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Ribosomes translate the genetic code into proteins. Recent technical advances have facilitated in situ structural analyses of ribosome functional states inside eukaryotic cells and the minimal bacterium Mycoplasma. However, such analyses of Gram-negative bacteria are lacking, despite their ribosomes being major antimicrobial drug targets. Here we compare two E. coli strains, a lab E. coli K-12 and human gut isolate E. coli ED1a, for which tetracycline exhibits bacteriostatic and bactericidal action, respectively. The in situ ribosome structures upon tetracycline treatment show a virtually identical drug binding-site in both strains, yet the distribution of ribosomal complexes clearly differs. While K-12 retains ribosomes in a translation competent state, tRNAs are lost in the vast majority of ED1a ribosomes. A differential response is also reflected in proteome-wide abundance and thermal stability assessment. Our study underlines the need to include molecular analyses and to consider gut bacteria when addressing antibiotic mode of action.
Cyclophilins, or immunophilins, are proteins found in many organisms including bacteria, plants and humans. Most of them display peptidyl-prolyl cis-trans isomerase activity, and play roles as chaperones or in signal transduction. Here, we show that cyclophilin anaCyp40 from the cyanobacterium Anabaena sp. PCC 7120 is enzymatically active, and seems to be involved in general stress responses and in assembly of photosynthetic complexes. The protein is associated with the thylakoid membrane and interacts with phycobilisome and photosystem components. Knockdown of anacyp40 leads to growth defects under high-salt and high-light conditions, and reduced energy transfer from phycobilisomes to photosystems. Elucidation of the anaCyp40 crystal structure at 1.2-Å resolution reveals an N-terminal helical domain with similarity to PsbQ components of plant photosystem II, and a C-terminal cyclophilin domain with a substrate-binding site. The anaCyp40 structure is distinct from that of other multi-domain cyclophilins (such as Arabidopsis thaliana Cyp38), and presents features that are absent in single-domain cyclophilins.
Das aufgefundene Ribonucleoprotein besitzt die Eigenschaft eines Vollantigens. Die durchgeführten Immunisierungsversuche ergaben eine Artspezifität des Antigens. Eine Organspezifität war nicht nachweisbar. Erste Lokalisierungsversuche mit Hilfe der Immunfluoreszenz-Methode ergaben in der Niere eine besondere Anreicherung des Nucleoproteins in den Glomerulokapseln.
Zusammenfassend läßt sich sagen, daß im menschlichen Serum ein Serumprotein vorhanden ist, welches das aus menschlichem Gewebe gewonnene Nucleoprotein spezifisch bindet. Zur Zeit sind Untersuchungen im Gang. den Serumgehalt des Nucleoproteins und des spezifisch bindenden Serumproteins bei verschiedenen Erkrankungen des Bindegewebsapparates quantitativ zu erfassen.
In weiteren Versuchen wurden Streptokokken der Gruppe A und C dem in der I. Mitteilung beschriebenen Trennungsgang unterworfen. Es gelang dabei, aus diesen Streptokokken ein Nucleoprotein zu isolieren, das in bezug auf seine elektrophoretischen Eigenschaften, Farbreaktionen, UV-Absorptionskurve, Zuckeranalyse mit dem aus menschlichen und tierischen Organen gewonnenen Nucleoprotein identisch ist.
Das in der I. Mitteilung beschriebene Nucleoprotein führt bei Zugabe zu Tropokollagen-Lösungen zur Bildung von kollagenen Fibrillen. Die Fibrillen-bildende Potenz des Nucleoproteins ist sehr groß. Die gebildeten Fibrillen zeigen eine sehr gute Feinstruktur und weisen eine 640 Å Hauptperiodik auf. Es ist zu vermuten, daß dem ubiquitär im Organismus vorkommenden Nucleoprotein eine bedeutende oder gar ausschlaggebende Rolle bei der Bildung von kollagenen Fibrillen aus Tropokollagen im Organismus zukommt.
