A Method for the Structural Investigation of Membrane Proteins

We investigated in meso crystallization of membrane proteins to develop a fast screening technology which combines features of the well established classical vapor diffusion experiment with the batch meso phase crystallization, but without premixing of protein and monoolein. It inherits the advantages of both methods, namely (i) the stabilization of membrane proteins in the meso phase, (ii) the control of hydration level and additive concentration by vapor diffusion. The new technology (iii) significantly simplifies in meso crystallization experiments and allows the use of standard liquid handling robots suitable for 96 well formats. CIMP crystallization furthermore allows (iv) direct monitoring of phase transformation and crystallization events. Bacteriorhodopsin (BR) crystals of high quality and diffraction up to 1.3 Å resolution have been obtained in this approach. CIMP and the developed consumables and protocols have been successfully applied to obtain crystals of sensory rhodopsin II (SRII) from Halobacterium salinarum for the first time

Multimedia in learning and instruction. State of the art and research perspectives

Mit den neuen Medien sind Hoffnungen auf eine Verbesserung der Lernwirksamkeit von Unterricht verbunden. Der Beitrag gibt einen zusammenfassenden Überblick über den Stand der empirischen Forschung zum Lernen mit neuen Medien. Berücksichtigung finden Erkenntnisse kognitionspsychologischer, instruktionspsychologischer und mediendidaktischer Provenienz. Im Anschluss an eine methodenkritische Einschätzung der Forschungslage erfolgen zunächst Aussagen über die Lernwirksamkeit neuer Medien im Hinblick auf ihre Gestaltungsmerkmale (i. e. das Zusammenspiel von Text, Bild und Ton, das Zusammenspiel der Codierung von Lernaufgaben und Testaufgaben sowie Ablaufgeschwindigkeit und Strukturierung der präsentierten Informationen). Anschließend wird auf die Lernwirksamkeit der neuen Medien unter Beachtung von Lehr-Lernzielen und von Persönlichkeitsmerkmalen der Schüler (i. e. Interessen und Einstellungen, themenspezifische Vorkenntnisse und medienspezifische Fertigkeiten sowie Lernstrategien) sowie im Hinblick auf mögliche instruktionale Unterstützung näher eingegangen. Auf dieser Basis werden Forschungsperspektiven entwickelt, die insbesondere auf eine Ergänzung der dominierenden Laborstudien um Forschung im realen Alltag von Lehrerinnen und Lehrer zielen. (DIPF/Orig.

Flexibility of the Cytoplasmic Domain of the Phototaxis Transducer II from Natronomonas pharaonis

Chemo- and phototaxis systems in bacteria and archaea serve as models for more complex signal transduction mechanisms in higher eukaryotes. Previous studies of the cytoplasmic fragment of the phototaxis transducer (pHtrII-cyt) from the halophilic archaeon Natronomonas pharaonis showed that it takes the shape of a monomeric or dimeric rod under low or high salt conditions, respectively. CD spectra revealed only approximately 24% helical structure, even in 4 M KCl, leaving it an open question how the rod-like shape is achieved. Here, we conducted CD, FTIR, and NMR spectroscopic studies under different conditions to address this question. We provide evidence that pHtrII-cyt is highly dynamic with strong helical propensity, which allows it to change from monomeric to dimeric helical coiled-coil states without undergoing dramatic shape changes. A statistical analysis of predicted disorder for homologous sequences suggests that structural flexibility is evolutionarily conserved within the methyl-accepting chemotaxis protein family

Controlled In Meso Phase Crystallization – A Method for the Structural Investigation of Membrane Proteins

We investigated in meso crystallization of membrane proteins to develop a fast screening technology which combines features of the well established classical vapor diffusion experiment with the batch meso phase crystallization, but without premixing of protein and monoolein. It inherits the advantages of both methods, namely (i) the stabilization of membrane proteins in the meso phase, (ii) the control of hydration level and additive concentration by vapor diffusion. The new technology (iii) significantly simplifies in meso crystallization experiments and allows the use of standard liquid handling robots suitable for 96 well formats. CIMP crystallization furthermore allows (iv) direct monitoring of phase transformation and crystallization events. Bacteriorhodopsin (BR) crystals of high quality and diffraction up to 1.3 Å resolution have been obtained in this approach. CIMP and the developed consumables and protocols have been successfully applied to obtain crystals of sensory rhodopsin II (SRII) from Halobacterium salinarum for the first time

