Maintenance of apico-basal polarity is essential for epithelial integrity and requires particular reinforcement during tissue morphogenesis, when cells are reorganised, undergo shape changes and remodel their junctions. It is well established that epithelial integrity during morphogenetic processes depends on the dynamic exchange of adherens junction components, but our knowledge on the dynamics of other proteins and their dynamics during these processes is still limited. The early Drosophila embryo is an ideal system to study membrane dynamics during morphogenesis. Here, morphogenetic activities differ along the anterior-posterior axis, with the extending germband showing a high degree of epithelial remodelling. We developed a Fluorescence Recovery After Photobleaching (FRAP) assay with a higher temporal resolution, which allowed the distinction between a fast and a slow component of recovery of membrane proteins during the germband extension stage. We show for the first time that the recovery kinetics of a general membrane marker, SpiderGFP, differs in the anterior and posterior parts of the embryo, which correlates well with the different morphogenetic activities of the respective embryonic regions. Interestingly, absence of crumbs, a polarity regulator essential for epithelial integrity in the Drosophila embryo, decreases the fast component of SpiderGFP and of the apical marker Stranded at Second-Venus specifically in the anterior region. We suggest that the defects in kinetics observed in crumbs mutant embryos are the first signs of tissue instability in this region, explaining the earlier breakdown of the head epidermis in comparison to that of the trunk, and that diffusion in the plasma membrane is affected by the absence of Crumbs.

Das Auffinden spezifischer Strukturmerkmale integraler Membranproteine ist eine wichtige Grundlage zum Verständnis der Stabilität und Faltung und die notwendige Voraussetzung zur Modellierung ihrer Raumstruktur. Die Aminosäurezusammensetzung der innerhalb des hydrophoben Bereichs der Membranabschnitte befindlichen alpha-Helices wird entscheidend durch dieses Milieu determiniert, wie der Vergleich mit beta-Barrel Membranproteinen und alpha-Helices globulärer Proteine zeigt. Die Untersuchung der Phi-, Psi- und Chi1-Winkel lieferte gleichwohl eine Reihe von signifikanten Besonderheiten welche speziell bei einigen polaren (Asn, Asp) bzw. aromatischen Aminosäuren (Trp, Tyr) deutlich miteinander korrelieren. Der Anteil kürzerer, der für alpha-Helices typischen i, i+4 Wasserstoffbrückenbindungen ist innerhalb der Membran insgesamt höher, was ebenfalls mit der Verschiebung der Phi- und Psi- Winkel korreliert. Die Geometrie von alpha-Helices integraler Membranproteine ist damit näherungsweise ideal. An den Enden von Membranhelices und in den Helixturns werden neben Pro und Gly häufig polare, amphiphile oder aromatische Aminosäuren gefunden. Die polaren und amphiphilen Aminosäuren liegen überwiegend in den Helixturns und im Bereich der polaren Lipidköpfchen auf der Seite der Helix, welche der Membran zugewandten ist. Zur Fettschicht im Zentrum der Membran hin ragen umgekehrt vorherrschend lipophile Aminosäureseitenketten. Dieser Gradient ist folgerichtig auch an der Aminosäurezusammensetzung der Helixcaps erkennbar, welche die transmembranen Helices zumeist intra- und extrazellulär abschließen. Helixcaps alpha-helikaler Membrandomänen sind somit spezifische, von den klassischen Caps globulärer Proteine unterscheidbare Strukturmotive. Die konsequente Trennung der Untersuchungen der alpha-helikalen Abschnitte innerhalb der hydrophoben Lipiddoppelschicht von den Helixenden im polar-wässrigen Milieu ermöglicht außerdem die Identifikation vieler Aminosäurepräferenzen für exponierte (Leu, Ile), oder verdeckte Positionen (Ser, Asn, Cys). Umgekehrt wie bei den alpha-Helices globulärer Proteine ist die Atomzusammensetzung der Solvent exponierten Aminosäureseitenketten im Zentrum der Membran doppelt so hydrophob wie im Proteininnern. / Sorting out structural patterns that are specific for integral membrane proteins is a crucial base for understanding their stability and folding and a valuable source for the modelling of their tertiary structure. The detailed comparison of alpha-helical with beta-barrel membrane proteins and alpha-helices of globular proteins pointed out, that the amino acid composition of integral membrane proteins is overwhelmingly directed by the influence of the surrounding hydrophobic milieu. Nevertheless the investigation of the phi-, psi und chi1-angles yielded remarkable peculiarities of alpha-helical membrane proteins that correlate strongly in particular for some polar (asn, asp) and aromatic (trp, tyr) amino acids. The portion of the shorter and stronger i, i+4 H-bonds, that is typically found in alpha-helices is higher within the borders of the membrane. This observation confirms the observed shift of the phi- and psi- angles as well. Consequently the geometry of alpha-helices in membrane proteins is nearly ideal. At the ends of alpha-helices and in the turns of integral membrane proteins pro, gly and those amino acids are predominant that contain polar, amphiphilic or aromatic side chains. Whilst the aromats are equally positioned at the protein inside, the polar or amphiphilic amino acids are largely found at the helix turns, or at the side of the helix that contacts the polar lipid head groups. In contrast merely hydrophobic side chains face the lipophilic tails of the fatty acids in the core of the membrane. This gradient consequently shapes the amino acid composition of the helix caps that frequently coat the transmembrane helices on both sides of the membrane, too. Hence helix caps of transmembrane domains are substantially specific structural patterns that must be distinguished from the classical caps, known from alpha-helices of globular proteins. The clear distinction of the alpha-helical parts that are in contact with the lipophilic tails of the fatty acids, from the helix ends that stretch out into the polar aqueous solvent, advances the identification of amino acid preferences either lipid-exposed (Leu, Ile) or protein-buried (Ser, Asn, Cys). Finally, in sharp contrast to the helices of globular proteins, in the core of the membrane, the atomic composition of the solvent exposed amino acid side chains is twice as hydrophobic as inside the protein.