Es wird auf die Mannigfaltigkeit der Kontraste hingewiesen, die sich bei mit Aldehyden fixiertem, in Vestopal W oder Durcupan ACM eingebettetem Gewebe durch gesteuerte Elektronenbestrahlung erreichen läßt. Voraussetzung für einen einwandfreien Vergleich von Kontrasten bei z. B. verschieden gefärbten Schnitten ist daher eine Bestrahlung, die zu ausreichend definierten Objektveränderungen führt. Brauchbar in diesem Sinne ist eine Bestrahlung, die zum maximalen „reinen Strahlenverlust“ führt, bei der aber thermische Substanzverluste vermieden werden. Der Negativkontrast von Chromatin und Nucleolen und die offenbar physikalisch bedingte Färbbarkeit der nucleinsäurehaltigen Zellbestandteile mittels der „negative-staining“-Methode werden diskutiert.
The TOM complex is the main entry point for precursor proteins into mitochondria. Precursor proteins containing targeting sequences are recognized by the TOM complex and imported into the mitochondria. We have determined the structure of the TOM core complex from Neurospora crassa by single-particle cryoEM at 3.3 Å resolution, showing its interaction with a bound presequence at 4 Å resolution, and of the TOM holo complex including the Tom20 receptor at 6-7 Å resolution. TOM is a transmembrane complex consisting of two β-barrels, three receptor subunits and three short transmembrane subunits. Tom20 has a transmembrane helix and a receptor domain on the cytoplasmic side. We propose that Tom20 acts as a dynamic gatekeeper, guiding precursor proteins into the pores of the TOM complex. We analyze the interactions of Tom20 with other TOM subunits, present insights into the structure of the TOM holo complex, and suggest a translocation mechanism.
Um die von RAJEWSKY und WOLF aufgeworfene Frage nach dem Einfluß der DNS-Struktur auf die radiationschemische Veränderung der Basen zu untersuchen, wurde die DNS-Spirale bei einem Teil der Untersuchungen in dest. Wasser aufgelöst und mit Röntgenstrahlen bestrahlt. Es ergab sich eine Erhöhung der Strahlenempfindlichkeit der Basen, vor allem zu Beginn der Bestrahlung auf den Wert, den man bei der Bestrahlung der Monomerlösungen beobachtet. Bei Bestrahlung in 0,1 und 1-n. NaCl gelöster DNS sind dagegen die Basen gegen die Einwirkung der im Wasser gebildeten Radikale geschützt, solange sie innerhalb der intakten Spirale gebunden sind. Dieser strukturbedingte Schutzeffekt besteht nicht gegenüber der direkten Strahlenwirkung von UV-Licht. Dieses Ergebnis ist von strahlenbiologischem Interesse, da das Optimum der Strahlenwirkung auf den Mitoseablauf nach Arbeiten von CARLSON und GRAY in der frühen Prophase liegt17, also ebenfalls in einem Stadium, in dem die DNS-Spirale (vor der Verdoppelung) völlig aufgelöst ist. (Vgl. auch BACQ-ALEXANDER und FRITZ-NIGGLI.
Die von verschiedenen Autoren 2–8 experimentell bestimmten Kontrastdicken für Kohle stimmen nicht mit den heute of benutzten numerischen Werten aus der Lenz schen Theorie überein. Die Diskrepanz läßt sich beheben, wenn man zur Auswertung der Theorie einen anderen, schon von LENZ zur Diskussion gestellten Θ-Wert benutzt. Durch Experimente wird gezeigt, daß auch der Bereich, in dem das Exponentialgesetz nicht mehr gilt, gut durch eine aus der Lenz schen Theorie hergeleitete Formel dargestellt werden kann. Der Bereich, in dem das Exponentialgesetz verwandt werden darf, wird näher diskutiert.
Cells maintain membrane fluidity by regulating lipid saturation, but the molecular mechanisms of this homeoviscous adaptation remain poorly understood. We have reconstituted the core machinery for regulating lipid saturation in baker’s yeast to study its molecular mechanism. By combining molecular dynamics simulations with experiments, we uncover a remarkable sensitivity of the transcriptional regulator Mga2 to the abundance, position, and configuration of double bonds in lipid acyl chains, and provide insights into the molecular rules of membrane adaptation. Our data challenge the prevailing hypothesis that membrane fluidity serves as the measured variable for regulating lipid saturation. Rather, we show that Mga2 senses the molecular lipid-packing density in a defined region of the membrane. Our findings suggest that membrane property sensors have evolved remarkable sensitivities to highly specific aspects of membrane structure and dynamics, thus paving the way toward the development of genetically encoded reporters for such properties in the future.
