Christoph G. Baumann

Metabolism of the viable mammalian embryo: quietness revisited

This review examines the ‘Quiet Embryo Hypothesis’ which proposes that viable preimplantation embryos operate at metabolite or nutrient turnover rates distributed within lower ranges than those of their less viable counterparts. The ‘quieter’ metabolism consistent with this hypothesis is considered in terms of (i) ‘functional’ quietness; the contrasting levels of intrinsic metabolic activity in different cell types as a consequence of their specialized functions, (ii) inter-individual embryo/cell differences in metabolism and (iii) loss of quietness in response to environmental stress. Data are reviewed which indicate that gametes and early embryos function in vivo at a lower temperature than core body temperature, which could encourage the expression of a quiet metabolism. We call for research to determine the optimum temperature for mammalian gamete/embryo culture. The review concludes by examining the key role of reactive oxygen species, which can induce molecular damage, trigger a cellular stress response and lead to a loss of quietness.

Supramolecular assemblies underpin turnover of outer membrane proteins in bacteria

Gram-negative bacteria inhabit a broad range of ecological niches. For Escherichia coli, this includes river water as well as humans and animals, where it can be both a commensal and a pathogen. Intricate regulatory mechanisms ensure that bacteria have the right complement of β-barrel outer membrane proteins (OMPs) to enable adaptation to a particular habitat. Yet no mechanism is known for replacing OMPs in the outer membrane, an issue that is further confounded by the lack of an energy source and the high stability and abundance of OMPs. Here we uncover the process underpinning OMP turnover in E. coli and show it to be passive and binary in nature, in which old OMPs are displaced to the poles of growing cells as new OMPs take their place. Using fluorescent colicins as OMP-specific probes, in combination with ensemble and single-molecule fluorescence microscopy in vivo and in vitro, as well as molecular dynamics simulations, we established the mechanism for binary OMP partitioning. OMPs clustered to form ∼0.5-μm diameter islands, where their diffusion is restricted by promiscuous interactions with other OMPs. OMP islands were distributed throughout the cell and contained the Bam complex, which catalyses the insertion of OMPs in the outer membrane. However, OMP biogenesis occurred as a gradient that was highest at mid-cell but largely absent at cell poles. The cumulative effect is to push old OMP islands towards the poles of growing cells, leading to a binary distribution when cells divide. Hence, the outer membrane of a Gram-negative bacterium is a spatially and temporally organized structure, and this organization lies at the heart of how OMPs are turned over in the membrane.

Direct C-H/C-X Coupling Methodologies Mediated by Pd/Cu or Cu: An Examination of the Synthetic Applications and Mechanistic Findings

Organocopper chemistry has long been known to be powerful synthetic methodology leading to the generation of C-C, C-O, C-N, C-S and C-X bonds. Advances in catalytic and stoichiometric organometallic reactions for the formation of C-C bonds from unfunctionalised arenes, e.g. by activation-functionalisation of a C-H bond, provides diverse and applicable reaction conditions for direct functionalisation. This review covers the background, applications and recent advances made in the area of aromatic C-X/C-H-bond couplings promoted by copper and copper/palladium mixed systems. The efficacy and scope of these reactions are discussed and the different methodologies are placed into context. Mechanistic considerations and evidence are highlighted at appropriate junctures in the review.

Mode of drug binding to DNA determined by optical tweezers force spectroscopy

Abstract Optical tweezers were employed to investigate the effects of small DNA-binding molecules on the low-force (≤ 15 pN) stretching behaviour of single DNA molecules. As the canonical B-DNA helix is not perturbed in this force regime, the effects on DNA elasticity observed upon drug binding provide useful insight into how DNA-binding drugs may alter in vivo processes. In this study, the effects of agents with different DNA binding modes were analysed. DNA force—extension curves were recorded in the presence of netropsin, a purely minor groove-binding antibiotic drug, ethidium bromide, an intercalating fluorescent dye, and berenil, an antiprotozoal drug proposed to exhibit both intercalative and minor groove-binding modes. Applying an approximation of the worm-like chain model, which describes the low-force stretching behaviour, the results were analysed in terms of the DNA contour length and persistence length. From these single molecule studies it was observed that minor groove-binding and intercalating modes of DNA-binding could be distinguished based on changes to DNA elasticity.

On the appearance of nitrite anion in [PdX(OAc)L2] and [Pd(X)(C^N)L] syntheses (X = OAc or NO2): photocrystallographic identification of metastable Pd(η1-ONO)(C^N)PPh3

Pd3(OAc)5NO2, an impurity in “Pd(OAc)2” {formally Pd3(OAc)6}, emerges as a serious issue in the synthesis of pure PdII complexes derived from Pd(OAc)2, for example in our C–H activation precatalyst, Pd(OAc)2(pip)2 (pip = piperidine). A previous proposal that nitrite anion can be formed by oxidation of CH3CN by metallic Pd and air, leading to cyclo(ortho)palladated complexes containing nitrite anion, e.g. Pd(NO2)(C^N)L (C^N = papaverine; L = CH3CN or DMSO) can be explained by Pd3(OAc)5NO2 acting as the nitrite source. Finally, photocrystallographic metastable linkage isomerisation and complete conversion to an oxygen-bound nitrito complex Pd(η1-ONO)(C^N)PPh3 has been observed.