No description available.

Maintenance of apico-basal polarity is essential for epithelial integrity and requires particular reinforcement during tissue morphogenesis, when cells are reorganised, undergo shape changes and remodel their junctions. It is well established that epithelial integrity during morphogenetic processes depends on the dynamic exchange of adherens junction components, but our knowledge on the dynamics of other proteins and their dynamics during these processes is still limited. The early Drosophila embryo is an ideal system to study membrane dynamics during morphogenesis. Here, morphogenetic activities differ along the anterior-posterior axis, with the extending germband showing a high degree of epithelial remodelling. We developed a Fluorescence Recovery After Photobleaching (FRAP) assay with a higher temporal resolution, which allowed the distinction between a fast and a slow component of recovery of membrane proteins during the germband extension stage. We show for the first time that the recovery kinetics of a general membrane marker, SpiderGFP, differs in the anterior and posterior parts of the embryo, which correlates well with the different morphogenetic activities of the respective embryonic regions. Interestingly, absence of crumbs, a polarity regulator essential for epithelial integrity in the Drosophila embryo, decreases the fast component of SpiderGFP and of the apical marker Stranded at Second-Venus specifically in the anterior region. We suggest that the defects in kinetics observed in crumbs mutant embryos are the first signs of tissue instability in this region, explaining the earlier breakdown of the head epidermis in comparison to that of the trunk, and that diffusion in the plasma membrane is affected by the absence of Crumbs.