Eine einfache Methode wird erklärt, die es gestattet, genaue Aussagen über das Verteilungsgesetz elektrischer Relaxationszeiten in frequenzabhängigen Dielektrika zu machen. Die Methode setzt die Gültigkeit einer verallgemeinerten Form des von Cole und Cole formulierten Verteilungsgesetzes elektrischer Relaxationszeiten voraus. Sie basiert auf der Tatsache, daß dielektrische Verluste. die bei wesentlich kleineren Frequenzen als der mittleren charakteristischen Frequenz bestimmt werden, außerordentlich empfindlich sind gegen geringe Änderungen im Verteilungsgesetz. Die Methode wird am Beispiel dielektrischer Messungen an Wasser demonstriert. Die Auswertung eigener Messungen ergibt, daß sich im Rahmen der erzielbaren hohen Genauigkeit das dielektrische Verhalten von Wasser durch eine einzige Relaxationszeit charakterisieren läßt.
In der vorliegenden Arbeit wird die Frequenzabhängigkeit des dielektrischen Verhaltens einer Suspension von Kugeln mit Schale untersucht. Es werden die allgemeine Lösung sowie spezielle Näherungsformeln angegeben. Das Frequenzverhalten wird exakt durch 2 Relaxationsausdrücke vom Debye - Typ, die sich superponieren, charakterisiert.
Die vorgetragenen Formeln erlauben die Analyse der Impedanzkurven von Zellsuspensionen aus Erythrozyten, Bakterien, Seeigeleiern u. a. m., aber auch von Proteinlösungen und anderen Suspensionen.
Bei B. cadaveris, die in einem an organischen Substanzen reichen Medium kultiviert wurden, nimmt der O2-Verbrauch pro Zeiteinheit bei Glucoseveratmung mit der Röntgenstrahlendosis ab, während bei Bakterien, die in einem Salzmedium gewachsen sind, die Atmung bis zu einer Dosis von 2 - 3 Mr erst ansteigt, um erst bei höheren Dosen abzufallen. Die Atmung wird erst bei Dosen in der Größenordnung von 1 Million r merklich beeinflußt.
Die Atmung der Bakterien ist damit unter den hier untersuchten Bedingungen noch strahlenresistenter als die Gewebeatmung von Säugetierzellen.
Es wurde das Leitfähigkeitsverhalten von reinem, lufthaltigem Wasser bei kontinuierlicher und impulsgetasteter Röntgenbestrahlung (60 kV8) untersucht. Hierbei ergaben sich zwei einander überlagerte Effekte: 1. Ein der Röntgen-Dosisleistung proportionaler irreversibler Leitfähigkeitsanstieg, der vermutlich auf eine Strahlenreaktion des gelösten CO2 zurückzuführen ist, 2. eine reversible Leitfähigkeitserhöhung während der Bestrahlung, die sich mit der Entstehung einer Ionenart mit einer mittleren Lebensdauer von ca. 0,15 sec erklären läßt. Es wird angenommen, daß es sich dabei um Radikalionen O2⊖ handelt, welche durch die Reaktion der als Strahlungsprodukt entstehenden Η-Radikale mit dem gelösten Sauerstoff gebildet werden. Ein möglicher chemischer Reaktionsmechanismus wird angegeben, der zu befriedigender quantitativer Übereinstimmung der Versuchsergebnisse mit Ausbeutewerten und Reaktionskonstanten aus der Literatur führt.
