The MotA and MotB proteins of E. coli form a MotA4MotB2 complex. Proton flow through a transmembrane channel in the complex powers flagellar rotation. Protonation of Asp-32 of MotB within the channel is proposed to cause a conformational change in the large cytoplasmic loop of MotA, which pushes against FliG in the rotor. MotB is believed to anchor the complex to the cell wall via a conserved sequence that is found in many proteins that bind peptidoglycan. The research presented in this dissertation focused primarily on the formation and activation of the MotAB proton channel. A proposed amphipathic α-helical region, extending from residue 52 through 65 of the periplasmic domain of MotB, was discovered to block proton flow through the channel in its inactive state prior to incorporation into a flagellar motor. The plug is thought to lie parallel to the periplasmic face of the cell membrane and to be removed from the membrane by a conformational change triggered by contact with the motor. Negatively charged residues near the cytoplasmic C-terminus of MotA and positively charged residues at the cytoplasmic N-terminus of MotB were identified as being important for motility. A mutational analysis and subsequent suppressor analysis suggest that these residues may align MotA and MotB to form the MotA4MotB2 complex in the proper position relative to FliG and the rotor. The underlying mechanism for producing MotA and MotB in a 2:1 ratio was also investigated and found to be primarily due to translational coupling of motA and motB. The stop codon of motA and the start codon of motB overlap, allowing the ribosome that has just completed translation of motA to reinitiate and translate motB. The efficiency of reinitiation is about 66%; presumably degradation of excess MotB not in the MotA4MotB2 complex produces the final 2:1 ratio. Research was also conducted to determine whether MotB binds directly to the peptidoglycan layer of the cell wall. Although inconclusive, the preliminary results appear to support this notion. The overall work provides insights into several aspects of the assembly and subsequent activation of the stator component of the bacterial flagellar motor.

The MotA and MotB proteins of E. coli form a MotA4MotB2 complex. Proton flow through a transmembrane channel in the complex powers flagellar rotation. Protonation of Asp-32 of MotB within the channel is proposed to cause a conformational change in the large cytoplasmic loop of MotA, which pushes against FliG in the rotor. MotB is believed to anchor the complex to the cell wall via a conserved sequence that is found in many proteins that bind peptidoglycan. The research presented in this dissertation focused primarily on the formation and activation of the MotAB proton channel. A proposed amphipathic α-helical region, extending from residue 52 through 65 of the periplasmic domain of MotB, was discovered to block proton flow through the channel in its inactive state prior to incorporation into a flagellar motor. The plug is thought to lie parallel to the periplasmic face of the cell membrane and to be removed from the membrane by a conformational change triggered by contact with the motor. Negatively charged residues near the cytoplasmic C-terminus of MotA and positively charged residues at the cytoplasmic N-terminus of MotB were identified as being important for motility. A mutational analysis and subsequent suppressor analysis suggest that these residues may align MotA and MotB to form the MotA4MotB2 complex in the proper position relative to FliG and the rotor. The underlying mechanism for producing MotA and MotB in a 2:1 ratio was also investigated and found to be primarily due to translational coupling of motA and motB. The stop codon of motA and the start codon of motB overlap, allowing the ribosome that has just completed translation of motA to reinitiate and translate motB. The efficiency of reinitiation is about 66%; presumably degradation of excess MotB not in the MotA4MotB2 complex produces the final 2:1 ratio. Research was also conducted to determine whether MotB binds directly to the peptidoglycan layer of the cell wall. Although inconclusive, the preliminary results appear to support this notion. The overall work provides insights into several aspects of the assembly and subsequent activation of the stator component of the bacterial flagellar motor.

