Mohrenfalter (Genus: Erebia) als Indikatoren des Klima- und Landnutzungswandels in Südwest-Deutschland

Die an kalte und kontinental geprägte Klimate angepasste Tagfaltergattung der Mohrenfalter (Genus: Erebia) ist im südwestlichen Deutschland mit einigen Arten vertreten. Drei Arten aus dieser Gattung – Rundaugen-Mohrenfalter (Erebia medusa), Graubindiger Mohrenfalter (Erebia aethiops) und Weißbindiger Mohrenfalter (Erebia ligea) - erweisen sich als ideale Indikatorspezies zur Erforschung des Einflusses von Klima- und Landnutzungswandel auf die Verbreitungsbilder kälte-stenotoper Arten. Im Rahmen der Promotion wurden mithilfe unterschiedlicher Analysemethoden – Auswertung archivierter Funddaten, Kartierungen in verschiedenen Naturräumen Baden-Württembergs, Aufnahme habitatbeschreibender Parameter, Klimakammerversuche, Klimadatenauswertung, Durchführung komplexer statistischer Analysemethoden - die Gründe für den seit Jahrzehnten zu beobachtenden Rückgang der drei Arten in Südwest-Deutschland erforscht. Dadurch gelang es die Lebensraumansprüche dieser Erebia-Arten sowohl auf Habitatebene, als auch auf mikrostruktureller und –klimatischer Ebene besser zu verstehen. Eine kritische Phase durchlaufen die Arten während der Zeit der relativ immobilen Präimaginalstadien (Eier, Larven), die empfindlich auf Klimaextreme (Fehlen einer Schneedecke, Frostwechsel, Trockenheit, Hitze) – wie sie in Zeiten atlantischer werdenden Großklimas häufig auftreten - reagieren. Doch auch der Landnutzungswandel trägt in Form von Lebensraumverlusten durch Nutzungsintensivierung und –aufgabe zum Rückgang der Erebia-Arten bei. Die kombinierte Wirkung beider Faktoren führte bei diesen drei ehemals häufigen Arten regional bereits zu Extinktionsprozessen. Die Promotionsarbeit versucht wirkungsvolle Ansätze für einen nachhaltigen Schutz dieser und weiterer kälte-stenotoper Arten in einer sich ständig wandelnden Umwelt zu geben

Atomistic Insight into the Role of Threonine 127 in the Functional Mechanism of Channelrhodopsin-2

Channelrhodopsins (ChRs) belong to the unique class of light-gated ion channels. The structure of channelrhodopsin-2 from Chlamydomonas reinhardtii (CrChR2) has been resolved, but the mechanistic link between light-induced isomerization of the chromophore retinal and channel gating remains elusive. Replacements of residues C128 and D156 (DC gate) resulted in drastic effects in channel closure. T127 is localized close to the retinal Schiff base and links the DC gate to the Schiff base. The homologous residue in bacteriorhodopsin (T89) has been shown to be crucial for the visible absorption maximum and dark–light adaptation, suggesting an interaction with the retinylidene chromophore, but the replacement had little effect on photocycle kinetics and proton pumping activity. Here, we show that the T127A and T127S variants of CrChR2 leave the visible absorption maximum unaffected. We inferred from hybrid quantum mechanics/molecular mechanics (QM/MM) calculations and resonance Raman spectroscopy that the hydroxylic side chain of T127 is hydrogen-bonded to E123 and the latter is hydrogen-bonded to the retinal Schiff base. The C=N–H vibration of the Schiff base in the T127A variant was 1674 cm−1, the highest among all rhodopsins reported to date. We also found heterogeneity in the Schiff base ground state vibrational properties due to different rotamer conformations of E123. The photoreaction of T127A is characterized by a long-lived P2380 state during which the Schiff base is deprotonated. The conservative replacement of T127S hardly affected the photocycle kinetics. Thus, we inferred that the hydroxyl group at position 127 is part of the proton transfer pathway from D156 to the Schiff base during rise of the P3530 intermediate. This finding provides molecular reasons for the evolutionary conservation of the chemically homologous residues threonine, serine, and cysteine at this position in all channelrhodopsins known so far