Trockene Mildisäure-Dehydrogenase wurde mit Protonen verschiedener Energie bis maximal 80 keV in dünnen Schichten bestrahlt. Die Inaktivierungsquerschnitte betrugen bei Zimmertemperatur ca. 0,4·10-12 cm2 und waren in dem gemessenen Energiebereich konstant. Der Einfluß der Teilchenenergie auf den Inaktivierungsverlauf wurde insbesondere bei sehr kleinen Energien abgeschätzt. Bestrahlungen bei verschiedenen Temperaturen zeigten eine Abnahme des Strahleneffektes nach niedrigeren Temperaturen.
Es wird der Einfluß von Röntgenstrahlen und ultravioletten Strahlen verschiedener Wellenlängen auf kernhaltige Zellteile von Acetabularia mediterranea untersucht. Die Röntgenbestrahlung führt zu einer Verminderung des Regenerationsvermögens der Zellteile, zu einer Verringerung der Cystenbildung der Regenerate und zu einer Herabsetzung der Lebensfähigkeit der Cysten. Erst nach einer Dosis von 400 kr ist das Regenerationsvermögen fast völlig zerstört. Die Fähigkeit zur Bildung fortpflanzungsfähiger Gameten geht bereits nach 40 kr verloren. Die Wirkung von UV-Bestrahlungen ist demgegenüber sehr gering. Es wird geschlossen, daß die beobachteten Leistungen der kernhaltigen Zellteile wesentlich durch den Zellkern bestimmt sind, der im Rhizoid gegen die UV-Strahlung weitgehend abgeschirmt ist. Erfolgt eine vollständige Regeneration bis zur Hutbildung, so scheinen Größe und Gestalt der ausgewachsenen Regenerate nicht wesentlich von der Röntgenbestrahlung beeinflußt zu sein. In den meisten Fällen bilden Hutregenerate auch Cysten.
Wir berichten im folgenden über histologische Befunde und physikalische Messungen, die zeigen, daß unter besonderen Beschallungsbedingungen in verschiedenen Säugetiergeweben Vorgänge ablaufen, die im Sinne einer Pseudokavitation gedeutet werden müssen. Die Einzelheiten der Untersuchungen sind teils in der Habilitationsschrift von O. Hug, Frankfurt a. M. 1953, teils in der Dissertation von R.Pape, Frankfurt a. M. 1953, niedergelegt.
Die Inaktivierung durch Röntgenstrahlen der an Lebermitochondrien gebundenen Bernsteinsäure-Oxydase wurde untersucht. Ihre Halbwertsdosis beträgt 3,5 · 106 r. Bernsteinsäure-Oxydase, die an Hepatommitochondrien gebunden ist, ist empfindlicher als die normaler Mitochondrien. Die Bernsteinsäure-Oxydase an kleinsten Partikeln zeigt dagegen in beiden Fällen eine größere Strahlenresistenz.
Die in der vorliegenden Arbeit beschriebenen Messungen von Dielektrizitätskonstanten und Leitfähigkeiten biologischer Substanzen im Bereich von 9 bis 180 cm Wellenlänge werden nach einem Resonanzverfahren durchgeführt. Dies ermöglicht trotz der starken Absorption der untersuchten Materialien eine genügende Meßgenauigkeit und einen relativ einfachen Aufbau der Meßanordnung. Die beschriebene Methode zur Auswertung der Messung ist so vereinfacht, daß sie auch von angelerntem Hilfspersonal leicht durchgeführt werden kann. Für den Wellenlängenbereich von 40 bis 180 cm wird eine Lecher-Leitung in Paralleldraht-Aus-führung benutzt, während sie für den Bereich von 9 bis 40 cm Wellenlänge konzentrisch aufgebaut ist. Die Meßfehler werden eingehend diskutiert und in Kurven anschaulich dargestellt. Anschließend werden die Ergebnisse der Messungen von DK und Leitfähigkeit an Blut, Leber, Muskel und Fettgewebe mitgeteilt. Bei allen Substanzen konnte unterhalb etwa 30 cm Wellenlänge eine Abnahme der DK und vor allem eine eindeutige Zunahme der Leitfähigkeit festgestellt werden. Diese Dispersion wird den polaren Molekülen in den Substanzen zugeschrieben und dürfte zumindest bei Blut und Leber im wesentlichen durch die Dispersion des Wassers verursacht werden.
