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Center for Synthetic Biology > Scientific Articles

Scientific articles published by UNIK Synthetic Biology


Synthetic biology researchers that are affiliated with the Center for Synthetic Biology, publish their findings in scientific journals. A full list of these publications between 2009 and 2013 can be found here.

A selection of the most recent publications are listed below with their abstracts. Please contact Rebecca Neale at rebn@life.ku.dk for further information.

MicroRNAs (miRNAs) are small regulatory RNAs that have important regulatory roles in numerous developmental and metabolic processes in most eukaryotes. In Arabidopsis, DICER-LIKE1 (DCL1), HYPONASTIC LEAVES 1, SERRATE, HUA ENHANCER1 and HASTY are involved in processing of primary miRNAs (pri-miRNAs) to yield precursor miRNAs (pre-miRNAs) and eventually miRNAs. In addition to these components, mRNA cap-binding proteins, CBP80/ABA HYPERSENSITIVE1 and CBP20, also participate in miRNA biogenesis. Here, we show that STABILIZED1(STA1), an Arabidopsis pre-mRNA processing factor 6 homolog, is also involved in the biogenesis of miRNAs. Similar to other miRNA biogenesis-defective mutants, sta1-1 accumulated significantly lower levels of mature miRNAs and concurrently higher levels of pri-miRNAs than wild type. The dramatic reductions of mature miRNAs were associated with the accumulation of their target gene transcripts and developmental defects. Furthermore, sta1-1 impaired splicing of intron containing pri-miRNAs and decreased transcript levels of DCL1. These results suggest that STA1is involved in miRNA biogenesis directly by functioning in pri-miRNA splicing and indirectly by modulating the DCL1 transcript level.
  • Chen YZ, Trier, F, Christensen, DV, Bovet, N , Jespersen, TS, Nygård, J, Pryds, N  A high-mobility two-dimensional electron gas at the spinel/perovskite interface of Al 2 O 3 /SrTiO 3 Nature Communications  4: 1371. (2013) 

The discovery of two-dimensional electron gases at the heterointerface between two insulating perovskite-type oxides, such as LaAlO3 and SrTiO3, provides opportunities for a new generation of all-oxide electronic devices. Key challenges remain for achieving interfacial electron mobilities much beyond the current value of approximately 1,000 cm2 V-1 s-1 (at low temperatures). Here we create a new type of two-dimensional electron gas at the heterointerface between SrTiO3 and a spinel γ-Al2O3 epitaxial film with compatible oxygen ions sublattices. Electron mobilities more than one order of magnitude higher than those of hitherto-investigated perovskite-type interfaces are obtained. The spinel/perovskite two-dimensional electron gas, where the two-dimensional conduction character is revealed by quantum magnetoresistance oscillations, is found to result from interface-stabilized oxygen vacancies confined within a layer of 0.9 nm in proximity to the interface. Our findings pave the way for studies of mesoscopic physics with complex oxides and design of high-mobility all-oxide electronic devices.

    • F. Mumm,  K. Müller Beckwith, S. Bonde, K. L. Martinez,  and P.  Sikorski: A transparent nanowire-based cell impalement device suitable for detailed cell-nanowire interaction studies SMALL  9 (2): 263–272 (2013)
    A method to fabricate inexpensive and transparent nanowire impalement devices is invented based on CuO nanowire arrays grown by thermal oxidation. By employing a novel process the nanowires are transferred to a transparent, cell-compatible epoxy membrane. Cargo delivery and detailed cell-nanowire interaction studies are performed, revealing that the cell plasma membrane tightly wraps the nanowires, while cell membrane penetration is not observed. The presented device offers an efficient investigation platform for further optimization, leading towards a simple and versatile impalement delivery system
    • M. Wadsater, T. Laursen, A. Singha, N.S. Hatzakis, D. Stamou, R. Barker, K. Mortensen, R. Feidenhans'l, B.L. Møller and M. Cardenas: Monitoring Shifts in the Conformation Equilibrium of the Membrane Protein Cytochrome P450 Reductase (POR) in Nanodiscs. Journal of Biological Chemistry 287 (41): 34596-34603 (2013) 
    Nanodiscs are self-assembled ∼50-nm(2) patches of lipid bilayers stabilized by amphipathic belt proteins. We demonstrate that a well ordered dense film of nanodiscs serves for non-destructive, label-free studies of isolated membrane proteins in a native like environment using neutron reflectometry (NR). This method exceeds studies of membrane proteins in vesicle or supported lipid bilayer because membrane proteins can be selectively adsorbed with controlled orientation. As a proof of concept, the mechanism of action of the membrane-anchored cytochrome P450 reductase (POR) is studied here. This enzyme is responsible for catalyzing the transfer of electrons from NADPH to cytochrome P450s and thus is a key enzyme in the biosynthesis of numerous primary and secondary metabolites in plants. Neutron reflectometry shows a coexistence of two different POR conformations, a compact and an extended form with a thickness of 44 and 79 Å, respectively. Upon complete reduction by NADPH, the conformational equilibrium shifts toward the compact form protecting the reduced FMN cofactor from engaging in unspecific electron transfer reaction

