Christian Grundahl Frankær

The structures of T6, T3R3 and R6 bovine insulin: combining X-ray diffraction and absorption spectroscopy.

The crystal structures of three conformations, T(6), T(3)R(3) and R(6), of bovine insulin were solved at 1.40, 1.30 and 1.80 Å resolution, respectively. All conformations crystallized in space group R3. In contrast to the T(6) and T(3)R(3) structures, different conformations of the N-terminal B-chain residue PheB1 were observed in the R(6) insulin structure, resulting in an eightfold doubling of the unit-cell volume upon cooling. The zinc coordination in each conformation was studied by X-ray absorption spectroscopy (XAS), including both EXAFS and XANES. Zinc adopts a tetrahedral coordination in all R(3) sites and an octahedral coordination in T(3) sites. The coordination distances were refined from XAS with a standard deviation of <0.01 Å. In contrast to the distances determined from the medium-resolution crystal structures, the XAS results were in good agreement with similar coordination geometries found in small molecules, as well as in other high-resolution insulin structures. As the radiation dose for XRD experiments is two orders of magnitude higher compared with that of XAS experiments, the single crystals were exposed to a higher degree of radiation damage that affected the zinc coordination in the T(3) sites in particular. Furthermore, XANES spectra for the zinc sites in T(6) and R(6) insulin were successfully calculated using finite difference methods and the bond distances and angles were optimized from a quantitative XANES analysis.

Seawater as Alternative to Freshwater in Pretreatment of Date Palm Residues for Bioethanol Production in Coastal and/or Arid Areas

The large water consumption (1.9-5.9 m(3) water per m(3) of biofuel) required by biomass processing plants has become an emerging concern, which is particularly critical in arid/semiarid regions. Seawater, as a widely available water source, could be an interesting option. This work was to study the technical feasibility of using seawater to replace freshwater in the pretreatment of date palm leaflets, a lignocellulosic biomass from arid regions, for bioethanol production. It was shown that leaflets pretreated with seawater exhibited lower cellulose crystallinity than those pretreated with freshwater. Pretreatment with seawater produced comparably digestible and fermentable solids to those obtained with freshwater. Moreover, no significant difference of inhibition to Saccharomyces cerevisiae was observed between liquids from pretreatment with seawater and freshwater. The results showed that seawater could be a promising alternative to freshwater for lignocellulose biorefineries in coastal and/or arid/semiarid areas.

Remote Loading of 64Cu2+ into Liposomes without the Use of Ion Transport Enhancers

Due to low ion permeability of lipid bilayers, it has been and still is common practice to use transporter molecules such as ionophores or lipophilic chelators to increase transmembrane diffusion rates and loading efficiencies of radionuclides into liposomes. Here, we report a novel and very simple method for loading the positron emitter (64)Cu(2+) into liposomes, which is important for in vivo positron emission tomography (PET) imaging. By this approach, copper is added to liposomes entrapping a chelator, which causes spontaneous diffusion of copper across the lipid bilayer where it is trapped. Using this method, we achieve highly efficient (64)Cu(2+) loading (>95%), high radionuclide retention (>95%), and favorable loading kinetics, excluding the use of transporter molecule additives. Therefore, clinically relevant activities of 200-400 MBq/patient can be loaded fast (60-75 min) and efficiently into preformed stealth liposomes avoiding subsequent purification steps. We investigate the molecular coordination of entrapped copper using X-ray absorption spectroscopy and demonstrate high adaptability of the loading method to pegylated, nonpegylated, gel- or fluid-like, cholesterol rich or cholesterol depleted, cationic, anionic, and zwitterionic lipid compositions. We demonstrate high in vivo stability of (64)Cu-liposomes in a large canine model observing a blood circulation half-life of 24 h and show a tumor accumulation of 6% ID/g in FaDu xenograft mice using PET imaging. With this work, it is demonstrated that copper ions are capable of crossing a lipid membrane unassisted. This method is highly valuable for characterizing the in vivo performance of liposome-based nanomedicine with great potential in diagnostic imaging applications.

Hydrothermal Pretreatment of Date Palm (Phoenix dactylifera L.) Leaflets and Rachis to Enhance Enzymatic Digestibility and Bioethanol Potential

Date palm residues are one of the most promising lignocellulosic biomass for bioethanol production in the Middle East. In this study, leaflets and rachis were subjected to hydrothermal pretreatment to overcome the recalcitrance of the biomass for enzymatic conversion. Evident morphological, structural, and chemical changes were observed by scanning electron microscopy, X-ray diffraction, and infrared spectroscopy after pretreatment. High glucan (>90% for both leaflets and rachis) and xylan (>75% for leaflets and >79% for rachis) recovery were achieved. Under the optimal condition of hydrothermal pretreatment (210°C/10 min) highly digestible (glucan convertibility, 100% to leaflets, 78% to rachis) and fermentable (ethanol yield, 96% to leaflets, 80% to rachis) solid fractions were obtained. Fermentability test of the liquid fractions proved that no considerable inhibitors to Saccharomyces cerevisiae were produced in hydrothermal pretreatment. Given the high sugar recovery, enzymatic digestibility, and ethanol yield, production of bioethanol by hydrothermal pretreatment could be a promising way of valorization of date palm residues in this region.