Adenosine triphosphate (ATP) is the energy currency of all living cells and its production is a key reaction in the energy metabolism of living organisms. Cells produce most of the ATP they require through ATP synthase, a unique molecular rotary motor driven by the movement of protons across the lipid membrane. In E.coli, ATP synthase is composed of a soluble domain called F1, which houses the catalytic sites, and a transmembrane domain called F0 that shuttles protons across the membrane to drive ATP production in the F1 sector. The F0 domain is built of three subunit types: subunit a and a dimer of subunit b form the stator of the motor, while a decameric c ring forms the rotor. The dynamic interface between a and c10 forms the proton channel. The ultimate goal of this work is to determine the structure of the proton transport machinery and understand the molecular mechanism of proton translocation in ATP synthase. We have characterized some of the key events in the stepwise assembly of the F0--complex. We have designed and validated a model protein, consisting of genetically fused subunits a and c, for structural studies. We have made progress towards determining the structure of the proton channel, including the development of a novel procedure for purification of subunit a and the a/c fusion protein, and crystallization of subunit a. Medical applications of this work include the potential development of novel antibiotic compounds, as well as the characterization and potential treatment of three human diseases caused by disruptions in proton transport through F0.

A novel truncate of Influenza A M2 protein (residues 22-62), incorporated into a uniquely tailored proteoliposome proton uptake assay, demonstrated proton flux more characteristic of an ion transporter than a traditional ion "channel." The liposome paradigm was essential for testing the conductance activity of this M2 truncate at a range of extraphysiological pHs appropriate for channel vs. transport function determination. In addition to transporter-typical proton flux, M2(22-62) showed the key characteristics of functional integrity: selective proton uptake into liposomes and block of uptake by amantadine. Two sets of proteoliposome proton flux assays were carried out, Set 1 at pH values of 6.5, 6.0. 5.5, 5.0, and 4.5; Set 2 at pH values of 6.25, 6.0, 5.75, 5.5, 5.25, 5.0, and 4.75. Observed flux rates followed a proton transport saturation curve similar to that observed in mouse erythroleukemia cells1. Proton transport was maximal at pH 5.5 in Set 1 (139 H+/second/tetramer) and at pH 5.75 in Set 2 (43 H+/second/tetramer). Amantadine block was strongest at pH 5.5 in Set 1 and 6.25 in Set 2, and apparent desensitization of the protein severely reduced proton flux and amantadine sensitivity below pH 5.5 in both sets of experiments. Decreased external pH increased proton uptake with an apparent pKa of 6 (Set 1) or 6.5 (Set 2). These data indicate acid activation of M2(22-62) between pH 5.5-6, optimal amantadine block between pH 5.5-6.25, and a loss of peptide functionality between pH 5.9-4.7.

The bacterial flagellum is a fascinating corkscrew-shaped macromolecular rotary machine used primarily to propel bacterial cells through their environment via the conversion of chemical potential energy into rotational power and thrust. Flagella are the principal targets of complex chemotaxis systems, which allow microbes to navigate their habitats to locate favorable conditions and avoid harmful ones by continuous sampling of environmental compounds and cues. Flagella serve as surface and temperature sensors, mediators of host cell adherence by bacterial pathogens and symbionts alike, and important virulence factors for disease-causing microbes. They play several essential roles in accelerating the foundational stages of biofilm formation, during which bacteria build highly intricate microbial communities with increased resistance to predation and environmental assaults. Flagellum-mediated chemotaxis has broad and impactful implications in fields of bioremediation, targeted drug delivery, bacterial-mediated cancer therapy and diagnostics, and cross-kingdom horizontal gene transfer. While the core structural and functional components of flagella have been well characterized in the closely related enteric bacteria, Escherichia coli and Salmonella typhimurium, major departures from this paradigm have been identified in other diverse species that merit further investigation. Many bacteria employ additional reinforcement modules to surround and stabilize their more powerful flagellar motors and provide increased contact points in the inner membrane, the peptidoglycan sacculus, and, in Gram-negative bacteria, the outer membrane. Additionally, the soil-dwelling bacterium Sinorhizobium meliloti exhibits marked distinctions in the regulation, structure, and function of its navigation systems. S. meliloti is a nitrogen-fixing symbiont of the agronomically valuable leguminous plant, Medicago sativa Lucerne, and uses its coupled chemotaxis and flagellar motility systems to search for host plant roots to colonize. Following root colonization, the bacterium converts to a nitrogen-fixing factory for the plant and the combined influences of this symbiosis can quadruple the yields of the host. This dissertation is aimed at delivering a