    • O. Bornert, T. C. Møller, J. Boeuf, M.-P. Candusso, R. Wagner, K. L. Martinez and F. Simonin;  Identification of a novel protein-protein interaction motif mediating interaction of GASPs with G protein-coupled receptors" PLoS One 8 (2) e56336 (2013) 
    GPCR desensitization and down-regulation are considered key molecular events underlying the development of tolerance in vivo. Among the many regulatory proteins that are involved in these complex processes, GASP-1 have been shown to participate to the sorting of several receptors toward the degradation pathway. This protein belongs to the recently identified GPCR-associated sorting proteins (GASPs) family that comprises ten members for which structural and functional details are poorly documented. We present here a detailed structure-function relationship analysis of the molecular interaction between GASPs and a panel of GPCRs. In a first step, GST-pull down experiments revealed that all the tested GASPs display significant interactions with a wide range of GPCRs. Importantly, the different GASP members exhibiting the strongest interaction properties were also characterized by the presence of a small, highly conserved and repeated "GASP motif" of 15 amino acids. We further showed using GST-pull down, surface plasmon resonance and co-immunoprecipitation experiments that the central domain of GASP-1, which contains 22 GASP motifs, is essential for the interaction with GPCRs. We then used site directed mutagenesis and competition experiments with synthetic peptides to demonstrate that the GASP motif, and particularly its highly conserved core sequence SWFW, is critically involved in the interaction with GPCRs. Overall, our data show that several members of the GASP family interact with GPCRs and highlight the presence within GASPs of a novel protein-protein interaction motif that might represent a new target to investigate the involvement of GASPs in the modulation of the activity of GPCRs.
    • Christensen, DV, Trier, F, Chen, YZ, Smith, A , Nygård, J & Pryds, N: Controlling interfacial states in amorphous/crystalline LaAlO 3 /SrTiO 3 heterostructures by electric fields Applied Physics Letters  102 (2): 021602 (2013) 
    The tunable metal-insulator transition in crystalline LaAlO3/SrTiO3 heterostructures constitutes a central element in the range of remarkable interface properties that has made this oxide system subject to extensive research. Recently, metallic interfaces have also been realized when depositing amorphous LaAlO3 films on SrTiO3. Here, we present a non-volatile and reversible tuning of the interface conductivity by more than 3 orders of magnitude at room temperature by applying an electric field to such amorphous/crystalline heterostructures with amorphous LaAlO3 film thicknesses of ∼2 nm. We show that the tunability is strongly temperature dependent, and demonstrate a simple protocol for enhancing the tunability.

    • Gasadei, A , Krogstrup, P , Heiss, M, Colombo, C, Röhr, JA, Ruelle, T , Upadhyay, S , Sørensen, CB , Nygård, J & i Morral, AF Doping incorporation paths in catalyst-free Be-doped GaAs nanowires 'Applied Physics Letters 102 (1): 013117 (2013)
    The incorporation paths of Be in GaAs nanowires grown by the Ga-assisted method in molecular beam epitaxy has been investigated by electrical measurements of nanowires with different doping profiles. We find that Be atoms incorporate preferentially via the nanowire side facets, while the incorporation path through the Ga droplet is negligible. We also demonstrate that Be can diffuse into the volume of the nanowire giving an alternative incorporation path. This work is an important step towards controlled doping of nanowires and will serve as a help for designing future devices based on nanowires.
    • T. Laursen, P. Naur and B.L. Møller: Amphipol assisted trapping of a functional Cytochrome P450 system. Biotechnology and Applied Biochemistry. 60 (1): 119-127 (2013)
    In plants, some enzymes of the cytochrome P450 (CYP) superfamily are thought to organize into transient dynamic metabolons to optimize the biosynthesis of bioactive natural products. Metabolon formation may facilitate efficient turnover of labile and toxic intermediates and prevent undesired metabolic cross talk. Two CYPs, CYP79A1 and CYP71E1 involved in the synthesis of dhurrin, were used to assess the possibility to use amphipols (APols) to trap these membrane‐bound enzymes in a soluble form in a detergent‐free environment. APol surfactants are short polymers composed of a hydrophilic backbone randomly grafted with hydrophobic side chains. An optimal ratio of 1:2 w/w of protein to APol (A8‐35) was required for trapping the single transmembrane helices of CYP79A1, CYP71E1, and the electron partner cytochrome P450 oxidoreductase (POR). CYP79A1 and POR retained their individual activity upon A8‐35 trapping, whereas a direct interaction between CYP79A1 and POR was hampered, probably due to electrostatic repulsion caused by the negatively charged APol molecules. Upon substitution of POR with NADPH‐ferredoxin oxidoreductase and ferredoxin as an electron donor system, the CYPs were shown to be catalytically active. The use of APol surfactants in functional and structural studies of membrane proteins is discussed.
    • P. Zhang, T.N. Jørgensen, C.J. Loland, A.H. Newman “A Rhodamine-Labeled Citalopram Analogue as a High-Affinity Fluorescent Probe for the Serotonin Transporter” Bioorganic & Medicinal Chemistry Letters 23(1): 323-6 (2013)
    A novel fluorescent ligand was synthesized as a high-affinity, high specificity probe for visualizing the serotonin transporter (SERT). The rhodamine fluorophore was extended from an aniline substitution on the 5-position of the dihydroisobenzofuran ring of citalopram (2, 1-(3-(dimethylamino)propyl)-1-(4-fluorophenyl)-1,3-dihydroisobenzofuran-5-carbonitrile), using an ethylamino linker. The resulting rhodamine-labeled ligand 8inhibited [3H]5-HT uptake in COS-7 cells (Ki = 225 nM) with similar potency to the tropane-based JHC 1-064 (1), but with higher specificity towards the SERT relative to the transporters for dopamine and norepinephrine. Visualization of the SERT with compound 8 was demonstrated by confocal microscopy in HEK293 cells stably expressing EGFP–SERT.
    • P.S. Chae, R.R. Rana, K. Gotfryd, S.G.F. Rasmussen, A.C. Kruse, K.H Cho, S. Capaldi, B. Kobilka, C.J. Loland, U. Gether, S. Banerjee, B. Byrne, J.K. Lee,  S.H. Gellman “Glucose-Neopentyl Glycol (GNG) Amphiphiles for Membrane Protein Solubilization, Stabilization and Crystallization” Chemical Communications 49(23):2287-9 (2013)
    The development of a new class of surfactants for membrane protein manipulation, “GNG amphiphiles”, is reported. These amphiphiles display promising behavior for membrane proteins, as demonstrated recently by the high resolution structure of a sodium-pumping pyrophosphatase reported by Kellosalo et al. (Science, 2012, 337, 473).
    • Irannejad R., Tomshine J.C., Tomshine J.R., Chevalier M., Mahoney J.P., Steyaert J., Rasmussen S.G.F., Sunahara R.K., El-Samad H., Huang B., von Zastrow M. Conformational biosensors reveal GPCR signaling from endosomes, Nature (2013), doi:10.1038/nature12000, published online 20 March 2013
    A long-held tenet of molecular pharmacology is that canonical signal transduction mediated by G-protein-coupled receptor (GPCR) coupling to heterotrimeric G proteins is confined to the plasma membrane. Evidence supporting this traditional view is based on analytical methods that provide limited or no subcellular resolution1. It has been subsequently proposed that signalling by internalized GPCRs is restricted to G-protein-independent mechanisms such as scaffolding by arrestins2, 3, or GPCR activation elicits a discrete form of persistent G protein signalling4, 5, 6, 7, 8, 9, or that internalized GPCRs can indeed contribute to the acute G-protein-mediated response10. Evidence supporting these various latter hypotheses is indirect or subject to alternative interpretation, and it remains unknown if endosome-localized GPCRs are even present in an active form. Here we describe the application of conformation-specific single-domain antibodies (nanobodies) to directly probe activation of the β2-adrenoceptor, a prototypical GPCR11, and its cognate G protein, Gs (ref. 12), in living mammalian cells. We show that the adrenergic agonist isoprenaline promotes receptor and G protein activation in the plasma membrane as expected, but also in the early endosome membrane, and that internalized receptors contribute to the overall cellular cyclic AMP response within several minutes after agonist application. These findings provide direct support for the hypothesis that canonical GPCR signalling occurs from endosomes as well as the plasma membrane, and suggest a versatile strategy for probing dynamic conformational changein vivo.
    Plants are sessile organisms and dependent on deployment of secondary metabolites for their response to biotic and abiotic challenges. A trade-off is envisioned between resources allocated to growth, development, and reproduction and to the biosynthesis, storage, and maintenance of secondary metabolites. However, increasing evidence suggests that secondary metabolites serve auxiliary roles, including functions associated with primary metabolism. In this opinion article, we examine how the costs of plant chemical defense can be offset by multifunctional biosynthesis and the optimization of primary metabolism. These additional benefits may negate the trade-off between primary and secondary metabolism, and provide plants with an innate plasticity required for growth, development, and interactions with their environment.