A sample holder for in-house X-ray powder diffraction studies of protein powders

A sample holder for handling samples of protein for in-house X-ray powder diffraction (XRPD) analysis has been made and tested on lysozyme. The use of an integrated pinhole reduced the background, and good signal-to-noise ratios were obtained from only 7 µl of sample, corresponding to approximately 2–3 mg of dry protein. The sample holder is further adaptable to X-ray absorption spectroscopy (XAS) measurements. Both XRPD and XAS at the Zn K-edge were tested with hexameric Zn insulin.

Towards accurate structural characterization of metal centres in protein crystals: the structures of Ni and Cu T6 bovine insulin derivatives.

The level of structural detail around the metal sites in Ni2+ and Cu2+ T6 insulin derivatives was significantly improved by using a combination of single-crystal X-ray crystallography and X-ray absorption spectroscopy. Photoreduction and subsequent radiation damage of the Cu2+ sites in Cu insulin was followed by XANES spectroscopy.

Polymeric strontium ranelate nona­hydrate

The title compound, poly[[μ-aqua-tetraaqua{μ-5-[bis(carboxylatomethyl)amino]-3-carboxylatomethyl-4-cyanothiophene-2-carboxylato}distrontium(II)] tetrahydrate], [Sr2(C12H6N2O8S)(H2O)5]·3.79H2O, crystallizes with nine- and eight-coordinated Sr2+ cations. They are bound to seven of the eight ranelate O atoms and five of the water molecules. The SrO8 and SrO9 polyhedra are interconnected by edge-sharing, forming hollow layers parallel to (011). The layers are, in turn, interconnected by ranelate anions, forming a metal–organic framework (MOF) structure with channels along the a axis. The four water molecules not coordinated to strontium are located in these channels and hydrogen bonded to each other and to the ranelates. Part of the water H atoms are disordered. The compound dehydrates very easily and 0.210 (4) water molecules out of nine were lost during crystal mounting causing additional disorder in the water structure.

Biocompatible Microporous Organically Modified Silicate Material with Rapid Internal Diffusion of Protons

A new four-component organically modified silicate (ORMOSIL) material was developed with optical pH sensors in mind. Through a sol-gel process, the porosity of an ORMOSIL framework was optimized to allow rapid diffusion of protons, ideal for fast response to pH in an optical sensor. The optically transparent material was produced by catalyzing the dual polymerization of 3-(glycidoxy)propyltrimethoxysilane (GPTMS) and propyltriethoxysilane (PrTES) with boron trifluoride diethyl etherate. The performance of the resulting material in fluorescence based optical pH sensors was evaluated by incorporation of active dye components in the inorganic polymer framework. It is demonstrated that the material has a short response time ( t90 < 30 s) and high stability in medium and during storage, and resulting sensor spots are biocompatible. It is concluded that this ORMOSIL material has excellent properties for optical pH sensors.

Insulin fibrillation: The influence and coordination of Zn2+

Protein amyloid fibrillation is obtaining much focus because it is connected with amyloid-related human diseases such as Alzheimers disease, diabetes mellitus type 2, or Parkinsons disease. The influence of metal ions on the fibrillation process and whether it is implemented in the amyloid fibrils has been debated for some years. We have therefore investigated the influence and binding geometry of zinc in fibrillated insulin using extended X-ray absorption fine-structure and X-ray absorption near-edge structure spectroscopy. The results were validated with fibre diffraction, Transmission Electron Microscopy and Thioflavin T fluorescence measurements. It is well-known that Zn2+ ions coordinate and stabilize the hexameric forms of insulin. However, this study is the first to show that zinc indeed binds to the insulin fibrils. Furthermore, zinc influences the kinetics and the morphology of the fibrils. It also shows that zinc coordinates to histidine residues in an environment, which is similar to the coordination seen in the insulin R6 hexamers, where three histidine residues and a chloride ion is coordinating the zinc.

Monitoring protein precipitates by in-house X-ray powder diffraction

Powder diffraction from protein powders using in-house diffractometers is an effective tool for identification and monitoring of protein crystal forms and artifacts. As an alternative to conventional powder diffractometers a single crystal diffractometer equipped with an X-ray micro-source can be used to collect powder patterns from 1 μl samples. Using a small-angle Xray scattering (SAXS) camera it is possible to collect data within minutes. A streamlined program has been developed for the calculation of powder patterns from pdb-coordinates, and includes correction for bulk-solvent. A number of such calculated powder patterns from insulin and lysozyme have been included in the powder diffraction database and successfully used for search-match identification. However, the fit could be much improved if peak asymmetry and multiple bulk-solvent corrections were included. When including a large number of protein data sets in the database some problems can be foreseen due to the large number of overlapping peaks in the low-angle region, and small differences in unit cell parameters between pdb-data and powder data. It is suggested that protein entries are supplied with more searchable keywords as protein name, protein type, molecular weight, source organism etc. in order to limit possible hits.