Der Einfluß von Elektrodenpolarisation auf die Bestimmung der elektrischen Konstanten leitfähiger Substanzen wird untersucht; es wird gezeigt, daß der durch Polarisation bedingte Effekt auf die Kapazität um mehrere Zehnerpotenzen größer ist als der auf die Wirkwiderstandskomponente. Die Bestimmung der Dielektrizitätskonstante stark leitfähiger Materialien bei Niederfrequenz wird dadurch sehr erschwert. Die verschiedenen Möglichkeiten, Polarisationseinflüsse herabzusetzen, werden diskutiert und es wird gezeigt, daß einzig eine Messung mit verschiedenem Elektrodenabstand einwandfreie Ergebnisse gewährleistet, wenn Polarisation merklich auftritt. Eine bei biologischen Arbeiten öfter angewandte Methodik geht von der Voraussetzung aus, daß beim Austausch biologischen Materials gegen eine Salzlösung gleicher Beschaffenheit, wie sie intrazellular im biologischen Material vorliegt und in Kontakt mit den Elektroden steht, die Polarisationsimpedanz keiner Änderung unterliegt. Die Analyse eigener experimenteller Untersuchungen, über die berichtet wird, zeigt, daß diese Annahme nur berechtigt ist, wenn das biologische Material in so hinreichend großem Abstand von den Elektroden angeordnet wird, daß keine Schattenwirkung auftreten kann. In allen anderen Fällen ist sie falsch, und Arbeiten, die dem nicht Rechnung tragen, sind kritisch zu bewerten.
Single-particle electron cryo-microscopy (cryoEM) has undergone a “resolution revolution” that makes it possible to characterize megadalton (MDa) complexes at atomic resolution without crystals. To fully exploit the new opportunities in molecular microscopy, new procedures for the cloning, expression and purification of macromolecular complexes need to be explored. Macromolecular assemblies are often unstable, and invasive construct design or inadequate purification conditions or sample preparation methods can result in disassembly or denaturation. The structure of the 2.6 MDa yeast fatty acid synthase (FAS) has been studied by electron microscopy since the 1960s. We report a new, streamlined protocol for the rapid production of purified yeast FAS for structure determination by high-resolution cryoEM. Together with a companion protocol for preparing cryoEM specimens on a hydrophilized graphene layer, our new protocol has yielded a 3.1 Å map of yeast FAS from 15,000 automatically picked particles within a day. The high map quality enabled us to build a complete atomic model of an intact fungal FAS.
Es wurde der Einfluß von Röntgenbestrahlungen auf die Lebensfähigkeit und das Formbildungs-Vermögen kernloser Zellteile von Acetabularia mediterranea in Abhängigkeit von der Strahlendosis untersucht. Dabei erwies sich die Hutbildung stets als der am strahlenempfindlichste Prozeß. Die gefundenen Ergebnisse werden diskutiert und mit den entsprechenden Befunden nach UV-Bestrahlung verglichen.
Es wurde die Einwirkung monochromatischer ultravioletter Strahlen der Wellenlänge 254 mμ, 281 mμ und 297 mμ auf kernlose Zellteile von Acetabularia untersucht. Dazu wurden in Abhängigkeit von der Strahlendosis die mittlere Lebensdauer und das Formbildungs-Vermögen der kernlosen Teile bestimmt. Der 254-mμ-Strahlung kam dabei stets die größte biologische Wirksamkeit zu, während die 297-mμ-Strahlung im untersuchten Dosisbereich fast ohne Wirkung blieb. Die gefundene Wellenlängen-Abhängigkeit weist darauf hin, daß der UV-Absorption durch die Purine und Pyrimidine für den Wirkungsmechanismus der beobachteten UV-Schädigungen kernloser Zellteile besondere Bedeutung zukommen muß.
Es wird versucht, die Treffertheorie auf die indirekte Strahlenwirkung auszudehnen. Dazu wird angenommen, daß durch die Strahlung „Energieträger“ erzeugt werden, die durch Diffusion zu den „empfindlichen Bereichen“ gelangen und diese verändern können.
