Defined interactions are crucial for the structure and function of the Synechocystis IM30 protein

Biogenesis and maintenance of the thylakoid membranes in cyanobacteria and chloroplasts is a vital process. Nevertheless, the mechanism of the biogenesis has stayed a mystery until today. The IM30 protein appears to play an important role in this process, as has been shown via depletion experiments. Recently, the ability of IM30 to bind to negatively charged lipids and to fuse liposomes in presence of Mg2+ has been demonstrated. This discovery indicates a capability of IM30 to fuse the cytoplasmic membrane in cyanobacteria or the inner envelope in chloroplasts with the constructive thylakoid membrane in order to create a transfer point for lipids and/or proteins. Additionally, connections between different thylakoid membranes through IM30 are thinkable. In the framework of this thesis, interactions of IM30 leading to membrane fusion and the fusion mechanism itself were investigated. Here, the interplay of IM30 and Mg2+ as well as negatively charged membranes were separately examined via in vitro approaches and finally combined in order to have a closer look at the IM30-mediated membrane fusion. It was observed that Mg2+ binds directly to IM30 and induces a structural alteration in the protein, which results in stabilization against different stressors and an increased tendency of IM30 to form double-rings. The IM30-membrane interaction was shown to depend on the electrostatic interaction with anionic lipids and is enhanced in presence of Mg2+. Furthermore, lower-ordered IM30 oligomerization states seem to have an increased membrane binding affinity. This circumstance potentially causes the dissociation of IM30 ring oligomers on membranes in absence of Mg2+. Since the presence of the ring structure is probably crucial for the IM30 function, dissociation of rings might be prevented by Mg2+. Stabilized by Mg2+, an IM30 double-ring might be able to fuse two membranes. While doing this, the double-ring seems to dissociate and most probably needs to be reactivated prior to another fusion. The results of this thesis provide hints concerning the in vivo function of IM30, which can now be investigated further in a more target-oriented manner.Die Biogenese und Instandhaltung der Thylakoidmembran in Cyanobakterien und Chloroplasten ist ein lebenswichtiger Prozess. Dennoch ist der Mechanismus der Biogenese bis heute ein Rätsel. Das IM30-Protein scheint in diesem Vorgang eine entscheidende Rolle zu spielen, wie Depletionsexperimente an den Tag gelegt haben. Kürzlich konnte gezeigt werden, dass IM30 an negativ geladene Lipide bindet und Liposomen in Gegenwart von Mg2+ miteinander fusionieren kann. Diese Entdeckung deutet die Fähigkeit des Proteins an, die Cytoplasmamembran in Cyanobakterien bzw. die innere Membran in Chloroplasten mit der sich aufbauenden Thylakoidmembran zu verbinden und eine Übertragung von Lipiden und/oder Proteinen zu ermöglichen. Weiterhin könnte IM30 verschiedene Thylakoidmembranen miteinander verbinden. Im Rahmen dieser Doktorarbeit sollten Interaktionen von IM30, welche zu der Membranfusion führen, und der Fusionsmechanismus selber näher untersucht werden. Hierbei wurden zunächst die Wechselwirkungen von IM30 mit Mg2+ und negativ geladenen Membranen mit Hilfe von biophysikalischen in vitro Experimenten jeweils einzeln untersucht und schließlich miteinander kombiniert, um die durch IM30 induzierte Membranfusion näher zu analysieren. Es konnte beobachtet werden, dass Mg2+ direkt an IM30 bindet und eine Strukturänderung auslöst, die zur Stabilisierung unter verschiedenen Stressbedingungen und einer erhöhten Tendenz zur Doppelringbildung des Proteins führt. Weiterhin wurde festgestellt, dass die Membranbindung von IM30 durch elektrostatische Wechselwirkungen mit anionischen Lipiden beding ist und in Anwesenheit von Mg2+ verstärkt wird. Außerdem scheinen niedere Oligomerisierungsstufen von IM30 eine erhöhte Membranaffinität zu besitzen. Dies trägt vermutlich dazu bei, dass IM30-Ringe in Abwesenheit von Mg2+ durch Membraninteraktion dissoziieren. Da das Vorliegen der Ringstruktur jedoch wichtig für die Ausübung der Fusionsfunktion ist, könnte die Dissoziation zunächst durch Mg2+ verhindert werden. Durch das Kation stabilisiert, könnte ein IM30-Doppelring zwei Membranen miteinander fusionieren. Dabei zerfällt der Doppelring und muss wahrscheinlich vor einer weiteren Fusion reaktiviert werden. Die Ergebnisse dieser Arbeit liefern Hinweise zur in vivo-Funktion von IM30, welche nun gezielter durch weitere Versuche untersucht werden kann.217 Seite