    • Lloret, N., Frederiksen, R.S., Møller, T.C et. al. Effects of buffer composition and dilution on nanowire field-effect biosensors. Nanotechnology 24: 035501-035509 (2013)

      Nanowire-based field-effect transistors (FETs) can be used as ultra sensitive and label-free biosensors for detecting protein–protein interactions. A way to increase the performance of such sensors is to dilute the sensing buffer drastically. However, we show here that this can have an important effect on the function of the proteins. Moreover, it is demonstrated that this dilution significantly affects the pH stability of the sensing buffer, which consequently impacts the charge of the protein and thus the response and signal-to-noise ratio in the sensing experiments. Three model systems are investigated experimentally to illustrate the impact on ligand–protein and protein–protein interactions. Simulations are performed to illustrate the effect on the performance of the sensors. Combining various parameters, the current study provides a means for evaluating and selecting the most appropriate buffer composition for bioFET measurements.

    • Christensen, S.M., Bolinger, P.Y., Hatzakis, N.S., Mortensen, M.W. and Dimitrios Stamou. Mixing subattolitre volumes in a quantitative and highly parallel manner with soft matter nanofluidics. Nature Nanotechnology 7: 51–55 (2012) I.F. 30.324

      Handling and mixing ultrasmall volumes of reactants in parallel can increase the throughput and complexity of screening assays while simultaneously reducing reagent consumption. Microfabricated silicon and plastic can provide reliable fluidic devices, but cannot typically handle total volumes smaller than ~1 × 10–12 l. Self-assembled soft matter nanocontainers can in principle significantly improve miniaturization and biocompatibility, but exploiting their full potential is a challenge due to their small dimensions. Here, we show that small unilamellar lipid vesicles can be used to mix volumes as small as 1 × 10–19 l in a reproducible and highly parallelized fashion. The self-enclosed nanoreactors are functionalized with lipids of opposite charge to achieve reliable fusion. Single vesicles encapsulating one set of reactants are immobilized on a glass surface and then fused with diffusing vesicles of opposite charge that carry a complementary set of reactants. We find that ~85% of the ~1 × 106 cm–2 surface-tethered nanoreactors undergo non-deterministic fusion, which is leakage-free in all cases, and the system allows up to three to four consecutive mixing events per nanoreactor.

