Impedance analysis of Al2O3/H-terminated diamond metal-oxide-semiconductor structures

Impedance spectroscopy (IS) analysis is carried out to investigate the electrical properties of the metal-oxide-semiconductor (MOS) structure fabricated on hydrogen-terminated single crystal diamond. The low-temperature atomic layer deposition Al2O3 is employed as the insulator in the MOS structure. By numerically analysing the impedance of the MOS structure at various biases, the equivalent circuit of the diamond MOS structure is derived, which is composed of two parallel capacitive and resistance pairs, in series connection with both resistance and inductance. The two capacitive components are resulted from the insulator, the hydrogenated-diamond surface, and their interface. The physical parameters such as the insulator capacitance are obtained, circumventing the series resistance and inductance effect. By comparing the IS and capacitance-voltage measurements, the frequency dispersion of the capacitance-voltage characteristic is discussed.

Structural studies in tellurium chemistry

The primary theme of this research was the characterisation of new and novel organo-tellurium complexes, using the technique of single crystal X-ray analysis to establish more firmly the various coordination modes of tellurium. In each study the unit cell dimensions and intensity data were collected using an Enraf-Nonius CAD-4, four circle diffractometer. The raw data collected in turn was transferred to the Birmingham University Honeywell Multics System and processed using the appropriate computer packages for the determination of crystal structures. The molecular and crystal structures of: bis[2-(2-pyridyl)phenyl]tritelluride, bis[2-(N-hydroxy)iminophenyl] ditelluride, 2-(2-pyridyl)phenyltellurium(IV) tribromide, (2-N,N-dimethylbenzylamine-C,N')tellurium(IV)tribromide, 2-dichloro(butyl)tellurobenzaldehyde, 2-dichlorobutotelluro-N-dimethylbenzyl ammonium chloride, dimethyldithiocarbamato[2-(2-pyridyl)phenyl]tellurium(II), dimethyldithiocarbamato[2-(2-quinolinyl)phenyl]tellurium(II) and para-ethoxypheny[2-(2-pyridyl)phenyl]telluride are described. In each structure, the Lewis acidity of tellurium appears to be satisfied by autocomplex formation, through short-range intramolecular secondary bonds between tellurium and an electron denoting species, (generally nitrogen in these structures) with long range weak inter molecular contacts forming in the majority of the tellurium(IV) structures. The order of Lewis acidity in each structure can be considered to be reflected by the length of the short range intramolecular secondary bond, identified, that is, when tellurium has a low Lewis acidity this interaction is long. Interestingly, no primary bonds are found trans to a Te-C covalent bond in any of the above structures, highlighting the strong trans effect of aromatic and aryl groups in tellurium complexes.

The growth, structure and properties of sputtered barium titanate thin films

The design and construction of a sputtering system for the deposition of barium titanate thin films is described. The growth and structure of barium titanate films deposited on a variety of substrates including amorphous carbon fi1ms, potassium bromide single crystals, and polycrystalline gold films has been studied. Films deposited on all substrates at room temperature were amorphous. Polycrystalline titanate films were formed on polycrystalline and amorphous substrates at temperatures above 450°C while films with a pronounced texture could be expitaxially deposited on single crystal potassium bromide above a temperature of only 200°C. Results of dielectric measurements made on the films are reported. Amorphous films were highly insulating (resistivities ~1014 ohm.cm with dielectric constants of between 10 and 20.

catena-[μ-tris(1,2-bis(tetrazol-1-yl)ethane-N4,N4')iron(II)] bis(tetrafluoroborate): Synthesis, structure, spectroscopic and magnetic characterisation of a chain-type coordination polymer spin-crossover compound.

