNS)Ph2Cl (NNS = anionic forms of the 2-quinolinecarboxaldehyde Schiff bases of S-methyl- and S-benzyldithiocarbazate) have been prepared and characterized by IR, electronic, I H NMR and ES mass spectroscopic techniques. The molecular structures of the 2-quinolinecarboxaldehyde Schiff base of S-methyldithiocarbazate (Hqaldsme) and its diphenyltin(IV) complex, Sn(qaldsme)Ph2Cl, have been determined by X-ray diffraction. In the solid state, the ligand remains as the thione tautomer in which the dithiocarbazate chain adopts an E,E configuration and is almost coplanar with the quinoline ring. The Sn(qaldsme)Ph2Cl complex crystallizes in two distinctly different conformationally isomeric forms, each having the same space group but different lattice parameters. X-ray analysis shows that in each polymorph, the tin atom adopts a distorted octahedral geometry with the Schiff base coordinated to it as a uninegatively charged tridentate chelating agent via the quinoline nitrogen atom, the azomethine nitrogen atom and the thiolate sulfur atom. The two phenyl groups occupy axial positions and the chloride ligand occupies the sixth coordination position of the tin atom. The deprotonated ligand adopts an E,E,Z configuration in the complex. (C) 2004 Elsevier Ltd. All rights reserved.

Implications of restructuring and privatisation for competitiveness and perfomance:two Brazilian steel companies

The thesis examines the effects of the privatisation process on productivity, competitiveness and performance in two major Brazilian steel companies, which were privatised in between 1991 and 1993. The case study method was adopted in this research due to its strengths as a useful technique allowing in-depth examination of the privatisation process, the context in which it happened and its effects on the companies. The thesis has developed a company analysis framework consisting of three components: management, competitiveness/productivity and performance and examined the evidence on the companies within this framework.The research indicates that there is no straightforward relationship between privatisation, competitiveness and performance. There were many significant differences in the management and technological capabilities, products and performance of the two companies, and these have largely influenced the effects of privatisation on each company. Company Alpha's strengths in technological and management capabilities and high value added products explain strong productivity and financial performance during and after privatisation. Company Beta's performance was weak before the privatisation and remained weak immediately after. Before the privatisation, weaknesses in management, commodity type low value added products and shortage of funds for investment were the major problems. These were compounded by greater government interference. Despite major restructuring, the poor performance has continued after privatisation largely because the company has not been able to improve its productivity sufficiently to be cost competitive in commodity type markets. Both companies state that their strategies have changed significantly. They claim to be more responsive to market conditions and customers and are attempting to develop closer links with major customers. It is not possible to assess the consequences of these changes in the short time that has elapsed since privatisation but Alpha appears to be more effective in developing a coherent strategy because of its strengths. Both companies accelerated their programme of organisational restructuring and reducing the number of their employees during the privatisation process to improve productivity and performance. Alpha has attained standards comparable to major international steel companies. Beta has had to make much bigger organisational changes and cuts in its labour force but its productivity levels still remain low in comparison with Alpha and international competitors.

Observations of diffraction and self diffraction effects during two wave mixing in a nematic liquid crystal

We report observations of the diffraction pattern resulting when a nematic liquid crystal is illuminated with two equal power, high intensity beams of light from an Ar+ laser. The time evolution of the pattern is followed from the initial production of higher diffraction orders to a final striking display arising as a result of the self-diffraction of the two incident beams. The experimental results are described with good approximation by a model assuming a phase distribution at the output plane of the liquid crystal in the form of the sum of a gaussian and a sinusoid.

Extended range interferometry using a coherence tuned synthesised dual wavelength technique with multimode fibres

We describe in the letter a technique for making extended-range interferometric measurements with a coherence multiplexed system by means of a variation on the dual-wavelength technique. The interferometer is illuminated with a single source and the two wavelengths are synthesised at the output by means of an interference filter.

Observations of diffraction and self diffraction effects during two wave mixing in a nematic liquid crystal

We report observations of the diffraction pattern resulting when a nematic liquid crystal is illuminated with two equal power, high intensity beams of light from an Ar+ laser. The time evolution of the pattern is followed from the initial production of higher diffraction orders to a final striking display arising as a result of the self-diffraction of the two incident beams. The experimental results are described with good approximation by a model assuming a phase distribution at the output plane of the liquid crystal in the form of the sum of a gaussian and a sinusoid.

Microstructural study of aluminide surface coatings on single crystal nickel base superalloy substrates

Aluminide diffusion coatings are frequently employed to enhance the oxidation resistance of nickel base superalloys. However, there is a concern that the presence of an aluminide coating could influence the properties of the coated superalloy, especially in respect of fatigue behaviour. To understand the nature of the effects of surface coatings on the fatigue properties of superalloys, an understanding of microstructural development within both the coating and the coating/substrate interfacial zone during high temperature fatigue testing is necessary. This paper is concerned with microstructural changes in aluminide diffusion coatings on single crystal γ′ strengthened superalloy substrates during the course of high temperature fatigue testing. The 'edge on' transmission electron microscopy technique is employed to study cross-sections of two stage (aluminization plus diffusion treatment) coated superalloy samples. The paper examines the degradation of the coating produced by phase transformations induced by loss of aluminum from the coating and/or aging of the coating. Aluminum removal both by interdiffusion with the substrate and by oxidation of the coating surface is considered. Microstructural development in the portion of the substrate influenced by interdiffusion with the coating is also discussed.

Initial nucleation study and new technique for sublimation growth of AlN on SiC substrate

Single crystal platelets of AlN were successfully grown on 6H-SiC(0001) by a novel technique designed to suppress SiC decomposition, promote two-dimensional growth, and eliminate cracking in the AlN. X-ray diffractometry and synchrotron white beam X-ray topography demonstrate that the final AlN single crystal is of high structural quality.