La dynamique moléculaire dans les globules rouges

Les techniques de diffusion incohérente élastique et quasiélastique de neutrons ont été utilisées pour mesurer la dynamique de la protéine et de l'eau cellulaire sur les échelles de quelques picosecondes et de quelques Ângstroms. La dynamique de l'hémoglobine a été mesurée dans les globules rouges, in vivo. La dynamique interne de la protéine montre un changement de régime à 36.9C, la température physiologique. À des températures plus élevées que la température physiologique, les chaînes latérales des amino acides occupent des volumes plus grands que prévu par la dépendance normale sur la température. La diffusion globale de 1 'hémoglobine a été interprétée avec la théorie pour la diffusion des particules colloïdales à temps de courte durée. L'influence de l'hydratation sur la dynamique de l'hémoglobine a été étudiée avec la diffusion de neutrons. Les temps de résidence entre les sauts locaux dans l'ordre de quelques picosecondes sont réduits en solution concentrée et augmentés en poudre d'hémoglobine hydratée. La transition dans la géométrie des mouvements internes à la température du corps a été trouvée dans la solution concentrée, mais pas dans la poudre hydratée. Il a été conclu que les poudres hydratées ne représentent pas un bon modèle pour la dynamique de la protéine dans l'ordre de quelques picosecondes, qui est corrélée à la fonction biologique. La dynamique de l'eau cellulaire dans les globules rouges a été mesurée avec la technique de diffusion incohérente quasiélastique de neutrons. Une fraction de l'eau cellulaire d'environ 90% est caractérisée par un coefficient de diffusion translationnel similaire à celui de l'eau volumique. Les 10% restant présentent une dynamique ralentie de façon significative. La fraction ralentie a été attribuée à l'eau en interaction avec l'hémoglobine. Elle correspond à environ la moitié de l'eau dans la première couche d'hydratation de la protéine.Incoherent quasielastic and elastic neutron scattering were used to measure protein and cell water dynamics in the picosecond time and Ângstrom length scale. Hemoglobin dynamics was measured in human red blood cells, in vivo. The experiments revealed a change in the geometry of internaI protein dynamics at 36.9C, human body temperature. Above this temperature amino acid side-chain dynamics occupy larger volumes than expected from normal temperature dependence. Global macromolecular diffusion was interpreted according to theoretical concepts for short-time self-diffusion of non-charged hard sphere colloids. The influence of hydration on hemoglobin dynamics \yas studied with neutron scattering. The residence times of localized jumps in the order of a few picoseconds were found to be significantly reduced in concentrated solution compared to fully hydrated powder. The change in the geometry of amino acid side chain dynamics at body temperature was found in concentrated hemoglobin solution, but the body temperature transition in protein dynamics was absent in fully hydrated powder. This indicated that picosecond protein motions responsible for the body temperature transition are activated only at a sufficient level of hydration, and that hydrated powders may not reflect fully aIl functional protein dynamics. The dynamics of cell water in human red blood cells was measured with quasielastic incoherent neutron scattering. A major fraction of around 90% of cell water is characterized by a translational diffusion coefficient similar to bulk water. A minor fraction of around 10% of cellular water exhibits reduced dynamics. The slow water fraction was attributed to dynamically bound water on the surface ofhemoglobin, which accounts for approximate1y half of the hydration layer.GRENOBLE1-BU Sciences (384212103) / SudocSudocFranceF

Nanosecond Dynamics of Calmodulin and Ribosome-Bound Nascent Chains Studied by Time-Resolved Fluorescence Anisotropy

We report a time-resolved fluorescence anisotropy study of ribosome-bound nascent chains (RNCs) of calmodulin (CaM), a prototypical member of the EF-hand family of calcium-sensing proteins. As shown in numerous studies, in vitro protein refolding can differ substantially from biosynthetic protein folding, which takes place cotranslationally and depends on the rate of polypeptide chain elongation. A challenge in this respect is to characterize the adopted conformations of nascent chains before their release from the ribosome. CaM RNCs (full-length, half-length, and first EF-hand only) were synthesized in vitro. All constructs contained a tetracysteine motif site-specifically incorporated in the first N-terminal helix; this motif is known to react with FlAsH, a biarsenic fluorescein derivative. As the dye is rotationally locked to this helix, we characterized the structural properties and folding states of polypeptide chains tethered to ribosomes and compared these with released chains. Importantly, we observed decelerated tumbling motions of ribosome-tethered and partially folded nascent chains, compared to released chains. This indicates a pronounced interaction between nascent chains and the ribosome surface, and might reflect chaperone activity of the ribosome

Sensory Rhodopsin II: Signal Development and Transduction

International audienc

Translation and folding of single proteins in real time

Protein biosynthesis is inherently coupled to cotranslational protein folding. Folding of the nascent chain already occurs during synthesis and is mediated by spatial constraints imposed by the ribosomal exit tunnel as well as self-interactions. The polypeptide’s vectorial emergence from the ribosomal tunnel establishes the possible folding pathways leading to its native tertiary structure. How cotranslational protein folding and the rate of synthesis are linked to a protein’s amino acid sequence is still not well defined. Here, we follow synthesis by individual ribosomes using dual-trap optical tweezers and observe simultaneous folding of the nascent polypeptide chain in real time. We show that observed stalling during translation correlates with slowed peptide bond formation at successive proline sequence positions and electrostatic interactions between positively charged amino acids and the ribosomal tunnel. We also determine possible cotranslational folding sites initiated by hydrophobic collapse for an unstructured and two globular proteins while directly measuring initial cotranslational folding forces. Our study elucidates the intricate relationship among a protein’s amino acid sequence, its cotranslational nascent-chain elongation rate, and foldin

Spatial filter and its application in three-dimensional single molecule localization microscopy

Single molecule localization microscopy (SMLM) allows the imaging of cellular structures with resolutions five to ten times below the diffraction limit of optical microscopy. It was originally introduced as a two-dimensional technique based on the localization of single emitters as projection onto the x-y imaging plane. The determination of the axial position of a fluorescent emitter is only possible by additional information. Here we report a method (spatial filter SMLM (SFSMLM)) that allows to determine the axial positions of fluorescent molecules and nanoparticles on the nanometer scale by the usage of two spatial filters, which are placed in two otherwise identical emission detection channels. SFSMLM allows axial localization in a range of ca. 1.5 μm with a localization precision of 15 - 30 nm in axial direction. The technique was utilized for localizing and imaging small cellular structures – e.g. actin filaments, vesicles and mitochondria - in three dimensions