Kinetics of Proton Coupled Electron Transfer Reactions in Enzyme Complexes of the Respiratory Chain

Protonen gekoppelter Elektronentransfer ist ein zentraler Bestandteil der chemiosmotischen Theorie. Die Beschreibung seiner Natur ist aufgrund seines transienten Charakters eine komplexe Aufgabe. Elektrometrische Messungen stellen hier eine große Hilfe in der Erfassung von Ladungsverschiebungen dar. Sie erlauben die zeitliche Beschreibung des Elektronen- und Protonentransportes, der mit kaum einer geeigneten Methode beobachtet werden kann, und geben Einblick in deren molekularen Mechanismus. Diese Technik wurde hier an drei verschiedenen Membrankomplexen der Atmungskette angewandt: der Cytochrom c Oxidase (COX) aus Paracoccus denitrificans, der Quinol-Fumarat-Reduktase (QFR) aus Wolinella succinogenes und dem bc(1)-Komplex aus Saccharomyces cerevisiae. Hinsichtlich der experimentellen Vorgehensweise für kinetische Untersuchungen stellte sich ein übergreifendes Problem. Neben einer schnellen Aktivierung der Enzymsysteme bedurfte es eines definierten Ausgangszustands. Ziel dieser Arbeit war das Etablieren von Bedingungen, die elektrometrische Messungen am bc(1) und der QFR erlauben, sowie die Fortführung dieser Methodik am bereits vorhandenen System der COX. Cytochrom c Oxidase Elektrometrische Untersuchungen an der COX wurden basierend auf Vorarbeiten weitergeführt. Insbesondere die Ladungsverschiebungen nach Photoreduktion ausgehend vom völlig oxidierten Zustand rückten in den Fokus. In diesem Schritt wird ein Proton aufgenommen, während Häm a vom angeregten Zustand eines Rutheniumkomplexes reduziert wird. Dieses Verhalten ist unabhängig vom heterogenen Ausgangszustand der COX, er wird jedoch durch eine Änderung des pH-Wertes beeinflußt. Der heterogene Ausgangszustand im O-E-Übergang wurde in einem sequentiellen Modell diskutiert. Dabei wurde auf die Diskrepanz zwischen den elektrometrischen und den publizierten spektroskopischen Messungen hingewiesen. Während spektroskopisch die Cu(A)-Oxidation und Häm a-Reduktion in einer Phase verliefen, wurde in den elektrometrischen Messungen eine zweite deutlich langsamere Phase für den Protonentransfer beobachtet. Ein sequentielles Reaktionsmodell führte hier zu einem Widerspruch. Die Auswirkungen auf die Natur der Kopplung von Häm a und dem aufgenommenen Proton wurden diskutiert. Die Kinetik der Ladungsverschiebung wurde detailliert anhand der Temperaturabhängigkeit und des Isotopeneffektes untersucht und mit den Ergebnissen aus den Messungen an einem thermophilen Enzym, der ba(3)-Oxidase aus Thermus thermophilus, verglichen. Quinol-Fumarat-Reduktase Für eine schnelle Aktivierung der QFR wurde ein caged Fumarat synthetisiert, das nach Photolyse zu einer schnellen Erhöhung der Fumaratkonzentration führte. Die neue Substanz wurde bezüglich einer möglichen Verwendung für die QFR charakterisiert. Die Freisetzung erfolgte mit einer Zeitkonstante von 0,1 ms und aktivierte die QFR nur im photolysierten Zustand. Aufgrund der Photochemie der metallischen Kofaktoren in der QFR konnten jedoch keine kinetischen Messungen durchgeführt werden. Da die photochemisch induzierten Elektronenbewegungen in der QFR mit zunehmender Wellenlänge abnahmen, wurde eine neue Substanz vorgeschlagen, die im sichtbaren Spektralbereich gespalten werden kann. bc(1)-Komplex Der bc(1)-Komplex kann wie die COX durch einen Rutheniumkomplex aktiviert werden. Ein dimerer sowie ein an Cytochrom c gekoppelter Rutheniumkomplex wurde hierfür anhand von Literaturdaten synthetisiert, und die Verbindungen wurden hinsichtlich ihrer Eigenschaften nach Lichtanregung charakterisiert. Für die elektrometrischen Messungen wurde der bc(1)-Komplex in Proteoliposomen rekonstituiert, und der Ausgangszustand des bc(1)-Komplexes unter reduktiven und oxidativen Bedingungen eingestellt. Mit dem dimeren Rutheniumkomplex wurden elektrometrische Experimente durchgeführt, die zu zwei Phasen in der Spannungsantwort führten. Die Daten wurden zusammen mit den publizierten spektroskopischen diskutiert. Dabei wurde eine schnelle elektrogene Phase einem Elektronentransfers zwischen Häm c(1) und dem Eisen-Schwefel-Cluster des Rieske-Proteins zugeordnet. Eine langsamere Phase erwies sich sensitiv gegenüber Antimycin und spiegelt Vorgänge unter der Kontrolle