Der Berechnung des Wirkvolumens für derartige „indirekte Treffer“ folgt eine reaktionskinetische Betrachtung der indirekten Wirkung.
Durch die Einschaltung physikalisch-chemischer Prozesse zwischen Strahlenabsorption und „Treffer“ erscheint eine Berücksichtigung der physikalischen und chemischen Gegebenheiten im bestrahlten Objekt viel eher möglich als in der „klassischen“ Theorie der „direkten Trefferwirkung“.
Die Feldstärke- und Wärmequellenverteilung im ebenen Körper aus Muskel, Fett und Haut bei Anstrahlung wird für den Wellenbereich von 1 m bis 1,27 cm jeweils bei verschiedenen Fettschichtdicken berechnet und graphisch dargestellt. Die mit der Strahlungsfeldmethode erreichbare Tiefendosis-Leistung im Zusammenhang mit der Hautdosisleistung sowie die Frage einer geeigneten Anpassung zur Erreichung einer gleichmäßigen Tiefendosis-Leistung werden diskutiert.
Unter der Voraussetzung, daß ein Strahler benutzt wird, der ein annähernd ebenes Wellenfeld liefert und in dem bestrahlten Körpergebiet ebene, parallele Schichten Haut, Fett, Muskel senkrecht getroffen werden, ergibt sich aus der Betrachtung der berechneten Feldstärke- und Wärmequellenverteilungen für die verschiedenen Wellenlängen etwa folgendes Bild:
Das Problem einer Fettüberlastung tritt bei dm-Bestrahlung bis herab zu 10 cm Wellenlänge nicht auf. Man erhält im Gegensatz zu UKW-Kondensatorfeld-Durchflutung eine starke Wärmeentwicklung in der Haut sowie in den oberen Schichten von Muskel oder inneren Organen. Der Abfall der Dosisleistung im Muskel nach der Tiefe zu wird ab λ = 30 cm mit kürzer werdender Wellenlänge zunehmend steiler. Es kann bei dm-Bestrahlung gegenüber UKW-C-Feld eine um das Mehrfache höhere Dosisleistung an der inneren Oberfläche des Körpers auch bei großen Dicken der Fettschicht erreicht werden, wenn gegebenenfalls durch zusätzliche äußere Maßnahmen (Kühlung) eine Überlastung der Haut bei bestimmten Dicken der Fettschicht (λF/4) vermieden wird.
Bei dm-Bestrahlung paralleler Schichten mit angepaßtem Sender lassen sich weitere qualitative Aussagen machen: Bei konstanter Intensität des Strahlers erfolgt bei längeren Wellen (1 m) mit wachsender Dicke der Fettschicht nur ein monotoner Abfall der Tiefendosis-Leistung auf etwa 60%. Im unteren dm-Bereich (30 cm, 10 cm) ergibt sich bei konstanter Intensität bei Vergrößerung der Fettschichtdicke von 0 bis λF/4 ein Abfall der Tiefendosis-Leistung auf etwa ⅓ und bei weiterer Zunahme der Fettschichtdicke von λF/4 bis λF/2 wieder eine Zunahme der Tiefendosis-Leistung im Verhältnis 1:2. Diese Zahlen gelten größenordnungsmäßig bei Anstrahlung aus einem Anpassungsmedium ε-Fett. Bei Anstrahlung aus Luft ist die absolute Tiefendosis-Leistung geringer und sind die Schwankungen größer. Diese Schwankungen der Tiefendosis-Leistung bei konstanter Intensität des Senders einer Wellenlänge können durch äußere Maßnahmen weitgehend verringert werden.
