NMR studies of the leader protease of the Foot-and-Mouth Disease Virus

Die Leader Protease Lbpro des Maul-und-Klauenseuche Viruses gehört zu der Familie der Papain-Cysteinproteasen und ist ein wichtiger Virulenzfaktor des Viruses. Es ist das erste Protein das am Genom des Viruses codiert ist. Während der Expression in der Wirtszelle spaltet es sich autocatalytisch vom wachsenden Polypeptid ab. Danach stoppt es die eukaryotische Translation durch die proteolytische Spaltung des eukaryotischen Translations-initiationfaktors 4GΙ and ΙΙ, wodurch unter anderem die Synthese von IFN verhindert wird. Die Proteinsynthese des viralen Genoms ist nicht beeinträchtigt, da die Translation von einer 'Internal Ribosome Entry Site' (IRES) initiiert wird. Kristallstukturen von Lbpro (1QOL.pdb), als auch von einer verkürzten Mutante, sLbpro (1QMY.pdb), der sechs C-terminale Aminosäuren fehlen, existierten bereits. Diese zeigten, dass Lbpro durch die Bindung der C-terminalen Aminsäuren im aktiven Zentrum eines benachbarten Lbpro Moleküles und umgekehrt dimerisiert. Diese Dimerisierung konnte bei der verkürzten Mutante nicht beobachtet werden. Zusätzlich war bei dieser der C-Terminus nicht sichtbar, was daraufhin weiste, dass dieser unstrukturiert und flexible ist Untersuchungen mittels NMR von Lbpro (2JQF.pdb) und sLbpro (2JQG.pdb) bestätigten die Dimerisierung von Lbpro, zeigten aber, dass Lbpro in Lösung ein symmetrisches Dimer bildet. Zusätzlich konnte gezeigt werden, dass der C-Terminus von sLbpro wirklich unstrukturiert und flexibel ist. Das Ziel dieser Arbeit war die NMR Strukturen von Lbpro und sLbpro zu verfeinern. Vollständigere Zuordnung konnte durch die Verwendung von dreifach Resonanz TROSY Experimenten erzielt werden. TROSY Experimente haben ein verbessertes 15N Relaxationsverhalten. Dadurch haben die Peaks dieser Spektren eine geringere Peakbreite, wodurch diese Peaks einfacher und genauer zugeordnet werden können. Für die Verfeinerung der Strukturen wurden dipolare Kopplungen gemessen. Dipolare Kopplungen erstellten Orientierungsbeschränkungen über große Distanzen. Im Fall für Lbpro sind sie daher nicht nur nützlich um die Struktur zu verfeinern, sondern auch um die genaue Orientierung der beiden Hälften des Dimers relativ zueinander zu bestimmen. Für beide Proteine, Lbpro and sLbpro wurden unterschiedliche dipolare Kopplungen gemessen. Die Proteine wurden durch den Phagen PF1 im Magnetfeld orientiert. Die Elemente des Orientierungstensors wurden durch least square fitting der gemessenen dipolaren Kopplungen unter der Zuhilfenahme der Kristallstrukturen berechnet. Die dipolaren Kopplungen wurden dann als zusätzliche Orientierungsbeschränkungen für die Berechnung der Strukturen eingesetzt. Die erhaltenen Strukturen zeigen eine bessere Konvergenz verglichen mit den existierenden NMR Strukturen. Zusätzlich ist die Anzahl von Aminosäuren, welche energetisch günstigere dihedarale Winkel, und , aufweisen höher. Dies ist vor allem bei sLbpro der Fall. Die quartäre Struktur, die von Lbpro erhalten wurde, zeigt verglichen mit der NMR-Struktur (2JQF.pdb), dass beide Strukturen denselben Drehungswinkel haben, jedoch unterscheidet sich ihr Beugungswinkel um ca. 20-25°.The leader protease Lbpro of the Foot-and-Mouth Disease Virus (FMDV) belongs to the family of papaine-like cysteine proteinases and is an important virulence determinant of the virus. It is the first encoded protein on the viral genome. During expression in the host cell it is autocatalytically cleaved from the growing polypeptide. Subsequently, it shuts down eukaryotic translation by cleavage of eukaryotic initation factor 4GΙ and ΙΙ thus preventing among others IFN synthesis. The protein synthesis from the viral genome is not affected as translation is initiated from an internal ribosomal entry site (IRES). Crystal structures of Lbpro (1QOL.pdb) as well as of a shortened mutant sLbpro (1QMY.pdb), lacking six C-terminal residues, already existed. They revealed dimerisation of Lbpro by binding of the C-terminal residues to the active site of an adjacent Lbpro molecule and vica versa. This dimerisation was not observed for the shortened mutant. Furthermore the C-terminus was not observable indicating that it is fexible and disordered. NMR studies of Lbpro (2JQF.pdb) and sLbpro (2JQG.pdb) confirmed the dimerisation of Lbpro but they revealed that Lbpro forms a completely symetric dimer in solution. Additionally the C-terminus of sLbpro was shown to be indeed unstructured and flexible The aim was to refine the NMR structures of Lbpro as well as of sLbpro. More complete assignment could be achieved with the use of triple resonance TROSY experiments. TROSY experiments have a more favourable 15N relaxation behaviour resulting in spectra whose peaks have smaller widths allowing more accurate peak assignment. For the refinement of the structures, residual dipolar couplings (RDCs) were measured. RDCs provided long range orientational restraints. In the case of Lbpro they are not only useful to refine the structure but also to determine the precise orientation of the two halves of the dimer to each other. For both proteins, Lbpro and sLbpro, various dipolar couplings were measured. The proteins were oriented via the phage PF1 in the magnetic field. The alignment tensor elements were determined by least square fitting of the measured dipolar couplings using the coordinates from the crystal structures. The dipolar couplings were then used as additional restraints for structures calculation. The structures obtained show a better convergence compared with the existing NMR structures. Their precision is a least as good as the precision of the crystal structures. Additionally the frequency of amino acid residues with energetically favorable dihedral angles, and , is higher especially for sLbpro. The quarternary structure of the obtained Lbpro compared to the NMR structure (2JQF.pdb) has the same twist angle but differs in the bending angle of about 20-25°