Investigation and Characterization of Nitrazapropane‐, Oxapropane‐ and Trinitrazaheptane‐Bridged Nitro Esters

In this work, 1,3-dinitroxy-2-nitrazapropane and 1,7-dinitroxy-2,4,6-trinitrazaheptane were synthesized and investigated. Starting from hexamine, the open-chain compounds were prepared by a modified Bachmann procedure which is commonly used for the synthesis of hexogen (RDX) and octogen (HMX). All new compounds were characterized by NMR spectroscopy, X-ray diffraction, vibrational analysis and elemental analysis. Their thermal behavior was studied by differential thermal analysis (DTA) and thermogravimetric analysis (TGA). The sensitivities towards impact (IS) and friction (FS) were determined according to the BAM (Bundesanstalt für Materialforschung und -prüfung) standard method. Their energetic properties, which were determined from the energies of formation at the CBS-4 M level, were calculated using the EXPLO5 computer code. Moreover, 1,3-dinitroxy-2-oxapropane was synthesized and investigated. The various compounds were compared with each other and with the commonly used explosives in terms of their physicochemical properties and detonation behavior

TGFβ-signaling and FOXG1-expression are a hallmark of astrocyte lineage diversity in the murine ventral and dorsal forebrain

Heterogeneous astrocyte populations are defined by diversity in cellular environment, progenitor identity or function. Yet, little is known about the extent of the heterogeneity and how this diversity is acquired during development. To investigate the impact of TGF (transforming growth factor) β-signaling on astrocyte development in the telencephalon we deleted the TGFBR2 (transforming growth factor beta receptor 2) in early neural progenitor cells in mice using a FOXG1 (forkhead box G1)-driven CRE-recombinase. We used quantitative proteomics to characterize TGFBR2-deficient cells derived from the mouse telencephalon and identified differential protein expression of the astrocyte proteins GFAP (glial fibrillary acidic protein) and MFGE8 (milk fat globule-EGF factor 8). Biochemical and histological investigations revealed distinct populations of astrocytes in the dorsal and ventral telencephalon marked by GFAP or MFGE8 protein expression. The two subtypes differed in their response to TGFβ-signaling. Impaired TGFβ-signaling affected numbers of GFAP astrocytes in the ventral telencephalon. In contrast, TGFβ reduced MFGE8-expression in astrocytes deriving from both regions. Additionally, lineage tracing revealed that both GFAP and MFGE8 astrocyte subtypes derived partly from FOXG1-expressing neural precursor cells.Fil: Weise, Stefan Christopher. Universität Freiburg Im Breisgau; AlemaniaFil: Villarreal, Alejandro. Universität Freiburg Im Breisgau; Alemania. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; ArgentinaFil: Heidrich, Stefanie. Universität Freiburg Im Breisgau; AlemaniaFil: Dehghanian, Fariba. University Of Isfahan; Irán. Universität Freiburg Im Breisgau; AlemaniaFil: Schachtrup, Christian. Universität Freiburg Im Breisgau; AlemaniaFil: Nestel, Sigrun. Universität Freiburg Im Breisgau; AlemaniaFil: Schwarz, Jennifer. Universität Freiburg Im Breisgau; AlemaniaFil: Thedieck, Kathrin. Universität Oldenburg; Argentina. University of Groningen; Países BajosFil: Vogel, Tanja. Universität Freiburg Im Breisgau; Alemani

