On Biomineralization: Enzymes Switch on Mesocrystal Assembly

Cellular machineries guide the bottom-up pathways toward crystal superstructures based on the transport of inorganic precursors and their precise integration with organic frameworks. The biosynthesis of mesocrystalline spines entails concerted interactions between biomolecules and inorganic precursors; however, the bioinorganic interactions and interfaces that regulate material form and growth as well as the selective emergence of structural complexity in the form of nanostructured crystals are not clear. By investigating mineral nucleation under the regulation of recombinant proteins, we show that SpSM50, a matrix protein of the sea urchin spine, stabilizes mineral precursors via vesicle-confinement, a function conferred by a low-complexity, disordered region. Site-specific proteolysis of this domain by a collagenase initiates phase transformation of the confined mineral phase. The residual C-type lectin domain molds the fluidic mineral precursor into hierarchical mesocrystals identical to structural crystal modules constituting the biogenic mineral. Thus, the regulatory functions of proteolytic enzymes can guide biomacromolecular domain constitutions and interfaces, in turn determining inorganic phase transformations toward hybrid materials as well as integrating organic and inorganic components across hierarchical length scales. Bearing striking resemblance to biogenic mineralization, these hybrid materials recruit bioinorganic interactions which elegantly intertwine nucleation and crystallization phenomena with biomolecular structural dynamics, hence elucidating a long-sought key of how nature can orchestrate complex biomineralization processes

Assessing sedimentation equilibrium profiles in analytical ultracentrifugation experiments on macromolecules: from simple average molecular weight analysis to molecular weight distribution and interaction analysis

Molecular weights (molar masses), molecular weight distributions, dissociation constants and other interaction parameters are fundamental characteristics of proteins, nucleic acids, polysaccharides and glycoconjugates in solution. Sedimentation equilibrium in the analytical ultracentrifugation provides a powerful method with no supplementary immobilization, columns or membranes required. It is particularly powerful when used in conjunction with its sister technique, namely sedimentation velocity analysis. We describe key approaches now available and their application to the characterisation of antibodies polysaccharides and glycoconjugates. We indicate how major complications such as thermodynamic non-ideality can now be routinely dealt with, thanks to a great extent to the extensive contribution of Professor DonWinzor over several decades of research

The Open AUC Project

Progress in analytical ultracentrifugation (AUC) has been hindered by obstructions to hardware innovation and by software incompatibility. In this paper, we announce and outline the Open AUC Project. The goals of the Open AUC Project are to stimulate AUC innovation by improving instrumentation, detectors, acquisition and analysis software, and collaborative tools. These improvements are needed for the next generation of AUC-based research. The Open AUC Project combines on-going work from several different groups. A new base instrument is described, one that is designed from the ground up to be an analytical ultracentrifuge. This machine offers an open architecture, hardware standards, and application programming interfaces for detector developers. All software will use the GNU Public License to assure that intellectual property is available in open source format. The Open AUC strategy facilitates collaborations, encourages sharing, and eliminates the chronic impediments that have plagued AUC innovation for the last 20 years. This ultracentrifuge will be equipped with multiple and interchangeable optical tracks so that state-of-the-art electronics and improved detectors will be available for a variety of optical systems. The instrument will be complemented by a new rotor, enhanced data acquisition and analysis software, as well as collaboration software. Described here are the instrument, the modular software components, and a standardized database that will encourage and ease integration of data analysis and interpretation software

Purification of bacterial membrane sensor kinases and biophysical methods for determination of their ligand and inhibitor interactions

This article reviews current methods for the reliable heterologous overexpression in Escherichia coli and purification of milligram quantities of bacterial membrane sensor kinase (MSK) proteins belonging to the two-component signal transduction family of integral membrane proteins. Many of these methods were developedatLeedsalongsideProfessor SteveBaldwintowhomthisreviewisdedicated.Italsoreviewstwo biophysical methods that we have adapted successfully for studies of purified MSKs and other membrane proteins – synchrotron radiation circular dichroism (SRCD) spectroscopy and analytical ultracentrifugation (AUC), both of which are non-immobilization and matrix-free methods that require no labelling strategies. Other techniques such as isothermal titration calorimetry (ITC) also share these features but generally require high concentrations of material. In common with many other biophysical techniques, both of these biophysical methods provide information regarding membrane protein conformation, oligomerization state and ligand binding, but they possess the additional advantage of providing direct assessments of whether ligand binding interactions are accompanied by conformational changes. Therefore, both methods provide a powerful means by which to identify and characterize inhibitor binding and any associated protein conformational changes, thereby contributing valuable information for future drug intervention strategies directed towards bacterial MSKs

Controlled depolymerisation, as assessed by analytical ultracentrifugation, of low molecular weight chitosan for potential use in archaeological conservation

The heterogeneity and molecular weight of a chitosan of low molecular weight (molar mass) and low degree of acetylation (0.1), for potential use as a consolidant for decayed archaeological wood, has been examined by sedimentation velocity and sedimentation equilibriumin the analytical ultracentrifuge before and after depolymerisation. Sedimentation velocity before polymerisation revealed a uniform distribution of sedimentation coefficient with little concentration dependence. SEDFIT-MSTAR analysis revealed a weight average molecular weight Mw of (14.2 + 1.2) kDa, and polydispersity index of ~ 1.2. Further analysis using MULTISIG revealed a distribution of material between 2-20 kDa and consistent with the weight average Mw. Controlled depolymerisation using hydrogen peroxide and UV in an acetic acid medium reduced this to (4.9 + 0.7) kDa, with a similar polydispersity. The depolymerised material appears to be within the range that has been predicted to fully penetrate into archaeological wood. The consequences for this and the use of the analytical ultracentrifuge in wood conservation strategies is considered