Die Rolle des Mesozooplanktonfraßes im biogeochemischen Kreislauf von Silizium des Südpolarmeeres

The role of copepod grazing, particularly of :i:Calanus simillimus:/i: and :i:Rhincalanus gigas:/i:, in the biogeochemical cycles of silicon (Si) and carbon (C) in the Antarctic Circumpolar Current (ACC) of the Southern Ocean is investigated. The two grazers show differences in feeding behavior before and in response to a diatom bloom stimulated by :i:in situ:/i: iron fertilization. The continuously high feeding activity of :i:C. simillimus:/i: on diatoms is conducive to enhance the export of primary produced C and Si. The grazing impact of this key species is high enough to influence population dynamics in the microplankton communities of the ACC. In the pre-bloom situation, :i:R. gigas:/i: fulfils most of its carbon requirement through grazing on detritus and thereby effectively reduces the vertical fecal flux produced by :i:C. simillimus:/i:. It is proposed that a Copepod-Retention-System for organic material is put in place by the copepod community under High Nutrient Low Chlorophyll (HNLC) conditions. Prey switching by :i:R. gigas:/i: from detritus to diatoms in the bloom situation lifts the grazing check on the detritus flux and enables loss of particulate C and Si from the surface layer with fast sinking fecal pellets. Results from dissolution experiments indicate that the enclosure of biogenic silica (BSi) in copepod fecal pellets prevents the dissolution of diatom frustules. Diatoms submitted to grazing of copepods and krill (:i:Euphausia superba:/i:) dissolved 4 to 26 times slower than un-grazed controls. :br:Estimates of C ingestion from :i:in vitro:/i: incubations and from gut fluorescence measurements are compared to respiratory carbon needs of copepods. Methods yield generally similar estimates for :i:C. simillimus:/i: whereas methods differ strongly for :i:R. gigas:/i: in the pre-bloom situation, reflecting the above mentioned differences in feeding behavior

Laser Optical Plankton Counter and Zooscan intercomparison in tropical and subtropical marine ecosystems

Sabine Schultes received a postdoctoral fellowship from FAPESP (grant 06/06683-9) and from the CNRS (project VICOFLUX). Rubens Lopes received support from the CNPq (306266/2007-5). The study was part of the projects ANTARES (headed by S. Gaeta) and PROABROLHOS (CNPq grant 420219/2005-6 to Eurico Cabral de Oliveira), and linked to the SCOR Working Group 130 on Automatic Visual Plankton Identification.International audienceTwo recently developed instruments, the Laser Optical Plankton Counter (LOPC) and the Zooscan, have been applied to study zooplankton biomass size spectra in tropical and subtropical marine ecosystems off Brazil. Both technologies rely on optical measurements of particles and may potentially be used in zooplankton monitoring programs. Vertical profiles of the LOPC installed in a 200 mu m ring net have been obtained from diverse environmental settings ranging from turbid and nearshore waters to oligotrophic open ocean conditions. Net samples were analyzed on the Zooscan and counted under a microscope. Particle biovolume in the study area estimated with the LOPC correlated with plankton displacement volume from the net samples, but there was no significant relationship between total areal zooplankton biomass determined with LOPC and the Zooscan. Apparently, normalized biomass size spectra (NBSS) of LOPC and Zooscan overlapped for particles in the size range of 500 to 1500 mu m in equivalent spherical diameter (ESD), especially at open ocean stations. However, the distribution of particles into five size classes was statistically different between both instruments at 24 of 28 stations. The disparities arise from unequal flow estimates, from different sampling efficiencies of LOPC tunnel and net for large and small particles, and possibly from the interference of non-zooplankton material in the LOPC signal. Ecosystem properties and technical differences therefore limit the direct comparability of the NBSS slopes obtained with both instruments during this study, and their results should be regarded as complementary