Comparative 11A structure of two molluscan hemocyanins from 3D cryo-electron microscopy

Hemocyanins are giant extracellular proteins that transport oxygen in the hemolymph of many molluscs. Molluscan hemocyanins are cylindrical decamers or didecamers of a 350-400 kDa subunit that contains seven or eight different covalently linked globular functional units (FUs), arranged in a linear manner. Each FU carries a single copper active site and reversibly binds one dioxygen molecule. As a consequence, the decamer can carry up to 70 or 80 O(2) molecules. Although complete sequence information is now available from several molluscan hemocyanins, many details of the quaternary structure are still unclear, including the topology of the 10 subunits within the decamer. Here we show 3D reconstructions from cryo-electron micrographs of the hemocyanin decamer of Nautilus pompilius (Cephalopoda) and Haliotis tuberculata (Gastropoda) at a resolution of 11A (FSC(1/2-bit) criterion). The wall structure of both hemocyanins is very similar and shows, as in previous reconstructions, three tiers with 20 functional units each that encircle the cylinder wall, and the 10 oblique minor and major wall grooves. However, the six types of wall FUs of the polypeptide subunit, termed a-b-c-d-e-f, are now for the first time individually discernable by their specific orientation, shape, and connections. Also, the internal collar complex of the decamers shows superior resolution which, in this case, reveals striking differences between the two hemocyanins. The five arcs (FU-g pairs) of the central collar (in both hemocyanins) and the five slabs (FU-h pairs) of the peripheral collar (only present in Haliotis hemocyanin), as well as their connections to the wall and to each other are now more clearly defined. The arc is attached to the wall through a feature termed the anchor, a previously undescribed structural element of the hemocyanin wall.     

Synthesis and Biological Evaluation of a Class of Mitochondrially‐Targeted Gadolinium(III) Agents

A structure–activity relationship study of a library of novel bifunctional Gd^III complexes covalently linked to arylphosphonium cations is reported. Such complexes have been designed for potential application in binary cancer therapies such as neutron capture therapy and photon activation therapy. A positive correlation was found between lipophilicity and cytotoxicity of the complexes. Mitochondria uptake was determined by means of inductively coupled plasma mass spectrometry (ICP‐MS), and Gd uptake was determined by means of quantification using synchrotron X‐ray fluorescence (XRF) imaging. A negative correlation between lipophilicity and tumour selectivity of the Gd^III complexes was demonstrated. This study highlights the delicate balance required to minimise in vitro cytotoxicity and optimise in vitro tumour selectivity and mitochondrial localisation for this new class of mitochondrially‐targeted binary therapy agents. We also report the highest in vitro tumour selectivity for any Gd agent reported to date, with a T/N (tumour/normal cell) ratio of up to 23.5±6.6.     

Mechanistic Insights into RNA Transphosphorylation from Kinetic Isotope Effects and Linear Free Energy Relationships of Model Reactions

Phosphoryl transfer reactions are ubiquitous in biology and the understanding of the mechanisms whereby these reactions are catalyzed by protein and RNA enzymes is central to reveal design principles for new therapeutics. Two of the most powerful experimental probes of chemical mechanism involve the analysis of linear free energy relations (LFERs) and the measurement of kinetic isotope effects (KIEs). These experimental data report directly on differences in bonding between the ground state and the rate‐controlling transition state, which is the most critical point along the reaction free energy pathway. However, interpretation of LFER and KIE data in terms of transition‐state structure and bonding optimally requires the use of theoretical models. In this work, we apply density‐functional calculations to determine KIEs for a series of phosphoryl transfer reactions of direct relevance to the 2′‐O‐transphosphorylation that leads to cleavage of the phosphodiester backbone of RNA. We first examine a well‐studied series of phosphate and phosphorothioate mono‐, di‐ and triesters that are useful as mechanistic probes and for which KIEs have been measured. Close agreement is demonstrated between the calculated and measured KIEs, establishing the reliability of our quantum model calculations. Next, we examine a series of RNA transesterification model reactions with a wide range of leaving groups in order to provide a direct connection between observed Brønsted coefficients and KIEs with the structure and bonding in the transition state. These relations can be used for prediction or to aid in the interpretation of experimental data for similar non‐enzymatic and enzymatic reactions. Finally, we apply these relations to RNA phosphoryl transfer catalyzed by ribonuclease A, and demonstrate the reaction coordinate–KIE correlation is reasonably preserved. A prediction of the secondary deuterium KIE in this reaction is also provided. These results demonstrate the utility of building up knowledge of mechanism through the systematic study of model systems to provide insight into more complex biological systems such as phosphoryl transfer enzymes and ribozymes.     