Impact of tunable oligophosphonates on barium sulfate crystallization

Calixarenes can be used as well-defined scaffolds for investigating structure–activity relationships of additives and their impact on crystallization. In this work, we present the crystal growth modification of barium sulfate by p-phosphonic acid calix[n]arenes that vary in size (n = 4, 5, 6, and 8) and thus vary in the size of the internal cavity for the same functionality in the upper rim. The tetrameric, hexameric, and octameric macrocycles induce nanoparticle formation with clear superstructure. In the case of the hexameric calix[6]arene, the initial mesocrystalline superstructure fuses over time to form almost hollow spheres, while the mesocrystals formed in the presence of the tetramer and octamer are stable over an extended period. The pentameric calix[5]arene forms more disordered aggregates of single crystals. Thermogravimetric data shows that a significant proportion of the mass of the barium sulfate-containing solid is the macrocycle, regardless of the choice of macrocycle

A Multilaboratory Comparison of Calibration Accuracy and the Performance of External References in Analytical Ultracentrifugation

Analytical ultracentrifugation (AUC) is a first principles based method to determine absolute sedimentation coefficients and buoyant molar masses of macromolecules and their complexes, reporting on their size and shape in free solution. The purpose of this multi-laboratory study was to establish the precision and accuracy of basic data dimensions in AUC and validate previously proposed calibration techniques. Three kits of AUC cell assemblies containing radial and temperature calibration tools and a bovine serum albumin (BSA) reference sample were shared among 67 laboratories, generating 129 comprehensive data sets. These allowed for an assessment of many parameters of instrument performance, including accuracy of the reported scan time after the start of centrifugation, the accuracy of the temperature calibration, and the accuracy of the radial magnification. The range of sedimentation coefficients obtained for BSA monomer in different instruments and using different optical systems was from 3.655 S to 4.949 S, with a mean and standard deviation of (4.304 ± 0.188) S (4.4%). After the combined application of correction factors derived from the external calibration references for elapsed time, scan velocity, temperature, and radial magnification, the range of s-values was reduced 7-fold with a mean of 4.325 S and a 6-fold reduced standard deviation of ± 0.030 S (0.7%). In addition, the large data set provided an opportunity to determine the instrument-to-instrument variation of the absolute radial positions reported in the scan files, the precision of photometric or refractometric signal magnitudes, and the precision of the calculated apparent molar mass of BSA monomer and the fraction of BSA dimers. These results highlight the necessity and effectiveness of independent calibration of basic AUC data dimensions for reliable quantitative studies

Rate-Dependent Nucleation and Growth of NaO2 in Na-O2 Batteries

Understanding the oxygen reduction reaction kinetics in the presence of Na ions and the formation mechanism of discharge product(s) is key to enhancing Na–O2 battery performance. Here we show NaO2 as the only discharge product from Na–O2 cells with carbon nanotubes in 1,2-dimethoxyethane from X-ray diffraction and Raman spectroscopy. Sodium peroxide dihydrate was not detected in the discharged electrode with up to 6000 ppm of H2O added to the electrolyte, but it was detected with ambient air exposure. In addition, we show that the sizes and distributions of NaO2 can be highly dependent on the discharge rate, and we discuss the formation mechanisms responsible for this rate dependence. Micron-sized (∼500 nm) and nanometer-scale (∼50 nm) cubes were found on the top and bottom of a carbon nanotube (CNT) carpet electrode and along CNT sidewalls at 10 mA/g, while only micron-scale cubes (∼2 μm) were found on the top and bottom of the CNT carpet at 1000 mA/g, respectively.Seventh Framework Programme (European Commission) (Marie Curie International Outgoing Fellowship, 2007-2013))National Science Foundation (U.S.) (MRSEC Program, award number DMR-0819762)Robert Bosch GmbH (Bosch Energy Research Network (BERN) Grant)China Clean Energy Research Center-Clean Vehicles Consortium (CERC-CVC) (award number DE-PI0000012)Skolkovo Institute of Science and Technology (Skoltech-MIT Center for Electochemical Energy Storage

A multilaboratory comparison of calibration accuracy and the performance of external references in analytical ultracentrifugation.

Analytical ultracentrifugation (AUC) is a first principles based method to determine absolute sedimentation coefficients and buoyant molar masses of macromolecules and their complexes, reporting on their size and shape in free solution. The purpose of this multi-laboratory study was to establish the precision and accuracy of basic data dimensions in AUC and validate previously proposed calibration techniques. Three kits of AUC cell assemblies containing radial and temperature calibration tools and a bovine serum albumin (BSA) reference sample were shared among 67 laboratories, generating 129 comprehensive data sets. These allowed for an assessment of many parameters of instrument performance, including accuracy of the reported scan time after the start of centrifugation, the accuracy of the temperature calibration, and the accuracy of the radial magnification. The range of sedimentation coefficients obtained for BSA monomer in different instruments and using different optical systems was from 3.655 S to 4.949 S, with a mean and standard deviation of (4.304 ± 0.188) S (4.4%). After the combined application of correction factors derived from the external calibration references for elapsed time, scan velocity, temperature, and radial magnification, the range of s-values was reduced 7-fold with a mean of 4.325 S and a 6-fold reduced standard deviation of ± 0.030 S (0.7%). In addition, the large data set provided an opportunity to determine the instrument-to-instrument variation of the absolute radial positions reported in the scan files, the precision of photometric or refractometric signal magnitudes, and the precision of the calculated apparent molar mass of BSA monomer and the fraction of BSA dimers. These results highlight the necessity and effectiveness of independent calibration of basic AUC data dimensions for reliable quantitative studies