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Vienna, Austria

Herzberger J.,Johannes Gutenberg University Mainz | Kurzbach D.,Vienna Biocenter Campus | Werre M.,Johannes Gutenberg University Mainz | Fischer K.,Johannes Gutenberg University Mainz | And 2 more authors.
Macromolecules | Year: 2014

Amine-functional poly(ethylene glycol) (PEG) copolymers have been prepared that exhibit thermo- and pH- responsive behavior in aqueous solution. Three novel tertiary di(n-alkyl)glycidylamine monomers have been introduced for anionic ring-opening copolymerization (AROcP) with ethylene oxide (EO): N,N-di(n-butyl)glycidylamine (DButGA), N,N-di(n-hexyl)glycidylamine (DHexGA), and N,N-di(n-octyl)glycidylamine (DOctGA). Via controlled AROcP we synthesized well-defined (Mw/Mn = 1.05-1.14), water-soluble block- and gradient-type PEG copolymers, containing up to 25 mol % of the respective dialkylglycidylamine comonomer. Molecular weights ranged from 4900 to 12-000 g mol-1. Detailed in-situ 1H NMR kinetics and 13C triad analyses elucidate the microstructures of the copolymers and the relative reactivity of the novel comonomers. Notably, the n-alkyl chain length had no significant influence on the relative reactivity of the glycidylamine comonomers. Calculated reactivity ratios ranged from rEO = 1.84, rDButGA = 0.49 to rEO = 1.78, rDOctGA = 0.42, manifesting the formation of gradient copolymers. Thermo- and pH-responsive properties of these copolymers are precisely tunable by the comonomer ratio, and cloud points in aqueous solution can be adjusted between 21 and 93 °C. Electron paramagnetic resonance (EPR) spectroscopic studies with TEMPO as a spin probe were conducted to elucidate host-guest interactions of the copolymers. Unexpectedly, the n-alkyl chain length of the different glycidylamine comonomers only influences the inverse phase transition of the gradient copolymers, but not of the block copolymers on the nanoscale. Self-assembly of the block- and gradient-type copolymers in aqueous alkaline solution by both static and dynamic light scattering has also been investigated after confirming the existence of pure unimers in methanol. © 2014 American Chemical Society. Source


Kurzbach D.,Vienna Biocenter Campus | Schwarz T.C.,Vienna Biocenter Campus | Platzer G.,Vienna Biocenter Campus | Hofler S.,Vienna Biocenter Campus | And 2 more authors.
Angewandte Chemie - International Edition | Year: 2014

Intrinsically disordered proteins (IDPs) play crucial roles in protein interaction networks and in this context frequently constitute important hubs and interfaces. Here we show by a combination of NMR and EPR spectroscopy that the binding of the cytokine osteopontin (OPN) to its natural ligand, heparin, is accompanied by thermodynamically compensating structural adaptations. The core segment of OPN expands upon binding. This "unfolding-upon-binding" is governed primarily through electrostatic interactions between heparin and charged patches along the protein backbone and compensates for entropic penalties due to heparin-OPN binding. It is shown how structural unfolding compensates for entropic losses through ligand binding in IDPs and elucidates the interplay between structure and thermodynamics of rapid substrate-binding and -release events in IDP interaction networks. Well-balanced: The binding of heparin to the intrinsically disordered protein (IDP) osteopontin was studied by a combination of NMR and EPR spectroscopy. The results offer insight into the interplay between structure and thermodynamics which results in low energy barriers and facilitates rapid substrate-binding and -release events in IDP interaction networks. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. Source


Sara T.,Vienna Biocenter Campus | Schwarz T.C.,Vienna Biocenter Campus | Kurzbach D.,Vienna Biocenter Campus | Wunderlich C.H.,University of Innsbruck | And 2 more authors.
ChemistryOpen | Year: 2014

Protein-protein interactions are of utmost importance to an understanding of biological phenomena since non-covalent and therefore reversible couplings between basic proteins leads to the formation of complex regulatory and adaptive molecular systems. Such systems are capable of maintaining their integrity and respond to external stimuli, processes intimately related to living organisms. These interactions, however, span a wide range of dissociation constants, from sub-nanomolar affinities in tight complexes to high-micromolar or even millimolar affinities in weak, transiently formed protein complexes. Herein, we demonstrate how novel NMR and EPR techniques can be used for the characterization of weak protein-protein (ligand) complexes. Applications to intrinsically disordered proteins and transiently formed protein complexes illustrate the potential of these novel techniques to study hitherto unobserved (and unobservable) higher-order structures of proteins. © 2014 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA. Source


Badarau A.,Vienna Biocenter Campus | Rouha H.,Vienna Biocenter Campus | Malafa S.,Vienna Biocenter Campus | Logan D.T.,SARomics Biostructures AB | And 10 more authors.
Journal of Biological Chemistry | Year: 2015

The bi-component leukocidins of Staphylococcus aureus are important virulence factors that lyse human phagocytic cells and contribute to immune evasion. The γ-hemolysins (HlgAB and HlgCB) and Panton-Valentine leukocidin (PVL or LukSF) were shown to assemble from soluble subunits into membrane-bound oligomers on the surface of target cells, creating barrel-like pore structures that lead to cell lysis. LukGH is the most distantly related member of this toxin family, sharing only 30-40% amino acid sequence identity with the others. We observed that, unlike other leukocidin subunits, recombinant LukH and LukG had low solubility and were unable to bind to target cells, unless both components were present. Using biolayer interferometry and intrinsic tryptophan fluorescence we detected binding of LukH to LukG in solution with an affinity in the low nanomolar range and dynamic light scattering measurements confirmed formation of a heterodimer. We elucidated the structure of LukGH by x-ray crystallography at 2.8-A˚ resolution. This revealed an octameric structure that strongly resembles that reported for HlgAB, but with important structural differences. Structure guided mutagenesis studies demonstrated that three salt bridges, not found in other bi-component leukocidins, are essential for dimer formation in solution and receptor binding. We detected weak binding of LukH, but not LukG, to the cellular receptor CD11b by biolayer interferometry, suggesting that in common with other members of this toxin family, the S-component has the primary contact role with the receptor. Thesenewinsights provide the basis for novel strategies to counteract this powerful toxin and Staphylococcus aureus pathogenesis. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc. Source

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