thorough representative overview of the processes facilitating bacterial flagellum-mediated chemotaxis and motility. Chapter 1 describes the interplay between chemotaxis and flagellar motility pathways as well as the structure, function, and regulation of these systems in several model bacteria. Particular emphasis is placed on the comparison of flagellar systems from the soil-dwelling legume symbiont, Sinorhizobium meliloti with other model systems, and a brief introduction is provided for its primary counterpart, the agronomically valuable legume, Medicago sativa, more commonly referred to as alfalfa. Chapter 2 embodies the first report of a flagellar system to require two copies of a protein known as FliL for its function. FliL is found in all bacterial flagellar systems reported to date but is only essential for some to drive motility. The more conserved copy of the protein has retained the title of FliL and several experiments to assay the proficiency of flagellar motor function revealed that in the absence of FliL swimming is essentially abolished as is the presence of flagella on the cell body. Flagellar motor activity and swimming proficiency of mutants lacking the FliL-paralog MotF was nearly as abysmal as those without FliL but flagellation was essentially normal indicating distinct roles for the two proteins. FliL is implicated in initial stator recruitment to the motor while MotF was found to serve as a power or speed modulator. A model to accommodate and explain the roles of these proteins in the flagellar motor of S. meliloti is provided. Chapter 3 links a never-before characterized flagellar protein, currently named Orf23, to a role in promoting maximum swimming velocity and perhaps stator alignment with the rotor in a peptidoglycan-dependent manner. The loss of LdtR, a transcriptional regulator of peptidoglycan-modification genes, caused defects in swimming motility that are restored only by removal of Orf23 or by replacing a nonpolar glycine with a polar serine in the periphery of stator units. Bioinformatics analyses, immunoblotting, and membrane topology reporter assays revealed that Orf23 is likely embedded in the inner membrane and that the remainder of the protein extends into the periplasm. Building on findings from Chapter 2, Orf23 is anticipated to influence stator positioning through interactions with MotF, FliL, and/or stator units directly. The chapter is concluded with the description of future experiments aimed to more thoroughly characterize Orf23. Altogether, this work increases the depth and breadth of knowledge regarding the composition and function of the speed-variable bacterial flagellar motor. We have identified several components required for stator incorporation and function, as well as an accessory component that improves stator performance. A wise society will draw inspiration from these fascinating and powerful machines to inform new technologies to achieve modern goals including targeted drug delivery, bioremediation, and perhaps one day our own exploration. / Doctor of Philosophy / Bacteria are small autonomous single-celled organisms capable of existing and thriving in highly diverse environments. Motility is achieved by these organisms in various ways, but the most common approach is to produce one or more corkscrew-shaped propeller systems known as flagella that are constructed upon and anchored within the wall of the bacterial cell. Rotation of these propellers relies on power converters known as stators to transform the flow of ions down self-produced gradients into useful rotational energy. This process can be likened to the way that the stored energy of water behind a dam can be harnessed and used to power hydroelectric generators. While the core components of flagellar motors are well conserved and understood among distantly related bacteria, billions of years of evolution and refinement of additional structures have allowed bacteria to accommodate swimming in diverse habitats with e.g. low nutrient availability or high viscosity. Here we describe the discovery and characterization of additional components in the flagellar motor system of the soil-dwelling bacterium Sinorhizobium meliloti to navigate soil environments. We report the first identification of a flagellar motor that requires two copies of a pervasive flagellar motor protein known as FliL and have named the more distinct version of the protein MotF. We found that FliL is required for the power converter components to install into the motor and that MotF is necessary to activate them. Next, we identify another motor component, Orf23, that is dispensible for motility but appears to be required to achieve maximum swimming velocity and may serve to shift the motor into a "higher gear". We find that disruption of a regulator of cell wall modification systems leads to defects in motility that are only restored when Orf23 is removed or when the power converter is modified. Ideas are proposed for how FliL, MotF, and Orf23 are integrated into the motor and may contribute to stator function. An advanced understanding of the mechanisms governing flagellar motor structure and function will provide avenues for the improvement of bacteria-based agricultural improvements, development of optimized bacteria-mediated drug delivery systems, bioremediation techniques, and more.