Interaction of σ70 with Escherichia coli RNA polymerase core enzyme studied by surface plasmon resonance

The interaction between the core form of bacterial RNA polymerases and σ factors is essential for specific promoter recognition, and for coordinating the expression of different sets of genes in response to varying cellular needs. The interaction between Escherichia coli core RNA polymerase and σ70 has been investigated by surface plasmon resonance. The His‐tagged form of σ70 factor was immobilised on a Ni2+–NTA chip for monitoring its interaction with core polymerase. The binding constant for the interaction was found to be 1.9×10−7 M, and the dissociation rate constant for release of σ from core, in the absence of DNA or transcription, was 4×10−3 s−1, corresponding to a half‐life of about 200 s.

Protein-protein interactions and the spatiotemporal dynamics of bacterial outer membrane proteins.

Highlights • We discuss spatiotemporal patterning in the bacterial outer membrane.• Promiscuous interactions between outer membrane proteins govern their behaviour.• Turnover and biogenesis of outer membrane proteins linked to formation of clusters.• Implications of spatiotemporal patterning for bacterial physiology discussed.

CHAPTER 12:Catalytic C–H/C–X Bond Functionalisation of Nucleosides, Nucleotides, Nucleic Acids, Amino Acids, Peptides and Proteins

Pd‐catalysed cross‐coupling chemistry is widely used to access modified and unnatural derivatives of nucleosides, nucleotides and amino acids. Many biologically active, fluorescent or electrochemically‐labelled compounds, not easily accessible by alternate methods, have been synthesised using Pd‐catalysed coupling chemistry. In recent years, this area has expanded to include the use of direct arylation and C–H activation methodologies for the direct synthesis of C‐modified biomolecules. Modified nucleosides and amino acids represent some of the most complex compounds yet synthesised by Pd‐catalysed direct C–H functionalisation. Current research has also begun to focus on the challenging Pd‐catalysed C‐functionalisation of large biomolecules such as nucleic acids, peptides and proteins. This field therefore represents the current cutting‐edge of Pd‐catalysed C‐functionalisation chemistry, and highlights the utility and versatility of these methodologies in the synthesis of highly complex biomolecules.

Interrogation of a Sonogashira Cross-Coupling of 8-Bromoguanosine with Phenylacetylene on Amberlite: Evidence for Pd/Cu Ion Binding and Propagation of Pd/Cu Nanoparticles

The reactivity of Amberlite (IRA-67) base “heterogeneous” resin in Sonogashira cross-coupling of 8-bromoguanosine 1 with phenylacetylene 3 to give 2 has been examined. Both 1 and 2 coordinate to Pd and Cu ions, which explains why at equivalent catalyst loadings, the homogeneous reaction employing triethylamine base is poor yielding. X-ray photo-electron spectroscopy (XPS) has been used to probe and quantify the active nitrogen base sites of the Amberlite resin, and postreaction Pd and Cu species. The PdCl2(PPh3)2 precatalyst and CuI cocatalyst degrade to give Amberlite-supported metal nanoparticles (average size ∼2.7 nm). The guanosine product 2 formed using the Amberlite Pd/Cu catalyst system is of higher purity than reactions using a homogeneous Pd precatalyst, a prerequisite for use in biological applications.

Intermembrane crosstalk drives inner-membrane protein organization in Escherichia coli

Gram-negative bacteria depend on energised protein complexes that connect the two membranes of the cell envelope. However, β-barrel outer-membrane proteins (OMPs) and α-helical inner-membrane proteins (IMPs) display quite different organisation. OMPs cluster into islands that restrict their lateral mobility, while IMPs generally diffuse throughout the cell. Here, using live cell imaging of Escherichia coli, we demonstrate that when transient, energy-dependent transmembrane connections are formed, IMPs become subjugated by the inherent organisation of OMPs and that such connections impact IMP function. We show that while establishing a translocon for import, the colicin ColE9 sequesters the IMPs of the proton motive force (PMF)-linked Tol-Pal complex into islands mirroring those of colicin-bound OMPs. Through this imposed organisation, the bacteriocin subverts the outer-membrane stabilising role of Tol-Pal, blocking its recruitment to cell division sites and slowing membrane constriction. The ordering of IMPs by OMPs via an energised inter-membrane bridge represents an emerging functional paradigm in cell envelope biology.Outer membrane proteins (OMPs) in Gram-negative bacteria have restricted lateral mobility. Here, Rassam et al. show that the bacteriocin ColE9, via its interactions with OMPs, imposes this restricted mobility on the inner membrane proteins of the Tol-Pal complex.