Wenn der Sender bei verschiedenen Fettschichtdicken jeweils auf gleiche Hautdosisleistung eingestellt wird (Dosierung nach der Hautempfindung), nimmt die Tiefendosis-Leistung bei Vergrößerung der Fettschichtdicke von 0 bis λF/4 stark ab auf etwa ⅙, die Tiefendosis-Leistungen bei weiterer Vergrößerung der Fettschichtdicke von λF/4 bis λF/2 verhalten sich dann etwa wie 1 : 3. Diese Schwankungen sind unabhängig von der Wahl des Anpassungsmediums. Eine Tiefendosierung nach der Hautempfindung ohne genauere Berücksichtigung der Fettschichtdicke wird daher bei Vorliegen von zu den Wellenflächen parallelen Schichten unzuverlässig sein. Es ergibt sich jedoch die Möglichkeit einer instrumentellen, brauchbaren Dosierung durch Einstellung der Intensität des Senders ohne Rücksichtnahme auf die Hautempfindung, wenn man die Schwankungen der Tiefendosis-Leistung mit der Fettschichtdicke bei konstanter Intensität durch die erwähnten Anpassungsmaßnahmen reduziert.
Die kurzen Wellenlängen von 3 cm und 1 cm sind für eine Wärmetherapie in der Tiefe wegen der hohen Absorptionsverluste in Haut und Fett kaum brauchbar, können jedoch zu einer Wärmetherapie der Haut und damit indirekten Beeinflussung innerer Erkrankungen oder zur Erwärmung oberflächiger bzw. weniger leitender Schichten herangezogen werden. Die Oberflächendosis-Leistung steigt bei derselben Intensität mit abnehmender Wellenlänge stark an.
Bezüglich der Angaben über die Dosisleistungs-Verteilung ist zu berücksichtigen, daß das Interferenz-Feld vor der Strahleröffnung bei der Rechnung vernachlässigt wurde. Ferner entspricht die Dosisleistungs-Verteilung nur dann der Temperaturverteilung während der Bestrahlung, wenn die Unterschiede in den Anfangstemperaturen und in den spezifischen Wärmen der biologischen Schichten außer Acht gelassen werden sowie intensiv und kurzzeitig bestrahlt wird, so daß noch kein merklicher Wärmetransport während der Bestrahlung stattfindet. Über die Temperaturverteilung bei Berücksichtigung des Wärmetransports, der bei schwacher, langdauernder Bestrahlung eine merkliche Erwärmung auch tieferer innerer Schichten, eine höhere Erwärmung des Fetts und eine geringere der Haut zur Folge haben kann, sind weitere Betrachtungen erforderlich.
Es wird eine einfache und billig herzustellende Resonanzanordnung beschrieben, mit der genaue Bestimmungen elektrischer Impedanzwerte und Materialkonstanten im Dezimeterwellenbereich durchführbar sind. Prinzip der Methode, Meßbereich, Genauigkeit und Einzelheiten des Aufbaues werden angegeben und an einigen Beispielen erläutert.
Die Primärwirkung von Röntgenstrahlen einer Dosis von etwa 0,5 bis 150 Millionen r auf die kristallisierte Trockensubstanz von Aminosäuren und Peptiden wurde mit Hilfe chemischer, biochemischer und physikalisch-chemischer Arbeitsmethoden untersucht. Es wurde gefunden, daß in allen prinzipiell möglichen Fällen folgende Reaktionen stets wiederkehren : Aminbildung infolge Decarboxylierung; Bildung einer α-Imino- bzw. α-Ketocarbonsäure infolge einer Dehydrierung in α-,β-Stellung; Bildung von β,γ- bzw. γ-δ-ungesättigten α-Aminocarbonsäuren oder deren γ- bzw. δ-Lactonen; Bruch und Vernetzung der aliphatischen Kohlenstoffketten. Bei Peptiden treten die gleichen Reaktionen wie bei den Aminosäuren auf, jedoch in einem anderen Verhältnis; hinzu kommt die strahlenchemische Dehydrierung einer Peptidbindung an der Aminogruppe zu einer energiereichen Iminoacyl-Bindung, welche bei Gegenwart von Wasser sofort hydrolysiert wird. Endprodukt namentlich bei längerkettigen Peptiden : Zwei Bruchstücke (daneben NH3); das eine mit alter amino-endständiger und neuer carboxyl-endständiger Aminosäure und das andere mit der alten carboxy-endständigen Aminosäure, statt Aminogruppe am anderen Ende jetzt Ketogruppe. — In fast allen Fällen wurden die Ionenausbeuten auch quantitativ bestimmt. Die lonenausbeuten für die Bildung von α-Ketosäuren aus α-Aminosäuren fallen exponentiell mit der eingestrahlten Dosis. Eine relativ einfache Funktion erklärt diese Verhältnisse. Die Ionenausbeute für die Bildung von Brenztraubensäure aus Serin ist dagegen unabhängig von der Dosis.