Synthesis of Bridged Tetrazoles with Promising Properties and Potential Applications by a One‐Step Finkelstein Reaction

Numerous nitramine bridged compounds which show promising combinations of properties have already been identified in the area of energetic materials. In this work, four new nitrazapropane bridged tetrazoles, as well as four new trinitrazaheptane tetrazoles and three oxapropane bridged tetrazoles were synthesized and fully characterized. These new compounds can all be synthesized by a simple, one-step synthesis using Finkelstein conditions. All of these new energetic materials were characterized using NMR spectroscopy, single crystal X-ray diffraction, vibrational analysis and elemental analysis. The thermal behaviour of these compounds was studied by differential thermal analysis (DTA) and partly by thermogravimetric analysis (TGA). The BAM standard method was used to determine the sensitivities towards impact (IS) and friction (FS). The enthalpies of formation were calculated at the CBS-4M level, and the energetic performances were calculated using the EXPLO5 (V6.06.01) computer code. The properties of the new compounds were compared to each other as well as to the known energetic material RDX. Moreover, the iron(II) and copper(II) perchlorate complexes with 1,3-bis-1,1-tetrazolylnitrazapropane as ligand were prepared and investigated

TGFβ-Signaling and FOXG1-Expression Are a Hallmark of Astrocyte Lineage Diversity in the Murine Ventral and Dorsal Forebrain

Heterogeneous astrocyte populations are defined by diversity in cellular environment, progenitor identity or function. Yet, little is known about the extent of the heterogeneity and how this diversity is acquired during development. To investigate the impact of TGF (transforming growth factor) β-signaling on astrocyte development in the telencephalon we deleted the TGFBR2 (transforming growth factor beta receptor 2) in early neural progenitor cells in mice using a FOXG1 (forkhead box G1)-driven CRE-recombinase. We used quantitative proteomics to characterize TGFBR2-deficient cells derived from the mouse telencephalon and identified differential protein expression of the astrocyte proteins GFAP (glial fibrillary acidic protein) and MFGE8 (milk fat globule-EGF factor 8). Biochemical and histological investigations revealed distinct populations of astrocytes in the dorsal and ventral telencephalon marked by GFAP or MFGE8 protein expression. The two subtypes differed in their response to TGFβ-signaling. Impaired TGFβ-signaling affected numbers of GFAP astrocytes in the ventral telencephalon. In contrast, TGFβ reduced MFGE8-expression in astrocytes deriving from both regions. Additionally, lineage tracing revealed that both GFAP and MFGE8 astrocyte subtypes derived partly from FOXG1-expressing neural precursor cells

IM30 IDPs form a membrane-protective carpet upon super-complex disassembly

Members of the phage shock protein A (PspA) family, including the inner membrane-associated protein of 30 kDa (IM30), are suggested to stabilize stressed cellular membranes. Furthermore, IM30 is essential in thylakoid membrane-containing chloroplasts and cyanobacteria, where it is involved in membrane biogenesis and/or remodeling. While it is well known that PspA and IM30 bind to membranes, the mechanism of membrane stabilization is still enigmatic. Here we report that ring-shaped IM30 super-complexes disassemble on membranes, resulting in formation of a membrane-protecting protein carpet. Upon ring dissociation, the C-terminal domain of IM30 unfolds, and the protomers self-assemble on membranes. IM30 assemblies at membranes have been observed before in vivo and were associated with stress response in cyanobacteria and chloroplasts. These assemblies likely correspond to the here identified carpet structures. Our study defines the thus far enigmatic structural basis for the physiological function of IM30 and related proteins, including PspA, and highlights a hitherto unrecognized concept of membrane stabilization by intrinsically disordered proteins