    • Chaabane, S.B., Liu, R., Chinnusamy, V., Kwon, Y., Park, J.,Kim, S.Y., Zhu, J., Yang, S.S and Lee, B. STA1, an Arabidopsis pre-mRNA processing factor 6 homolog, is a new player involved in miRNA biogenesis. Nucleic Acids Research 1–14 (2013)

      MicroRNAs (miRNAs) are small regulatory RNAs that have important regulatory roles in numerous developmental and metabolic processes in most eukaryotes. In Arabidopsis, DICER-LIKE1 (DCL1), HYPONASTIC LEAVES 1, SERRATE, HUA ENHANCER1 and HASTY are involved in processing of primary miRNAs (pri-miRNAs) to yield precursor miRNAs (pre-miRNAs) and eventually miRNAs. In addition to these components, mRNA cap-binding proteins, CBP80/ABA HYPERSENSITIVE1 and CBP20, also participate in miRNA biogenesis. Here, we show that STABILIZED1 (STA1), an Arabidopsis pre-mRNA processing factor 6 homolog, is also involved in the biogenesis of miRNAs. Similar to other miRNA biogenesisdefective mutants, sta1-1 accumulated significantly lower levels of mature miRNAs and concurrently higher levels of pri-miRNAs than wild type. The dramatic reductions of mature miRNAs were associated with the accumulation of their target gene transcripts and developmental defects. Furthermore, sta1-1 impaired splicing of intron containing pri-miRNAs and decreased transcript levels of DCL1. These results suggest that STA1 is involved in miRNA biogenesis directly by functioning in pri-miRNA splicing and indirectly by modulating the DCL1 transcript level.

    • Li, T.; Hauptmann, J.R.; Wei, Z. Søren Petersen, Nicolas Bovet, Vosch, T., Nygård, J., Hu, W., Liu, Y.,  Bjørnholm, T., Nørgaard, K. and Laursen, B.W.: Solution-processed Ultrathin Chemically Derived Graphene Films as Soft Top Contacts for Solid-State Molecular Electronic Junctions. Advanced Materials 24: 1333-1339 (2012) I.F. 10.880

      A novel method using solution-processed ultrathin chemically derived graphene films as soft top contacts for the non-destructive fabrication of molecular junctions is demonstrated. We believe this protocol will greatly enrich the solid-state test beds for molecular electronics due to its low-cost, easy-processing and flexible nature.

    • Jensen K., Jensen P.E. and Møller B.L. Light-driven chemical synthesis Trends in Plant Science 17 (2): 60–63 (2012) I.F. 10.095

      Depletion of the fossil fuel reserves of the Earth has prompted research into sources of renewable and sustainable energy, and feedstock for the chemical and pharmaceutical industries to support the transition towards a bio-based society. Photosynthesis efficiently captures solar energy, but its subsequent conversion into chemical energy in the form of biomass is limited to a final output in the 1–4% range. Re-routing of photosynthetic electron transport and reducing power directly into desired biosynthetic pathways offers a new avenue for sustainable production of high-value products.

    • Elizondo. E., Larsen, J., Hatzakis, N.S., Cabrera, I., Bjørnholm, T., Veciana, J., Stamou, D. and  Ventosa, N. Influence of the Preparation Route on the Supramolecular Organization of Lipids in a Vesicular System. J. Am. Chem. Soc.,134 (4) 1918–1921 (2012) I.F. 9.023

      A confocal fluorescence microscopy-based assay was used for studying the influence of the preparation route on the supramolecular organization of lipids in a vesicular system. In this work, vesicles composed of cholesterol and CTAB (1/1 mol %) or cholesterol and DOPC (2/8 mol %) and incorporating two membrane dyes were prepared by either a compressed fluid (CF)-based method (DELOS-susp) or a conventional film hydration procedure. They were subsequently immobilized and imaged individually using a confocal fluorescence microscope. Two integrated fluorescence intensities, Idye1 and Idye2, were assigned to each tracked vesicle, and their ratio, Idye1/Idye2, was used for quantifying the degree of membrane inhomogeneity between individual vesicles within each sample. A distribution of Idye1/Idye2values was obtained for all the studied vesicular systems, indicating intrasample heterogeneity. The degree of inhomogeneity (DI) was similar for Chol/DOPC vesicles prepared by both procedures. In contrast, DI was more than double for the hydration method compared to the CF-based method in the case of Chol/CTAB vesicles, which can suffer from lipid demixing during film formation. These findings reveal a more homogeneous vesicle formation path by CFs, which warranted good homogeneity of the vesicular system, independently of the lipid mixture used.

    • Hatzakis, N.S., Wei, L., Jorgensen, S.K., Kunding, A.H., Bolinger, P., Ehrlich, N., Makarov, I., Skjot, M.,   Svendsen, A., Hedegård, P. and Stamou, D.: Single Enzyme Studies Reveal the Existence of Discrete Functional States for Monomeric Enzymes and How They Are “Selected” upon Allosteric Regulation. J. Am. Chem. Soc. 134(22): 9296 (2012) I.F. 9.023

      Allosteric regulation of enzymatic activity forms the basis for controlling a plethora of vital cellular processes. While the mechanism underlying regulation of multimeric enzymes is generally well understood and proposed to primarily operate via conformational selection, the mechanism underlying allosteric regulation of monomeric enzymes is poorly understood. Here we monitored for the first time allosteric regulation of enzymatic activity at the single molecule level. We measured single stochastic catalytic turnovers of a monomeric metabolic enzyme (Thermomyces lanuginosus Lipase) while titrating its proximity to a lipid membrane that acts as an allosteric effector. The single molecule measurements revealed the existence of discrete binary functional states that could not be identified in macroscopic measurements due to ensemble averaging. The discrete functional states correlate with the enzyme’s major conformational states and are redistributed in the presence of the regulatory effector. Thus, our data support allosteric regulation of monomeric enzymes to operate via selection of preexisting functional states and not via induction of new ones.