“CLASSIC NMR”: An In‐Situ NMR Strategy for Mapping the Time‐Evolution of Crystallization Processes by Combined Liquid‐State and Solid‐State Measurements

A new in‐situ NMR strategy (termed CLASSIC NMR) for mapping the evolution of crystallization processes is reported, involving simultaneous measurement of both liquid‐state and solid‐state NMR spectra as a function of time. This combined strategy allows complementary information to be obtained on the evolution of both the solid and liquid phases during the crystallization process. In particular, as crystallization proceeds (monitored by solid‐state NMR), the solution state becomes more dilute, leading to changes in solution‐state speciation and the modes of molecular aggregation in solution, which are monitored by liquid‐state NMR. The CLASSIC NMR experiment is applied here to yield new insights into the crystallization of m‐aminobenzoic acid.     

Synthesis,Structure, and Reactivity of a Gold Carbenoid Complex That Lacks Heteroatom Stabilization

Hydride abstraction from the neutral gold cycloheptatrienyl complex [( P )Au(η¹‐C₇H₇)] ( P =P(tBu)₂(o‐biphenyl)) with triphenylcarbenium tetrafluoroborate at ?80 °C led to the isolation of the cationic gold cycloheptatrienylidene complex [( P )Au(η¹‐C₇H₆)]⁺ BF₄^? in 52 % yield, which was characterized in solution and by single‐crystal X‐ray diffraction. This cycloheptatrienylidene complex represents the first example of a gold carbenoid complex that lacks conjugated heteroatom stabilization of the electron‐deficient C1 carbon atom. The cycloheptatrienylidene ligand of this complex is reactive; it can be reduced by mild hydride donors, and converted to tropone in the presence of pyridine N‐oxide.     

Synthesis of Novel C/D Ring Modified Bile Acids

Bile acid receptors have been identified as important targets for the development of new therapeutics to treat various metabolic and inflammatory diseases. The synthesis of new bile acid analogues can help elucidate structure–activity relationships and define compounds that activate these receptors selectively. Towards this, access to large quantities of a chenodeoxycholic acid derivative bearing a C-12 methyl and a C-13 to C-14 double bond provided an interesting scaffold to investigate the chemical manipulation of the C/D ring junction in bile acids. The reactivity of this alkene substrate with various zinc carbenoid species showed that those generated using the Furukawa methodology achieved selective α-cyclopropanation, whereas those generated using the Shi methodology reacted in an unexpected manner giving rise to a rearranged skeleton whereby the C ring has undergone contraction to form a novel spiro–furan ring system. Further derivatization of the cyclopropanated steroid included O-7 oxidation and epimerization to afford new bile acid derivatives for biological evaluation.     

Role of structure in amorphous magnetic materials: Dense random packing of hard spheres of two sizes

We investigate the structure of amorphous rare-earth transition-metal alloys and its influence on their magnetic properties in the context of a simple model. The packing fraction of the alloys is discussed using both ball-bearing assemblies and also computer-generated clusters of randomly close-packed hard spheres, and the latter clusters are used to discuss the radial distribution functions (RDF's) and the magnetic anisotropy. The computer-generated RDF's are compared with published neutron diffraction results on amorphous TbFe₂.     

Modelling of a novel high-impedance matching layer for high frequency (>30 MHz) ultrasonic transducers

This work describes a new approach to impedance matching for ultrasonic transducers. A single matching layer with high acoustic impedance of 16 MRayls is demonstrated to show a bandwidth of around 70%, compared with conventional single matching layer designs of around 50%. Although as a consequence of this improvement in bandwidth, there is a loss in sensitivity, this is found to be similar to an equivalent double matching layer design. Designs are calculated by using the KLM model and are then verified by FEA simulation, with very good agreement Considering the fabrication difficulties encountered in creating a high-frequency double matched design due to the requirement for materials with specific acoustic impedances, the need to accurately control the thickness of layers, and the relatively narrow bandwidths available for conventional single matched designs, the new approach shows advantages in that alternative (and perhaps more practical) materials become available, and offers a bandwidth close to that of a double layer design with the simplicity of a single layer design. The disadvantage is a trade-off in sensitivity. A typical example of a piezoceramic transducer matched to water can give a 70% fractional bandwidth (comparable to an ideal double matched design of 72%) with a 3 dB penalty in insertion loss.