Relatively little is known about the folding and stability of membrane proteins. Conventional thermal or chemical unfolding techniques probe the average behavior of large numbers of molecules and thus cannot resolve co-existing minor and major unfolding pathways and intermediates. Here, I applied single-molecule force measurements based on an atomic force microscope (AFM) to characterize the stability of the membrane protein bacteriorhodopsin (BR). In these mechanical unfolding experiments, an external pulling force played the role of the denaturant and lead to unfolding of the three-dimensional structure of individual proteins. It was found that single BRs unfold step-wise in a well-defined sequence of stable intermediates and in different unfolding pathways. Although single [alpha]-helices were sufficiently stable to unfold in individual steps they also exhibited certain probabilities to unfold in pairs. These observations support the "two-stage" and the "helical-hairpin" model of membrane protein folding. Dynamic force measurements showed that [alpha]-helices and helical hairpins are relatively rigid structures, which are stabilized by narrow energy barriers and have stabilities between 100-10?000 seconds. These forced unfolding experiments were complemented with the development of new force measurement techniques. It is demonstrated that hydrodynamic effects need to be considered to obtain more complete kinetic pictures of single molecules. In addition, two force spectroscopy approaches to measure the complex visco-elastic response of single molecules are presented and applied to BR. These experiments revealed that the unfolding patterns of single proteins are dominated by purely elastic polypeptide extension and determined the dissipative interactions associated with the unfolding of single [alpha]-helices. In addition, it was found that kinks result in a reduced unfolding cooperativity of [alpha]-helices.

Gleich einem Etikett dient die N-Glykokosylierung vom Ribosom neu synthetisierter Proteine durch die Oligosaccharyltransferase (OST) bei der kotranslationalen Translokation in das Endoplasmatische Retikulum (ER) als Startpunkt vielschichtiger Prozessierungen. Bisher fehlte der strukturelle Nachweis, dass die OST als mit dem Ribosom assoziierten Membranprotein (RAMP) Bestandteil des auf dem proteinleitenden Kanal, dem Sec61-Komplex, basierenden Translokons ist. In dieser Arbeit berichten wir von der kryoelektronenmikroskopischen 3D-Struktur eines definierten OST-Sec61-Ribosom-Komplexes aus Saccharomyces cerevisiae bei 15,4 Å Auflösung. Dazu wurden die Komponenten (OST, Sec61 und Ribosomen mit naszierender Proteinkette) affinitätschromatographisch gereinigt und das Bindungsverhalten mit 80S-Ribosomen in vitro untersucht. Die OST band mit einer KD von 12,8 nM hochaffin und spezifisch an den bekannten Sec61-Ribosomen-Komplex. Dieser in vitro rekonstituierte trimere Komplex zeigte eine neuartige eng an das Ribosom anschließende Translokonstruktur mit zwei bisher unbekannten ribosomalen Verbindungen, einer einzigen dezentralen porenförmigen Vertiefung und zusätzlichen luminalen Bereichen. Durch das Docken eines Sec61-Homologs in einer alternativen Bindeposition sowie das Docken eines Stt3p-Homologs (der katalytischen Untereinheit der OST) und mit Hilfe der mittels (Kryo-)Negativkontrastierung gewonnenen 3D-Struktur der OST konnten Hybridmodelle erstellt werden. Daraus wurde unter Einbeziehung von bekannten molekularbiologisch gewonnenen Interaktionsdaten das 3D-Modell eines aktiven Ribosomen-gebundenen OST-Sec61-Translokons entwickelt. / Like a label, N-glycosylation by the oligosaccharyltransferase (OST) of newly synthesized proteins emerging from the ribosome while being cotranslationally translocated into the endoplasmic reticulum (ER) provides a starting point for a multitude of processes. Hitherto no structural proof has been presented, that the OST as a ribosome associated membrane protein (RAMP) is a constituent of the translocon, based at its core on the protein conducting channel (Sec61-complex). In this work we report on the 3D-structure of a defined OST-Sec61-ribosome complex from Saccharomyces cerevisiae by cryo-electron microscopy at 15.4 Å resolution. Thereto, the components (OST, Sec61, ribosome nascent chain complexes) have been purified by affinity chromatography and the binding of 80S-ribosomes has been checked in vitro. The OST bound with high affinity by a KD of 12.8 nM specifically to the established Sec61-ribosome complex. This trimeric complex reconstituted in vitro exhibits a new kind of tightly bound ribosomal translocon showing two hitherto unknown connections to the ribosome, a single off-center pore-like indentation and an additional luminal domain. By docking of a Sec61 homologue at an alternative binding position plus the docking of a Stt3p homologue (the catalytic subunit of the OST) and by means of the 3D-structure of the OST using the (cryo-)negative staining technique, hybrid models could be created. Consequently, integrating known interaction data from molecular biology experiments has been used to develop a 3D-model of an active ribosome-bound OST-Sec61-translocon.