    • Annaka, M., Mortensen, K., Vigild, M.E., Matsuura, T., Tsuji, S., Ueda, T., and Tsujinaka, H. Design of an Injectable in Situ Gelation Biomaterials for Vitreous Substitutes Biomacromolecules 12: 4011−4021 (2012) I.F. 5.327

      To adapt the physical properties of living materials to their biological function, nature developed various types of polymers with outstanding physical behavior. One example is the vitreous body, which is important intraocular elements not only because of its optical and mechanical performances, but also due to its important role in the pathogenesis and treatment of conditions affecting adjacent tissues and eventually the whole eye. Here, we report a novel biocompatible material for injectable vitreous substitute, composed of thermosensitive amphiphilic polymer, which is capable of forming a transparent gel in the vitreous cavity. It is nontoxic, provides adequate support for the retina, and allows light to reach the sensory elements at the back of the eye. The amphiphilic polymer exhibits mechanical stability by assembling to form highly interconnected hydrophobic domains, which leads to the constitution of a network structure.

    • Madsen, K.L., Thorsen, T.S., Rahbek-Clemmensen, T., Eriksen, J. and Gether, U.: Protein Interacting with C Kinase 1 (PICK1) Reduces Reinsertion Rates of Interaction Partners Sorted to the RAB11-Dependent Slow Recycling Pathway. J. Biol. Chem 287: 12293-308 (2012) I.F. 5.328

      The scaffolding protein PICK1 (protein interacting with C kinase 1) contains an N-terminal PSD-95/Discs large/ZO-1 (PDZ) domain and a central lipid-binding Bin/amphiphysin/Rvs (BAR) domain. PICK1 is thought to regulate trafficking of its PDZ binding partners but different and even opposing functions have been suggested. Here, we apply ELISA-based assays and confocal microscopy in HEK293 cells with inducible PICK1 expression to assess in an isolated system the ability of PICK1 to regulate trafficking of natural and engineered PDZ binding partners. The dopamine transporter (DAT), which primarily sorts to degradation upon internalization, did not form perinuclear clusters with PICK1, and PICK1 did not affect DAT internalization/recycling. However, transfer of the PICK1-binding DAT C terminus to the β2-adrenergic receptor, which sorts to recycling upon internalization, led to formation of PICK1 co-clusters in Rab11-positive compartments. Furthermore, PICK1 inhibited Rab11-mediated recycling of the receptor in a BAR and PDZ domain-dependent manner. In contrast, transfer of the DAT C terminus to the δ-opioid receptor, which sorts to degradation, did not result in PICK1 co-clusters or any change in internalization/recycling. Further support for a role of PICK1 determined by its PDZ cargo was obtained for the PICK1 interaction partner prolactin-releasing peptide receptor (GPR10). GPR10 co-localized with Rab11 and clustered with PICK1 upon constitutive internalization but co-localized with the late endosomal marker Rab7 and did not cluster with PICK1 upon agonist-induced internalization. Our data suggest a selective role of PICK1 in clustering and reducing the recycling rates of PDZ domain binding partners sorted to the Rab11-dependent recycling pathway.

    • Skov Pedersen, J., Oliveira, C.L.P., Baun Hübschmann, H., Arleth, L., Manniche, S., Kirkby, N. and Mørck Nielsen, H.: Structure of Immune Stimulating Complex Matrices and Stimulating Complexes in Suspension Determined by Small-Angle X-Ray Scattering. Biophysical Journal 102: 2372-2380 (2012) I.F. 4.218

      Immune stimulating complex (ISCOM) particles consisting of a mixture of Quil-A, cholesterol, and phospholipids were structurally characterized by small-angle x-ray scattering (SAXS). The ISCOM particles are perforated vesicles of very well-defined structures. We developed and implemented a novel (to our knowledge) modeling method based on Monte Carlo simulation integrations to describe the SAXS data. This approach is similar to the traditional modeling of SAXS data, in which a structure is assumed, the scattering intensity is calculated, and structural parameters are optimized by weighted least-squares methods when the model scattering intensity is fitted to the experimental data. SAXS data from plain ISCOM matrix particles in aqueous suspension, as well as those from complete ISCOMs (i.e., with an antigen (tetanus toxoid) incorporated) can be modeled as a polydisperse distribution of perforated bilayer vesicles with icosahedral, football, or tennis ball structures. The dominating structure is the tennis ball structure, with an outer diameter of 40 nm and with 20 holes 5–6 nm in diameter. The lipid bilayer membrane is 4.6 nm thick, with a low-electron-density, 2.0-nm-thick hydrocarbon core. Surprisingly, in the ISCOMs, the tetanus toxoid is located just below the membrane inside the particles.

    • Krogstrup, P., Madsen, M.H. , Wen, H., Miwa, K., Nygård, J., Masamitu, T. and Feidenhans'l, R.K.:  In-situ x-ray characterization of wurtzite formation in GaAs nanowires.  Applied Physics Letters 100 (9): 093103 (2012) I.F. 3.841

      In-situ monitoring of the crystal structure formation during Ga-assisted GaAs nanowire growth on Si(111) substrates has been performed in a combined molecular beam epitaxy growth and x-ray characterization experiment. Under Ga rich conditions, we show that an increase in the V/III ratio increases the formation rate of the wurtzite structure. Moreover, the response time for changes in the structural phase formation to changes in the beam fluxes is observed to be much longer than predicted time scales of adatom kinetics and liquid diffusion. This suggests that the morphology of the growth interface plays the key role for the relative growth structure formation rates.