In analogy to a common synthesis of 1-substituted 5-H tetrazoles (Tetrahedron Lett. 36 (1995)1759; Beloruss. Gos. Univ., Minsk, USSR. Khim. Geterotsikl. Soedin. 11 (1985) 1521; Beloruss. Gos. Univ., Minsk, USSR. Khim. Geterotsikl. Soedin. 1 (1991) 66; BGU, Belarus. Vestsi Akad. Navuk Belarusi, Ser. Khim. Navuk 1 (1992) 73), the new bidentate ligand 1,2-bis(tetrazol-1-yl)ethane [endi] was synthesized and characterized by X-ray diffraction, NMR, IR and UV–Vis spectroscopy. By using iron(II) tetrafluoroborate hexahydrate the complexation with this ligand yields a 1-dimensional linear coordination polymer similar to the recently published chain compound (Inorg. Chem. 39 (2000) 1891) exhibiting a thermally induced spin-crossover phenomenon. Similar to the 1,2-bis(tetrazol-1-yl)propane-bridged compound, our 1,2-bis(tetrazol-1-yl)ethane-bridged compound shows a gradual spin transition, but the spin-crossover temperature T1/2≈140 K is found to be 10 K above the other T1/2. The T1/2 was determined by temperature-dependent 57Fe-Mössbauer, far FT-IR and UV–Vis spectroscopy as well as by temperature-dependent magnetic susceptibility measurements. Single crystals of the complex were grown in situ from a solution of the ligand and iron(II) tetrafluoroborate. The X-ray structure determinations of both the high spin as well as the low spin state of the compound revealed a solid state structure, which is comparable to that of catena-[Fe(1,2-bis(tetrazole-1-yl)propane)3](ClO4)2 (Inorg. Chem. 39 (2000) 1891; 2nd TMR-TOSS Meeting, 4th Spin Crossover Family Meeting, Lufthansa Training Center, Seeheim/Germany, April 30–May 2, 1999). Both the 1,2-bis(tetrazol-1-yl)propane-bridged and our compound do not show a thermal hysteresis effect (J. Am. Chem. Soc. 115 (1993) 9810; Inorg. Chim. Acta 37 (1979) 169; Chem. Phys. Lett. 93 (1982) 567). The synthesis of the complex described in the experimental section yielded a fine powdered product being poorly soluble in most common solvents. The single crystal measurements were done with crystals obtained by various diffusion methods. Most of them yielded either thin needles or small hexagonal prism crystals depending on the specific conditions.