Mikrobielle Rhodopsine sind lichtsensitive Membranproteine und agieren als Sensoren, Biokatalysatoren oder Ionentransporter. Die Ionentransporter unterteilen sich in lichtgetriebene Ionenpumpen und in lichtaktivierte Kanalrhodopsine. Besonders die Protonenpumpe Bakteriorhodopsin steht schon lange im Fokus biophysikalischer Untersuchungen. Obwohl die Protonenpumpen seit über 40 Jahren intensiv untersucht werden, ist das Wissen über deren elektrophysiologische Eigenschaften noch immer gering. Aus diesem Grund widmete sich diese Arbeit der elektrophysiologischen Charakterisierung der mikrobiellen Rhodopsine mit dem Fokus auf Protonenpumpen. Hierfür wurden vor allem „Two-Electrode Voltage Clamp“ -Messungen (TEVC) an Oozyten des afrikanischen Krallenfrosches Xenopus leavis durchgeführt. Die Untersuchung verschiedener Protonenpumpen hat gezeigt, dass diese eine unerwartet große Diversität in ihren elektrophysiologischen Eigenschaften aufweisen. Von besonderem Interesse war die Beobachtung, dass einige Protonenpumpen neben Pumpströmen auch passive einwärts gerichtete Photoströme zeigten. Besonders deutlich war der „Pump-Kanal-Dualismus“ bei dem Gloeobacter-Rhodopsin ausgeprägt. Andere Protonenpumpen, wie das Bakteriorhodopsin oder Coccomyxa-Rhodopsin, zeigten keine einwärts gerichteten Photoströme. Das Coccomyxa-Rhodopsin wurde aufgrund seiner hohen Photostrom-Amplituden in Oozyten für eine Mutationsanalyse ausgewählt. Diese Mutationsanalyse verhalf die strukturellen Ursachen für die funktionalen Unterschiede zu identifizieren, welche sowohl zwischen den Protonenpumpen untereinander als auch gegenüber Kanalrhodopsinen beobachtet wurden. Mutationen im Gegenion-Komplex führen zu rein passiven oder inaktiven Transportern. Dagegen übernimmt der extrazelluläre Halbkanal in Protonenpumpe die Aufgabe einen passiven Protonen-Rückfluss während des Pumpzyklus zu verhindern, denn Mutationen in dieser Region verursachen passive Photoströme zusätzlich zum aktiven Pumpstrom. / Microbial rhodopsins are light-sensitive membrane proteins and operate as sensors, enzymes or ion-transporters. The ion transporters are subdivided into light-driven ion pumps and light-gated channels. Biophysical research has put focus on the proton pump bacteriorhodopsin for long time. Despite the fact that light-driven proton pumps are investigated for over 40 years, the knowledge about their electrophysiological properties is surprisingly low. For this reason, this thesis is devoted to the electrophysiological characterization of microbial rhodopsins with special focus on light-driven proton pumps. For this purpose, “Two-Electrode Voltage Clamp”-recordings (TEVC) were primarily performed using oocytes from African clawed frog Xenopus leavis. The investigation of diverse proton pumps has shown that the differences in their electrophysiological behaviors are unexpectedly high. Special interest was laid on proton pumps which show passive inward directed photocurrents when the electrochemical load exceeds a certain level. The dualism of pump and channel activity was particularly pronounced in the proton pump Gloeobacter-rhodopsin. Other proton pumps, for instance bacteriorhodopsin or Coccomyxa-rhodopsin, do not show inward directed photocurrents. Due to high photocurrent amplitudes, the Coccomyxa-rhodopsin was selected for an efficient mutagenesis study. This study allowed the identification of structural key determinants for the differences among proton pumps themselves and for the differences of proton pumps in comparison with light-gated ion channels (channelrhodopsins). Therefore, mutations of the counter-ion-complex cause inactive or purely passive transporters. The extracellular half-channel is the key element in proton pumps which prevents passive proton-backflow during the pump-cycle. Mutations in this region lead to passive leak-currents in overlap with the remaining pump-activity.

