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Hamburg, Germany

Broeker N.K.,University of Potsdam | Gohlke U.,Max Delbruck Centrum fur Molekulare Medizin | Muller J.J.,Max Delbruck Centrum fur Molekulare Medizin | Uetrecht C.,University of Potsdam | And 6 more authors.
Glycobiology | Year: 2013

Bacteriophage HK620 recognizes and cleaves the O-antigen polysaccharide of Escherichia coli serogroup O18A1 with its tailspike protein (TSP). HK620TSP binds hexasaccharide fragments with low affinity, but single amino acid exchanges generated a set of high-affinity mutants with submicromolar dissociation constants. Isothermal titration calorimetry showed that only small amounts of heat were released upon complex formation via a large number of direct and solvent-mediated hydrogen bonds between carbohydrate and protein. At room temperature, association was both enthalpy- and entropy-driven emphasizing major solvent rearrangements upon complex formation. Crystal structure analysis showed identical protein and sugar conformers in the TSP complexes regardless of their hexasaccharide affinity. Only in one case, a TSP mutant bound a different hexasaccharide conformer. The extended sugar binding site could be dissected in two regions: first, a hydrophobic pocket at the reducing end with minor affinity contributions. Access to this site could be blocked by a single aspartate to asparagine exchange without major loss in hexasaccharide affinity. Second, a region where the specific exchange of glutamate for glutamine created a site for an additional water molecule. Side-chain rearrangements upon sugar binding led to desolvation and additional hydrogen bonding which define this region of the binding site as the high-affinity scaffold. © The Author 2012. Published by Oxford University Press. All rights reserved. Source


Tseng Y.-H.,National Dong Hwa University | Uetrecht C.,University Utrecht | Uetrecht C.,European XFEL GmbH | Yang S.-C.,National Dong Hwa University | And 3 more authors.
Analytical Chemistry | Year: 2013

Electrospray ionization coupled to native mass spectrometry (MS) has evolved into an important tool in structural biology to decipher the composition of protein complexes. However, the mass analysis of heterogeneous protein assemblies is hampered because of their overlapping charge state distributions, fine structure, and peak broadening. To facilitate the mass analysis, it is of importance to automate preprocessing raw mass spectra, assigning ion series to peaks and deciphering the subunit compositions. So far, the automation of preprocessing raw mass spectra has not been accomplished; Massign was introduced to simplify data analysis and decipher the subunit compositions. In this study, we develop a search engine, AutoMass, to automatically assign ion series to peaks without any additional user input, for example, limited ranges of charge states or ion mass. AutoMass includes an ion intensity-dependent method to check for Gaussian distributions of ion series and an ion intensity-independent method to address highly overlapping and non-Gaussian distributions. The minimax theorem from game theory is adopted to define the boundaries. With AutoMass, the boundaries of ion series in the well-resolved tandem mass spectra of the hepatitis B virus (HBV) capsids and those of the mass spectrum from CRISPR-related cascade protein complex are accurately assigned. Theoretical and experimental HBV ion masses are shown in agreement up to ∼0.03%. The analysis is finished within a minute on a regular workstation. Moreover, less well-resolved mass spectra, for example, complicated multimer mass spectra and norovirus capsid mass spectra at different levels of desolvation, are analyzed. In sum, this first-ever fully automatic program reveals the boundaries of overlapping ion peak series and can further aid developing high-throughput native MS and top-down proteomics. © 2013 American Chemical Society. Source


Pereira H.,CERN | Haug F.,CERN | Santos Silva P.,CERN | Kuster M.,European XFEL GmbH | Lang P.,TU Darmstadt
AIP Conference Proceedings | Year: 2012

The astroparticle physics experiment CERN Axion Solar Telescope (CAST) aims to detect hypothetical axions or axion-like particles produced in the Sun by the Primakoff process. A Large Hadron Collider (LHC) prototype superconducting dipole magnet provides a 9 T transverse magnetic field for the conversion of axions into detectable X-ray photons. These photons are detected with an X-ray telescope and a novel type of framestore CCD detector built from radio-pure materials, installed in the optics focal plane. A novel type of cooling system has been designed and built based on krypton-filled cryogenic heat pipes, made out of oxygen-free radiopure copper, and a Stirling cryocooler as cold source. The heat pipes provide an efficient thermal coupling between the cryocooler and the CCD which is kept at stable temperatures between 150 and 230 K within an accuracy of 0.1 K. A graded-Z radiation shield, also serving as a gas cold-trap operated at 120 K, is implemented to reduce the surface contamination of the CCD window and suppress background radiation. © 2012 American Institute of Physics. Source


Snijder J.,University Utrecht | Snijder J.,Netherlands Proteomics Center | Snijder J.,University | Uetrecht C.,University Utrecht | And 12 more authors.
Nature Chemistry | Year: 2013

The interaction between a viral capsid and its genome governs crucial steps in the life cycle of a virus, such as assembly and genome uncoating. Tuning cargo-capsid interactions is also essential for successful design and cargo delivery in engineered viral systems. Here we investigate the interplay between cargo and capsid for the picorna-like Triatoma virus using a combined native mass spectrometry and atomic force microscopy approach. We propose a topology and assembly model in which heterotrimeric pentons that consist of five copies of structural proteins VP1, VP2 and VP3 are the free principal units of assembly. The interpenton contacts are established primarily by VP2. The dual role of the genome is first to stabilize the densely packed virion and, second, on an increase in pH to trigger uncoating by relaxing the stabilizing interactions with the capsid. Uncoating occurs through a labile intermediate state of the virion that reversibly disassembles into pentons with the concomitant release of protein VP4. © 2013 Macmillan Publishers Limited. Source


Mallagaray A.,University of Lubeck | Lockhauserbaumer J.,Heinrich Pette Institute | Lockhauserbaumer J.,European XFEL GmbH | Hansman G.,University of Heidelberg | And 3 more authors.
Angewandte Chemie - International Edition | Year: 2015

Human noroviruses recognize histo blood group antigens (HBGAs) as cellular attachment factors. Recently, it has been discovered that norovirus infection can be significantly enhanced by HBGA binding. Yet the attachment process and how it promotes host-cell entry is only poorly understood. The binding of a norovirus protruding (P) domain of a predominant GII.4 Saga strain to HBGAs at atomic resolution was studied. So far, independent and equivalent multiple binding sites were held responsible for attachment. Using NMR experiments we show that norovirus-HBGA binding is a cooperative multi-step process, and native mass spectrometry reveals four instead of two HBGA binding sites per P-dimer. An accompanying crystallographic study has disclosed four instead of two L-fucose binding sites per P-dimer of a related GII.10 strain1 further supporting our findings. We have uncovered a novel paradigm for norovirus-HBGA recognition that will inspire further studies into norovirus-host interactions. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. Source

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