Editorial overview:new protein production tools for structural biology

Full text: The idea of producing proteins from recombinant DNA hatched almost half a century ago. In his PhD thesis, Peter Lobban foresaw the prospect of inserting foreign DNA (from any source, including mammalian cells) into the genome of a λ phage in order to detect and recover protein products from Escherichia coli [ 1 and 2]. Only a few years later, in 1977, Herbert Boyer and his colleagues succeeded in the first ever expression of a peptide-coding gene in E. coli — they produced recombinant somatostatin [ 3] followed shortly after by human insulin. The field has advanced enormously since those early days and today recombinant proteins have become indispensable in advancing research and development in all fields of the life sciences. Structural biology, in particular, has benefitted tremendously from recombinant protein biotechnology, and an overwhelming proportion of the entries in the Protein Data Bank (PDB) are based on heterologously expressed proteins. Nonetheless, synthesizing, purifying and stabilizing recombinant proteins can still be thoroughly challenging. For example, the soluble proteome is organized to a large part into multicomponent complexes (in humans often comprising ten or more subunits), posing critical challenges for recombinant production. A third of all proteins in cells are located in the membrane, and pose special challenges that require a more bespoke approach. Recent advances may now mean that even these most recalcitrant of proteins could become tenable structural biology targets on a more routine basis. In this special issue, we examine progress in key areas that suggests this is indeed the case. Our first contribution examines the importance of understanding quality control in the host cell during recombinant protein production, and pays particular attention to the synthesis of recombinant membrane proteins. A major challenge faced by any host cell factory is the balance it must strike between its own requirements for growth and the fact that its cellular machinery has essentially been hijacked by an expression construct. In this context, Bill and von der Haar examine emerging insights into the role of the dependent pathways of translation and protein folding in defining high-yielding recombinant membrane protein production experiments for the common prokaryotic and eukaryotic expression hosts. Rather than acting as isolated entities, many membrane proteins form complexes to carry out their functions. To understand their biological mechanisms, it is essential to study the molecular structure of the intact membrane protein assemblies. Recombinant production of membrane protein complexes is still a formidable, at times insurmountable, challenge. In these cases, extraction from natural sources is the only option to prepare samples for structural and functional studies. Zorman and co-workers, in our second contribution, provide an overview of recent advances in the production of multi-subunit membrane protein complexes and highlight recent achievements in membrane protein structural research brought about by state-of-the-art near-atomic resolution cryo-electron microscopy techniques. E. coli has been the dominant host cell for recombinant protein production. Nonetheless, eukaryotic expression systems, including yeasts, insect cells and mammalian cells, are increasingly gaining prominence in the field. The yeast species Pichia pastoris, is a well-established recombinant expression system for a number of applications, including the production of a range of different membrane proteins. Byrne reviews high-resolution structures that have been determined using this methylotroph as an expression host. Although it is not yet clear why P. pastoris is suited to producing such a wide range of membrane proteins, its ease of use and the availability of diverse tools that can be readily implemented in standard bioscience laboratories mean that it is likely to become an increasingly popular option in structural biology pipelines. The contribution by Columbus concludes the membrane protein section of this volume. In her overview of post-expression strategies, Columbus surveys the four most common biochemical approaches for the structural investigation of membrane proteins. Limited proteolysis has successfully aided structure determination of membrane proteins in many cases. Deglycosylation of membrane proteins following production and purification analysis has also facilitated membrane protein structure analysis. Moreover, chemical modifications, such as lysine methylation and cysteine alkylation, have proven their worth to facilitate crystallization of membrane proteins, as well as NMR investigations of membrane protein conformational sampling. Together these approaches have greatly facilitated the structure determination of more than 40 membrane proteins to date. It may be an advantage to produce a target protein in mammalian cells, especially if authentic post-translational modifications such as glycosylation are required for proper activity. Chinese Hamster Ovary (CHO) cells and Human Embryonic Kidney (HEK) 293 cell lines have emerged as excellent hosts for heterologous production. The generation of stable cell-lines is often an aspiration for synthesizing proteins expressed in mammalian cells, in particular if high volumetric yields are to be achieved. In his report, Buessow surveys recent structures of proteins produced using stable mammalian cells and summarizes both well-established and novel approaches to facilitate stable cell-line generation for structural biology applications. The ambition of many biologists is to observe a protein's structure in the native environment of the cell itself. Until recently, this seemed to be more of a dream than a reality. Advances in nuclear magnetic resonance (NMR) spectroscopy techniques, however, have now made possible the observation of mechanistic events at the molecular level of protein structure. Smith and colleagues, in an exciting contribution, review emerging ‘in-cell NMR’ techniques that demonstrate the potential to monitor biological activities by NMR in real time in native physiological environments. A current drawback of NMR as a structure determination tool derives from size limitations of the molecule under investigation and the structures of large proteins and their complexes are therefore typically intractable by NMR. A solution to this challenge is the use of selective isotope labeling of the target protein, which results in a marked reduction of the complexity of NMR spectra and allows dynamic processes even in very large proteins and even ribosomes to be investigated. Kerfah and co-workers introduce methyl-specific isotopic labeling as a molecular tool-box, and review its applications to the solution NMR analysis of large proteins. Tyagi and Lemke next examine single-molecule FRET and crosslinking following the co-translational incorporation of non-canonical amino acids (ncAAs); the goal here is to move beyond static snap-shots of proteins and their complexes and to observe them as dynamic entities. The encoding of ncAAs through codon-suppression technology allows biomolecules to be investigated with diverse structural biology methods. In their article, Tyagi and Lemke discuss these approaches and speculate on the design of improved host organisms for ‘integrative structural biology research’. Our volume concludes with two contributions that resolve particular bottlenecks in the protein structure determination pipeline. The contribution by Crepin and co-workers introduces the concept of polyproteins in contemporary structural biology. Polyproteins are widespread in nature. They represent long polypeptide chains in which individual smaller proteins with different biological function are covalently linked together. Highly specific proteases then tailor the polyprotein into its constituent proteins. Many viruses use polyproteins as a means of organizing their proteome. The concept of polyproteins has now been exploited successfully to produce hitherto inaccessible recombinant protein complexes. For instance, by means of a self-processing synthetic polyprotein, the influenza polymerase, a high-value drug target that had remained elusive for decades, has been produced, and its high-resolution structure determined. In the contribution by Desmyter and co-workers, a further, often imposing, bottleneck in high-resolution protein structure determination is addressed: The requirement to form stable three-dimensional crystal lattices that diffract incident X-ray radiation to high resolution. Nanobodies have proven to be uniquely useful as crystallization chaperones, to coax challenging targets into suitable crystal lattices. Desmyter and co-workers review the generation of nanobodies by immunization, and highlight the application of this powerful technology to the crystallography of important protein specimens including G protein-coupled receptors (GPCRs). Recombinant protein production has come a long way since Peter Lobban's hypothesis in the late 1960s, with recombinant proteins now a dominant force in structural biology. The contributions in this volume showcase an impressive array of inventive approaches that are being developed and implemented, ever increasing the scope of recombinant technology to facilitate the determination of elusive protein structures. Powerful new methods from synthetic biology are further accelerating progress. Structure determination is now reaching into the living cell with the ultimate goal of observing functional molecular architectures in action in their native physiological environment. We anticipate that even the most challenging protein assemblies will be tackled by recombinant technology in the near future.

Single-cell analysis for bioprocessing

Cell population heterogeneity has attracted great interest for understanding the individual cellular performances in their response to external stimuli and in the production of targeted products. Physical characterization of single cells and analysis of dynamic gene expression, synthesized proteins, and cellular metabolites from one single cell are reviewed. Advanced techniques have been developed to achieve high-throughput and ultrahigh resolution or sensitivity. Single cell capture methods are discussed as well. How to make use of cellular heterogeneities for maximizing cellular productivity is still in the infant stage, and control strategies will be formulated after the causes for heterogeneity have been elucidated.