der Q(i)-Bindungsstelle wider.Proton coupled electron transfer is an essential part of the chemi-osmotic theory. Its description poses a complex experimental challenge due to its transient character. Electrometric measurements are a great help in the acquisition of charge translocation processes. They allow to follow in a time-resolved manner the transport of electrons and protons, respectively. In particular the latter are difficult to detect with other techniques. Potential measurements at a black lipid membrane have been performed for three different enzyme complexes from the respiratory chain: the cytochrome c oxidase from Paracoccus denitrificans (COX), the quinol:fumarate reductase from Wolinella succinogenes (QFR) and the bc(1)-complex from Saccharomyces cerevisiae. There are common problems with regard of the experimental strategy for kinetic investigations: the need for a fast and efficient activation of a particular enzyme system starting from a controlled defined state. Therefore, the goal of this work was to establish experimental conditions for electrometric measurements on the bc(1)-complex and the QFR and the continuation on the already implemented system for the COX. Cytochrome c oxidase The electrometric measurements on the COX were pursued based on previous achievements. In particular the charge translocating steps after photoreduction of the fully oxidized state of the enzyme were in the focus of this work. Here, a proton is taken up after heme a is going to be reduced from the excited state of an organometallic ruthenium complex. This behaviour is independent from the heterogeneous starting state. However, it may be influenced by a change of the pH-value. The consequences of a heterogeneous starting state in the O-E transition is discussed in the framework of a sequential reaction model revealing a discrepancy between the electrometric and the spectroscopic data. While the oxidation of Cu(A) proceeds spectroscopically with the reduction of heme a in a single observable phase, a second much slower phase is observed for the proton uptake in the electrometric measurements. This leads to a conflict with a sequential reaction model. The implications of this result are discussed with respect to the nature of the coupling between electron and proton transfer. The kinetics of charge translocation processes have been examined in detail by means of the temperature dependence and the isotope effect for the electron and proton transfer in the O-E transition. The results are compared with the ones for a thermophilic enzyme, i.e. the ba(3)-oxidase from Thermus thermophilus. Quinol:fumarate reductase A caged fumarate has been synthesized with the aim of a fast activation of the QFR. Photolysis of this compound leads to a fast concentration jump of fumarate. The new compound has been characterized to explore the its suitability in measurements with the QFR. The release of fumarate takes place with a time constant of 0,1 ms and it starts the enzymatic reaction of the QFR only after photolysis. However, it was not possible to perform kinetic measurements due to the photochemistry of the metallic cofactors in the QFR. Because the light-induced redox reactions in the QFR are decreasing with increasing wavelengths, the synthesis of a new compound has been proposed that may be photolyzed in the visible spectral range. bc(1)-complex The bc(1)-complex as well can be activated by a ruthenium complex as the COX. Therefore, a dimeric complex and one coupled to cytochrome c were synthesized according to literature data. The compounds were characterized, in particular after light excitation. The bc(1)-complex was reconstituted in proteoliposomes for the electrometric measurements and the starting state was prepared under oxidizing or reducing experimental conditions. Usage of the dimeric ruthenium complex in the electrometric measurements leads to two distinct phases in the photopotential over the membrane. The results were discussed and compared to published data from spectroscopic experiments. A fast electrogenic phase was assigned to electron transfer between heme c(1) and the iron-sulfur cluster of the Rieske protein. A second slower one showed a sensitivity towards antimycin and reflects processes in the Q(i) binding site