    • Frisch, T. and Møller, B.L. Possible evolution of alliarinoside biosynthesis from the glucosinolate pathway in Alliaria petiolata  FEBS Journal 279: 1545–1562 (2012)  I.F. 3.129

      Nitrile formation in plants involves the activity of cytochrome P450s. Hydroxynitrile glucosides are widespread among plants but generally do not occur in glucosinolate producing species. Alliaria petiolata (garlic mustard, Brassicaceae) is the only species known to produce glucosinolates as well as a γ-hydroxynitrile glucoside. Furthermore, A. petiolata has been described to release diffusible cyanide, which indicates the presence of unidentified cyanogenic glucoside(s). Our research on A. petiolata addresses the molecular evolution of P450s. By integrating current knowledge about glucosinolate and hydroxynitrile glucoside biosynthesis in other species and new visions on recurrent evolution of hydroxynitrile glucoside biosynthesis, we propose a pathway for biosynthesis of the γ-hydroxynitrile glucoside, alliarinoside. Homomethionine and the corresponding oxime are suggested as shared intermediates in the biosynthesis of alliarinoside and 2-propenyl glucosinolate. The first committed step in the alliarinoside pathway is envisioned to be catalysed by a P450, which has been recruited to metabolize the oxime. Furthermore, alliarinoside biosynthesis is suggested to involve enzyme activities common to secondary modification of glucosinolates. Thus, we argue that biosynthesis of alliarinoside may be the first known case of a hydroxynitrile glucoside pathway having evolved from the glucosinolate pathway. An intriguing question is whether the proposed hydroxynitrile intermediate may also be converted to novel homomethionine-derived cyanogenic glucoside(s), which could release cyanide. Elucidation of the pathway for biosynthesis of alliarinoside and other putative hydroxynitrile glucosides in A. petiolata is envisioned to offer significant new knowledge on the emerging picture of P450 functional dynamics as a basis for recurrent evolution of pathways for bioactive natural product biosynthesis.


    • Jain,T., Roodbeen, R., Reeler, N.E.A., Vosch, T., Jensen, K.J., Bjørnholm, T., Nørgaard, K.: End-to-end assembly of gold nanorods via oligopeptide linking and surfactant control. Journal of Colloid and Interface Science 376: 83–90 (2012) I.F. 3.068

      We report two novel approaches for fabricating self-assembled chains of end-to-end linked Au nanorods separated by a nanogap. In one approach, bi-functional cysteine end-capped oligopeptides of different lengths are used as the linking agent. The widths of the produced nanogaps scale with the length and tertiary structure of the peptide linker. Functionalized oligopeptides containing an acetylene group are also employed as a linker, and the functional group is uniquely identified using surface-enhanced Raman spectroscopy. The development of an oligopeptide-linking platform is motivated by the ease of synthesis and high modularity of peptides; these features enable the possibility to integrate diverse functionality into molecular nanogap junctions – synthesized in water. The stepwise nanochain formation is followed via the evolution of the longitudinal plasmon absorption band in combination with transmission electron microscopy. The reaction rate and extent is tuned by controlling the concentration of the stabilizing CTAB surfactant in the solution. At very low surfactant concentrations, spontaneous end-to-end linking of the Au nanorods is observed even in the absence of linking peptide. The assembled AuNRs may act as next-generation electrodes in a platform for molecular electronics and synthetic biology.


    • Nilsson, L., Lundmark, M., Jensen, P.E. and Nielsen, T.H.: The Arabidopsis transcription factor PHR1 is essential for adaptation to high light and retaining functional photosynthesis during phosphate starvation. Physiol. Plant. 144: 35-37 (2012) I.F. 3.067

      The transcription factor PHR1 (PHOSPHATE STARVATION RESPONSE 1; encoded by gene At4g28610) is central for adaptation to phosphate deficiency in Arabidopsis (Arabidopsis thaliana). A rapid turnover of phosphate pools in the leaves is essential for energy transfer and metabolism within photosynthesis, and consequently, we hypothesized that PHR1 is needed for adaptation to high-light stress during P deficiency. We analyzed three Arabidopsis plant lines: wild-type, a transgenic PHR1 overexpressor line and a knockout mutant,phr1. The plants were grown under phosphate-limiting and sufficient conditions and exposed to different light conditions. Photosynthetic activity and light stress of the leaves were characterized by analyzing accumulation of carbohydrates, chlorophyll fluorescence, immunoblot detection of photosystem subunits and anthocyanin accumulation. Compared to the wild-type and the overexpressor line, the phr1 mutant has decreased levels of phosphate, anthocyanins and carbohydrates during combined P deficiency and light stress. The stressed mutant also has strongly decreased photosystem II (PSII) quantum efficiency, and shows degradation of the core units of PSII demonstrating extensive irreversible photodamage. We conclude that PHR1 is needed for the metabolic balance, for retaining Pi levels and for inducing anthocyanin production, and during P deficiency PHR1 is vital for adaptations to avoid permanent damage to photosystems during high-light conditions.


    • Kirkensgaard, J. J. K.: Striped networks and other hierarchical structures in AmBmCn (2m+n)-miktoarm star terpolymer melts. Physical Review E 85: 031802 (2012) I.F. 2.352

      Using dissipative particle dynamics simulations we give numerical evidence of the formation of “striped” (or AB alternating) diamond and gyroid network structures and other hierarchical morphologies in AmBmCn (2m+n)-miktoarm star terpolymers where the main variable is the ratio x=n/m with m,n being the number of equal length polymer arms of A and B and C, respectively. The formed networks are purely a result of the star topology, as clearly shown by direct comparison with parallel ABCmiktoarm star terpolymer simulations with matching overall composition. Progressively changing x, the system adopts the following phase sequence: three-colored lamellae, C spheres embedded in AB lamellae, C spheres decorating AB lamellae, three-colored [6.6.6] tiling, AB striped diamond network, AB striped gyroid network,AB striped hexagonally arranged cylinders, and finally AB striped globular aggregates. The striped gyroid is particularly interesting as it constitutes an inherently chiral structure made from achiral building blocks.