No description available.

Bei der lichtinduzierten Oxidation von Wasser im Photosystem II (PSII) werden zwei wassermoleküle im katalytischen Zyklus des Metallclusters (Mn4CaO5) benötigt, und vier Protonen aus dem Cluster in den Lumen abgegeben. Daher ist es für das Verständnis des Mechanismus´ der Wasseroxidation von entscheidender Bedeutung, die Veränderung der Protonierungszustände am cluster während der Katalyse zu untersuchen. Hierbei sollten sowohl die Wasserkanäle für die Zuführung der Substratwassermoleküle als auch die Transportwege für die Freisetzung der Protonen untersucht werden. Deshalb wurde in meiner ersten Veröffentlichung ein neues Protokoll entwickelt, um einzelne große Kristalle von dPSII mit einer Länge von ~3 mm in der Längsachse zu züchten. Diese Kristalle mit einer Auflösung von ca. 8 Å gemessen. Um eine höhere Auflösung zu erzielen, ist die Verbesserung der Kristallqualität essenziell. Daher wurde in meiner zweiten Veröffentlichung die Struktur des Detergens-Protein-Komplexes von dPSII mit βDM, durch Anwendung von SANS in Kombination mit SAXS untersucht. Die Ergebnisse zeigten, dass βDM eine monomolekulare Schicht um dPSII bildet. Darüber hinaus konnten freie Mizellen von βDM in der Lösung nachgewiesen werden. Damit ist eine weitere Optimierung der βDM-Konzentration in der Proteinlösung erforderlich, um die Bildung von freien Mizellen zu minimieren. In meiner dritten Veröffentlichung wurde die strukturelle Dynamik in den Wasserkanälen, während des S2-S3 Übergangs mit Hilfe der XFEL untersucht. Ein Datensatz mit einer hohen Auflösung von 1,89 Å wurde durch die Zusammenführung von Daten gewonnen, die während des S2-S3 Übergangs gesammelt wurden. In Anbetracht der Analyse der zusammengeführten Daten und der einzelnen Zeitpunkte, die während des S2-S3 Übergangs gesammelt wurden, ist es wahrscheinlich, dass ein Substratwasser durch den O1-Kanal geliefert wird. Im Gegensatz dazu wird ein Proton aus dem Cluster durch den Cl1 Transportweg in Richtung Lumen freigesetzt. / The light-induced oxidation of water in Photosystem II (PSII) requires incorporating two water molecules in the catalytic cycle of the active metal cluster (Mn4CaO5). Furthermore, four protons are released towards the bulk from the cluster. Therefore, tracking the change of protonation states at the active catalytic site and the surrounding protein side chains during catalysis and elucidating the pathways of water substrate insertion and proton release are crucial to understanding the water oxidation mechanism. Therefore, in the first study of my work, a new protocol was developed to grow single large dPSIIcc crystals with a length of ~3 mm in the long axis. These crystals, soaked in D2O containing buffer, diffracted to about 8 Å resolution. Improving the crystal quality is crucial for achieving a better resolution. Consequently, in the second study of my work, the structure of the detergent-protein complex of βDM-dPSIIcc has been investigated by applying SANS in combination with SAXS. The results showed that βDM is forming a monomolecular layer around the dimeric PSII core complex (dPSIIcc). Moreover, the SAXS data detected a peak assigned to the free micelles of βDM. These results raise the necessity to optimize the βDM concentration in the protein solution to avoid the possible excess of free micelles. In the third study of my work, the structural dynamics in the water channels connecting the cluster to the lumen during the S2  S3 transition were investigated using serial femtosecond XFEL. A high-resolution data set was obtained at a resolution of 1.89 Å by combining data collected at RT. Considering the analysis of the combined data and the individual time points collected during the S2  S3 transition, it is likely that the substrate water insertion into the open coordination site of the Mn1 ion is delivered through the O1 channel. In contrast, a proton from the cluster is released towards the bulk through the Cl1 A channel.