Resonance Raman and FTIR spectroscopic characterization of the closed and open states of channelrhodopsin-1.

Channelrhodopsin-1 from Chlamydomonas augustae (CaChR1) is a light-activated cation channel, which is a promising optogenetic tool. We show by resonance Raman spectroscopy and retinal extraction followed by high pressure liquid chromatography (HPLC) that the isomeric ratio of all-trans to 13-cis of solubilized channelrhodopsin-1 is with 70:30 identical to channelrhodopsin-2 from Chlamydomonas reinhardtii (CrChR2). Critical frequency shifts in the retinal vibrations are identified in the Raman spectrum upon transition to the open (conductive P2(380)) state. Fourier transform infrared spectroscopy (FTIR) spectra indicate different structures of the open states in the two channelrhodopsins as reflected by the amide I bands and the protonation pattern of acidic amino acids

Temporal evolution of helix hydration in a light-gated ion channel correlates with ion conductance

The discovery of channelrhodopsins introduced a new class of light-gated ion channels, which when genetically encoded in host cells resulted in the development of optogenetics. Channelrhodopsin-2 from Chlamydomonas reinhardtii, CrChR2, is the most widely used optogenetic tool in neuroscience. To explore the connection between the gating mechanism and the influx and efflux of water molecules in CrChR2, we have integrated light-induced time- resolved infrared spectroscopy and electrophysiology. Cross-correlation analysis revealed that ion conductance tallies with peptide backbone amide I vibrational changes at 1,665(−) and 1,648(+) cm−1. These two bands report on the hydration of transmembrane α-helices as concluded from vibrational coupling experiments. Lifetime distribution analysis shows that water influx proceeded in two temporally separated steps with time constants of 10 μs (30%) and 200 μs (70%), the latter phase concurrent with the start of ion conductance. Water efflux and the cessation of the ion conductance are synchronized as well, with a time constant of 10 ms. The temporal correlation between ion conductance and hydration of helices holds for fast (E123T) and slow (D156E) variants of CrChR2, strengthening its functional significance

New Channelrhodopsin with a Red-Shifted Spectrum and Rapid Kinetics from Mesostigma viride

Light control of motility behavior (phototaxis and photophobic responses) in green flagellate algae is mediated by sensory rhodopsins homologous to phototaxis receptors and light-driven ion transporters in prokaryotic organisms. In the phototaxis process, excitation of the algal sensory rhodopsins leads to generation of transmembrane photoreceptor currents. When expressed in animal cells, the algal phototaxis receptors function as light-gated cation channels, which has earned them the name “channelrhodopsins.” Channelrhodopsins have become useful molecular tools for light control of cellular activity. Only four channelrhodopsins, identified in Chlamydomonas reinhardtii and Volvox carteri, have been reported so far. By screening light-induced currents among algal species, we identified that the phylogenetically distant flagellate Mesostigma viride showed photoelectrical responses in vivo with properties suggesting a channelrhodopsin especially promising for optogenetic use. We cloned an M. viride channelrhodopsin, MChR1, and studied its channel activity upon heterologous expression. Action spectra in HEK293 cells match those of the photocurrents observed in M. viride cells. Comparison of the more divergent MChR1 sequence to the previously studied phylogenetically clustered homologs and study of several MChR1 mutants refine our understanding of the sequence determinants of channelrhodopsin function. We found that MChR1 has the most red-shifted and pH-independent spectral sensitivity so far reported, matches or surpasses known channelrhodopsins’ channel kinetics features, and undergoes minimal inactivation upon sustained illumination. This combination of properties makes MChR1 a promising candidate for optogenetic applications