    • Jensen, K., Johnston, J.B, Ortiz de Montellano, P.R. and Møller, B.L. Photosystem I from plants as a bacterial cytochrome P450 surrogate electron donor: terminal hydroxylation of branched hydrocarbon chains Biotechnology Letters 34:239–245 (2012) I.F. 1.768

      The ability of cytochrome P450 enzymes to catalyze highly regio- and stereospecific hydroxylations makes them attractive alternatives to approaches based on chemical synthesis but they require expensive cofactors, e.g. NAD(P)H, which limits their commercial potential. Ferredoxin (Fdx) is a multifunctional electron carrier that in plants accepts electrons from photosystem I (PSI) and facilitates photoreduction of NADP+ to NADPH mediated by ferredoxin-NAD(P)H oxidoreductase (FdR). In bacteria, the electron flow is reversed and Fdx accepts electrons from NADPH via FdR and serves as the direct electron donor to bacterial P450s. By combining the two systems, we demonstrate that irradiation of PSI can drive the activity of a bacterial P450, CYP124 fromMycobacterium tuberculosis. The substitution of the costly cofactor NADPH with sunlight illustrates the potential of the light-driven hydroxylation system for biotechnology applications.

    • Tikhomirov, V.K., Vosch, T., Fron, E., Rodríguez, C.V.D., Velázquez, J.J., Kirilenko, D., Van Tendeloo, G., Hofkens, J., Van der Auweraerc C.M., Moshchalkova V.V. Luminescence of oxyfluoride glasses co-doped with Ag nanoclusters and Yb3+ions RSC Advances 2: 1496–1501 (2012) I.F. not yet available

      Bulk oxyfluoride glasses co-doped with Ag nanoclusters and Yb3+ ions have been prepared by a melt quenching technique. When excited in the absorption band of the Ag nanoclusters between 300 to 500 nm, these glasses emit a broad band characteristic of the Ag nanoclusters between 400 to 750 nm as well as an emission band between 900 to 1100 nm, originating from Yb3+ ions. The intensity ratio of the Yb3+/Ag emission bands increases with the Ag doping level at a fixed concentration of Yb3+, indicating the presence of energy transfer mechanism from the Ag nanoclusters to the Yb3+ ions. Comparison of time-resolved decay kinetics of the luminescence in the respectively Ag nanocluster-Yb3+ co-doped and single Ag nanocluster doped glasses, hints towards an energy transfer from the red and infrared emitting Ag nanoclusters to the Yb3+ ions.


    • Åkesson, A., Lind, T., Ehrlich, N., Stamou, D., Wacklin, H. and Cárdenas, M. Composition and structure of mixed phospholipid supported bilayers formed by POPC and DPPC. Soft Matter 8:5658-5665 (2012)

      In this paper we present a systematic study of the morphology and composition of supported lipid bilayers (SLBs) formed by vesicle fusion using a wide variety of surface sensitive techniques that give information about the lateral as well as vertical structure and bilayer fluidity. SLBs of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) mixtures at five different bulk vesicle compositions were formed in such a way that the phase separation boundaries were crossed. For all compositions studied, the SLBs were systematically enriched with POPC compared to the nominal vesicle composition. Nevertheless, gel-fluid domain coexistence was observed for SLB compositions in which phase separation was expected based on the bulk phase diagram. The probable causes for the compositional difference in the SLBs are discussed in terms of the phase behaviour of the mixture and its effect on the membrane formation process by vesicle fusion.


    • Holm, S. The Scientific Aspirations of Synthetic Biology and the Need for Analytic Ethics. Ethics, Policy & Environment 15(1): 25-28 (2012)


    • Chae, P.S., Rasmussen, S.G.F.,  Rana, R.R., Gotfryd, K., Kruse, A.C., Nurva, S., Gether, U., Guan, L., Loland, C.J., Byrne, B., Kobilka, B. and Gellman, S.H. A New Class of Amphiphiles Bearing Rigid Hydrophobic Groups for Solubilization and Stabilization of Membrane Proteins. Chem. Eur. J. (2012) I.F. 5.476

      Non-traditional amphiphiles: Conferring aqueous solubility on membrane proteins generally requires the use of a detergent or other amphiphilic agent. A new class of amphiphiles was synthesized, based on steroidal lipophilic groups, and evaluated with several membrane proteins. The results show that the new amphiphile, “glyco-diosgenin” (GDN; see figure), confers enhanced stability to a variety of membrane proteins in solution relative to popular conventional detergents, such as dodecylmaltoside (DDM).