No description available.

Das Biopolymer-produzierende Knallgasbakterium Ralstonia eutropha H16 gilt mit seinem außergewöhnlichen Stoffwechsel als vielversprechender Produktionsstamm für die weiße Biotechnologie. Es wächst auf einer Vielzahl organischer Substrate sowie chemolithoautotroph mit H2 und CO2 als einzige Energie- bzw. Kohlenstoffquelle. Unter anaeroben Bedingungen ist es zudem zur Denitrifikation befähigt. In dieser Arbeit wurde das Proteinprofil von R. eutropha unter chemolithoautotrophen sowie anaeroben Bedingungen mittels GeLC-MS/MS untersucht. Beide Proteomstudien offenbarten, dass die Nutzung unterschiedlicher Elektronendonoren bzw. -akzeptoren mit zahlreichen Veränderungen im Proteinbestand der Zellen einherging. Hierbei waren neben Proteinen metabolischer und Transportprozesse auch jene der Zellbewegung betroffen. Die Ergebnisse stellen im Vergleich zu vorangegangenen Studien den bisher umfassendsten Überblick zum Proteinbestand beim H2-basierten sowie anaeroben Wachstum in R. eutropha dar. Von besonderer Bedeutung war dabei das Einbinden der Analyse der Membran als Ort wichtiger Energie- und Transportprozesse. Besonderes Interesse galt einem unter H2/CO2-Bedingungen abundanten Zweikomponentensystem. Sequenzvergleiche zeigten Ähnlichkeit zum Regulationssystem der Katabolitrepression des Biphenylabbaus in Acidovorax sp. KKS102. Die Deletion des Response-Regulator-Gens führte zu vielfältigen Wachstumseffekten auf Substraten wie Fructose, Glycerin sowie auf H2/CO2. Der pleiotrope Phänotyp sowie die Ergebnisse von Genexpressionsstudien und der Suche nach Regulator-Bindestellen lassen eine globale Rolle des Systems im Energie- und/oder Kohlenstoffmetabolismus von R. eutropha H16 annehmen. Histidin-Kinase und Response Regulator wurden in GloS bzw. GloR umbenannt. Die vorliegende Arbeit zeigt eindrucksvoll das Potential der Proteomik als Teil der funktionellen Genomik für den Anstoß neuer Forschungsansätze zur Evaluierung des biotechnologischen Potentials von Mikroorganismen. / Due to its remarkable metabolism the bioplastic-producing “Knallgas” bacterium Ralstonia eutropha H16 is ranked as a promising production strain for white biotechnology. It grows on a wide range of organic substrates as well as lithoautotrophically on H2 and CO2 as sole energy and carbon source, respectively. Under anaerobic conditions it thrives by denitrification. This thesis focused on characterizing the protein profiles of lithoautotrophically and anaerobically grown R. eutropha cells. Proteome analyses revealed an extensive protein repertoire adapting the organism to alternative electron donors and acceptors, respectively. Changes concerned proteins involved in metabolic and transport processes as well as in cell movement. Compared to previous studies the results reported here offer the most comprehensive proteomic survey regarding the H2-based as well as anaerobic lifestyle of R. eutropha so far. In this context analyzing the cell membrane as a place for a number of energy, transport and signal transduction processes was of particular importance. Special interest aroused the identification of a two-component system upregulated on H2/CO2. Sequence analysis offered high similarity to the regulatory system for catabolite control of biphenyl degradation in Acidovorax sp. KKS102. Deletion of the response regulator gene led to versatile growth effects on substrates such as fructose and glycerol as well as H2/CO2. This pleiotrophic phenotype as well as the results of gene expression studies and the search for regulator binding sites suggests that the two-component system is a global player in energy and/or carbon metabolism in R. eutropha and possibly other bacteria. Thus, histidine kinase and response regulator have been renamed GloS/R. Since their characterization was initiated by proteomic data this study impressively elucidates the power of functional genomics in terms of revealing new research approaches to evaluate the biotechnological use of microbes.

No description available.

No description available.