Optogenetics and deep brain stimulation neurotechnologies

Brain neural network is composed of densely packed, intricately wired neurons whose activity patterns ultimately give rise to every behavior, thought, or emotion that we experience. Over the past decade, a novel neurotechnique, optogenetics that combines light and genetic methods to control or monitor neural activity patterns, has proven to be revolutionary in understanding the functional role of specific neural circuits. We here briefly describe recent advance in optogenetics and compare optogenetics with deep brain stimulation technology that holds the promise for treating many neurological and psychiatric disorders

Future improvements in conjunction assessment and collision avoidance using a combined laser tracking/nudging network

The expected significant increase of space launch activities in the next years, both from spacefaring nations and in the private sector, yields an enhanced risk of space debris generation. In this regard, space situational awareness (SSA) is mandatory not only for the protection of active space missions, but as a prerequisite to prevent aggravation of the space debris environment by cascading effects of secondary debris generation due to in-space collisions. High accuracy in laser ranging to space objects (within a meter or better) has already been demonstrated, e.g., by the International Laser Ranging Service (ILRS) network. Thus, laser ranging can be considered as a highly promising sensor technology for space surveillance in the Low Earth Orbit (LEO) which has the potential to complement existing radar facilities in terms of achievable state vector accuracy. Furthermore several laser-based concepts on orbit modification have been proposed in the recent years. In particular, momentum transfer to space debris via photon pressure appears to become feasible, due to advancements in adaptive optics and the commercial availability of high power lasers with an average power output beyond the 10 kW level. This allows for the setup of a network of comparably cost-efficient laser stations for momentum transfer in the near future paving the way for the capability to remotely operate space debris in particular in terms of debris vs. debris collision avoidance maneuvers. In the scope of the conceptual study LARAMOTIONS (SSA P3-SST-XV) funded by the European Space Agency (ESA) simulations of a ground-based laser tracking and momentum transfer network have been carried out in order to estimate the subsequent improvements in conjunction assessment and collision avoidance for operational satellites as well as for debris vs. debris encounters. Therefore, the software generates reference trajectories from a Two-Line-Element (TLE) catalogue for any number of target objects in LEO. From these trajectories station passes as well as random measurement samples are computed and the orbit determination process is simulated yielding the collision rate and false alert rate of the given network. Special emphasis is taken on considering station downtimes due to weather conditions by introducing a station-specific duty cycle based on the analysis of historical weather data. Afterwards a momentum transfer network can be simulated. In order to achieve this, forces induced by photon pressure are computed from tabulated data of target-specific Laser-Matter-Interaction simulations and are applied to the object’s trajectories. A second laser tracking simulation based on the modified orbits eventually shows the advantages of the given system in terms of conjunction analysis and avoidance, in particular considering debris vs. debris collisions for which at present collision avoidance maneuvers are not yet available. The paper will outline the software architecture as well as the results for different network geometries considering the number of stations, their geographical distribution and different duty cycle values. Among the results, the effects of the network geometry and station distribution on the achievable orbit accuracy will be presented. Two operational scenarios will be compared: On-demand tracking in response to conjunction alerts and a laser catalog scenario yielding the maximum number of objects, which can continuously be tracked by a given network independently from radar-based SSA data. Finally, an outlook will be given regarding future simulations and possible enhancements of the simulation environment