Kanalrhodopsine (ChRs) sind lichtaktivierte Ionenkanäle motiler Algen. Heterolog exprimiert erlauben sie es, Ionenflüsse durch Licht zu steuern. Bevorzugt geleitet werden von den meisten ChRs Protonen. Ausprägung und Wirkung lichtaktivierter Protonenflüsse sowie der molekulare Mechanismus protonenselektiver ChRs werden in vorliegender Arbeit untersucht und zur Entwicklung neuer optogenetischer Werkzeuge genutzt. Eine besonders hohe Protonenselektivität zeigten die grün- und rotlicht-aktivierten Kanäle CsChR und Chrimson aus den Algen Chloromonas subdivisa und Chlamydomonas noctigama. Im spektroskopisch detailliert untersuchten CrChR2 aus Chlamydomonas reinhardtii änderte sich die Protonenselektivität nach Anregung mit einem ns-Laserblitz sogar innerhalb eines Aktivierungszyklus und war insbesondere nach Öffnung des Kanals sowie in Folge der Lichtadaptation hoch. Als unentbehrlich für eine effiziente Protonenleitung erwiesen sich in allen drei Kanälen konservierte, titrierbare Reste entlang der Pore, deren individuelle Bedeutung für die Protonenleitung sich je nach Protein wesentlich unterschied. Entsprechend genügte in Chrimson der Austausch einzelner Glutaminsäuren des extrazellulären Halbkanals, dieses in einen grün- oder rotlichtaktivierten Natriumkanal zu transformieren. Aminosäuresubstitutionen der unmittelbaren Retinalumgebung verschoben hingegen das Aktionsmaximum von Chrimson röter als 600 nm und damit röter als in allen bisher beschriebenen ChRs. In Chrimson versperrt hierbei ein zusätzliches äußeres Tor den extrazellulär Halbkanal, während die Retinalbindetasche in Struktur und funktionaler Bedeutung der einzelnen Reste wesentlich jener der Protonenpumpe Bacteriorhodopsin ähnelt. Als Zwei-Komponenten-Optogenetik wurden schließlich protonen-, kationen- und anionenleitende ChRs unterschiedlicher Farbsensitivität fusioniert sowie lichtgetriebene Protonenpumpen mit protonenaktivierten Ionenkanälen kombiniert und neue optogenetische Perspektiven eröffnet. / Channelrhodopsins (ChRs) are light-gated ion channels from green algae. Expressed in host cells they are used to control ion fluxes by light and are widely applied in Neurosciences. Although generally classified as either cation or anion channels, most ChRs preferentially conduct protons. This thesis compares proton conductance of different ChRs, examines the molecular mechanism of proton selective ChRs and explores the usage of light regulated proton fluxes in two-component-optogenetics. Proton selectivity varied strongly among different ChRs and was most pronounced for the green- and red-light activated channels CsChR and Chrimson from the algae Chloromonas subdivisa and Chlamydomonas noctigama, that conducted predominantly protons even at high pH. In CrChR2 from Chlamydomonas reinhardtii proton selectivity also changed during a single activation cycle and was especially high directly after channel opening and later on following light adaptation. In all three channels efficient proton conductance depended on conserved titratable residues along the pore with different contribution of the individual side chains in each protein. The substitution of single glutamic acids in the extracellular half pore converted Chrimson into a green or red-light activated sodium channel. A single point mutation close to the retinal chromophore shifted peak absorption of Chrimson beyond 600 nm - further red than all other cation conducting ChRs. Whereas the retinal binding pocket of Chrimson resembles the proton pump Bacteriorhodpsin, the overall pore structure corresponds to other ChRs, but features an additional outer gate, that occludes the extracellular half pore and is important for both, proton selectivity and red light absorption. Finally different Two-Component-Optogenetic approaches combined proton and anion selective ChRs of distinct colour as well as light-driven proton pumps and proton-activated ion channels with major prospect for future optogenetic applications.