    • Zerbe, P., Hamberger, B., Chiang, A., Hamberger, B., Draper, J.A., Britton, R. and Bohlmann J.: Bifunctional cis-abienol synthase from Abies balsamea discovered by transcriptome sequencing and its implications for diterpenoid fragrance production. J. Biol. Chem MS ID#: JBC/2011/317669 (2012) I.F. 5.328

      The labdanoid diterpene alcohol cis-abienol is a major component of the aromatic oleoresin of balsam fir (Abies balsamea) and serves as a valuable bioproduct material for the fragrance industry. Using high-throughput 454 transcriptome sequencing and metabolite profiling of balsam fir bark tissue, we identified candidate diterpene synthase sequences for full-length cDNA cloning and functional characterization. We discovered a bifunctional class I/II cis-abienol synthase (AbCAS), along with the paralogous levopimaradiene/abietadiene synthase and isopimaradiene synthase, all of which are members of the gymnosperm-specific TPS-d subfamily. The AbCAS-catalyzed formation of cis-abienol proceeds via cyclization and hydroxylation at carbon C-8 of a postulated carbocation intermediate in the class II active site, followed by cleavage of the diphosphate group and termination of the reaction sequence without further cyclization in the class I active site. This reaction mechanism is distinct from that of synthases of the isopimaradiene- or levopimaradiene/abietadiene synthase type, which employ deprotonation reactions in the class II active site and secondary cyclizations in the class I active site, leading to tricyclic diterpenes. Comparative homology modeling suggested the active site residues Asp-348, Leu-617, Phe-696, and Gly-723 as potentially important for the specificity ofAbCAS. As a class I/II bifunctional enzyme, AbCAS is a promising target for metabolic engineering of cis-abienol production.


    • Nygaard, J., Munch, H.K., Thulstrup, P.W., Christensen, N.J., Høeg-Jensen, T., Jensen, K.J and Arleth, L.: Metal ion controlled self-assembly of a chemically re- engineered protein drug studied by Small-Angle X-ray Scattering Langmuir, (2012) I.F. 4.269

      Precise control of the oligomeric state of proteins is of central importance for biological function and for the properties of biopharmaceutical drugs. Here, the self-assembly of 2,2′-bipyridine conjugated monomeric insulin analogues, induced through coordination to divalent metal ions, was studied. This protein drug system was designed to form non-native homo-oligomers through selective coordination of two divalent metal ions, Fe(II) and Zn(II), respectively. The insulin type chosen for this study is a variant designed for a reduced tendency toward native dimer formation at physiological concentrations. A small-angle X-ray scattering analysis of the bipyridine-modified insulin system confirmed an organization into a novel well-ordered structure based on insulin trimers, as induced by the addition of Fe(II). In contrast, unmodified monomeric insulin formed larger and more randomly structured assemblies upon addition of Fe(II). The addition of Zn(II), on the other hand, led to the formation of small quantities of insulin hexamers for both the bipyridine-modified and the unmodified monomeric insulin. Interestingly, the location of the bipyridine-modification significantly affects the tendency to hexamer formation as compared to the unmodified insulin. Our study shows how combining a structural study and chemical design can be used to obtain molecular understanding and control of the self-assembly of a protein drug. This knowledge may eventually be employed to develop an optimized in vivo drug release profile.


    • Saito, S., Motawia, M.S., Olsen, C.E., Møller, B.L. and Bak. S. Biosynthesis of rhodiocyanosides in Lotus japonicus; Rhodiocyanoside A is synthesized from (Z)-2-methylbutanaloxime via 2-methyl-2-butenenitrile  Phytochemistry (2012)    I.F. 3.150

      Lotus japonicus contains the two cyanogenic glucosides, linamarin and lotaustralin, and the non cyanogenic hydroxynitriles, rhodiocyanoside A and D, with rhodiocyanoside A as the major rhodiocyanoside. Rhodiocyanosides are structurally related to cyanogenic glucosides but are not cyanogenic. In vitro administration of intermediates of the lotaustralin pathway to microsomes prepared from selected L. japonicus accessions identified 2-methyl-2-butenenitrile as an intermediate in the rhodiocyanoside biosynthetic pathway. In vitro inhibitory studies with carbon monoxide and tetcyclacis indicate that the conversion of (Z)-2-methylbutanal oxime to 2-methyl-2-butenenitrile is catalyzed by cytochrome P450(s). Carbon monoxide inhibited cyanogenic glucosides as well as rhodiocyanosides synthesis, but inhibition of the latter pathway was much stronger. These results demonstrate that the cyanogenic glucoside and rhodiocyanosides pathways share CYP79Ds to obtain (Z)-2-methylbutanaloxime from l-isoleucine, whereas the subsequent conversions are catalyzed by different P450s. The aglycon of rhodiocyanoside A forms the cyclic product 3-methyl-2(5H)-furanone. Furanones are known to possess antimicrobial properties indicating that rhodiocyanoside A may have evolved to serve as a phytoanticipin that following β-glucosidase activation and cyclization of the aglycone formed, give rise to a potent defense compound.


    • Wadsater, M., Laursen, T., Singha, A., Hatzakis, N.S., Stamou, D., Barker, R., Mortensen, K., Feidenhans'l, R., Moller, B.L. and Cardenas, M. Monitoring Shifts in the Conformation Equilibrium of the Membrane Protein Cytochrome P450 Reductase (POR) in Nanodiscs. J Biol Chem (2012)

      Nanodiscs are self-assembled ∼50-nm(2) patches of lipid bilayers stabilized by amphipathic belt proteins. We demonstrate that a well ordered dense film of nanodiscs serves for non-destructive, label-free studies of isolated membrane proteins in a native like environment using neutron reflectometry (NR). This method exceeds studies of membrane proteins in vesicle or supported lipid bilayer because membrane proteins can be selectively adsorbed with controlled orientation. As a proof of concept, the mechanism of action of the membrane-anchored cytochrome P450 reductase (POR) is studied here. This enzyme is responsible for catalyzing the transfer of electrons from NADPH to cytochrome P450s and thus is a key enzyme in the biosynthesis of numerous primary and secondary metabolites in plants. Neutron reflectometry shows a coexistence of two different POR conformations, a compact and an extended form with a thickness of 44 and 79 Å, respectively. Upon complete reduction by NADPH, the conformational equilibrium shifts toward the compact form protecting the reduced FMN cofactor from engaging in unspecific electron transfer reaction.