I. X-irradiation of isolated rat diaphragm with 10 to 200 kr produces a change in tissue metabolism which we schematize in two successive phases:
1st phase: Increase of oxygen comsumption, proportional to the dosage; an even greater increase of CO2 production; QCO2/QO2 > 1, that is, aerobic glycolysis; inhibition of anaerobic glycolysis.
2nd phase: Reduction of oxygen consumption, proportional to the dosage (over 65 kr the Qo2 decreases below the control); an even greater decrease of CO2 production: QCO2/QO2 > 1; a greater inhibition of anaerobic glycolysis.
With 200 kr or more no increase of respiration appears, but instead from the beginning there is a reduction of the metabolism as described in the second phase.
II. A similar effect is found in rat liver and in frog heart tissue.
III. When the tissue was incubated in the homologus serum no change in the quality of the described effect was observed. Under our experimental conditions the tissue was X-irradiated within a small quantity of incubation medium and immediately afterwards placed in a fresh medium; this limits the effect of oxidative radicals (arising in the X-irradiated water) upon the tissue.
IV. We set forth the experimental hypothesis that all the described changes in the metabolism of the cell after X-irradiation depend upon a primary alteration of electrolyte balance in the cell, especially of the potassium/sodium relationship. The well known decrease of glycolysis after X-irradiation is a consequence of the loss of potassium from the X-irradiated cell.
Die Primärwirkung von Röntgenstrahlung einer Dosis von 2 — 30 Millionen r auf kristallisiertes Lysozym wurde mit Hilfe physikalisch-chemischer (Elektrophorese, Ultrazentrifuge), chemischer, biochemischer und biologischer Arbeitsmethoden untersucht. Es wurde gefunden, daß durch Bestrahlung eine Reihe nah verwandter, jedoch weniger basischer Proteine verschiedenen Mol.-Gew. entsteht, deren Aminosäure-Bausteine als Folge der Bestrahlung teilweise in andere Verbindungen umgewandelt wurden. Bei der Untersuchung der amino- und carboxyl-endständigen Aminosäuren des bestrahlten Proteins wurden Unterschiede gegenüber Lysozym nur bei den carboxyl-terminalen Gruppen festgestellt. Die biologische Aktivität des Proteins blieb auch nach Bestrahlung mit einer Dosis von 5 Millionen r praktisch unverändert.
Die indirekte Wirkung von Röntgenstrahlen einer Dosis von 0,08 bis 7.5 Millionen r auf eine 2 · 10-2-m. wäßrige Tryptophanlösung wurde mit Hilfe chemischer und physikalisch-chemischer Arbeitsmethoden (Hochspannungs-Elektrophorese) untersucht.
Der Einfluß der Dosis, Dosisleistung, Temperatur und die durch oxydierend wirkende Radikale (HO2) ausgelösten Reaktionsschritte bei Bestrahlung in Sauerstoffatmosphäre wurden in qualitativer Hinsicht geprüft.
Aus der Vielzahl der strahlenchemisch gebildeten Abbauprodukte konnten mit Sicherheit nachgewiesen werden: Glycin, α-Alanin, Asparaginsäure, Kynurenin, 3-Hydroxykynurenin, 3-Oxyanthranilsäure und Tryptamin.
Schließlich wurde versucht, die experimentell gewonnenen Ergebnisse mit Hilfe von Radikalwirkungen, die sich auf
a) Decarboxylierungen
b) und Veränderungen des Kohlenstoff-Gerüstes beziehen, zu deuten.
Elektronenresonanz-Untersuchungen von Nachreaktionen in einem röntgenbestrahlten Faserprotein
(1962)
Of the 16 non-structural proteins (Nsps) encoded by SARS CoV-2, Nsp3 is the largest and plays important roles in the viral life cycle. Being a large, multidomain, transmembrane protein, Nsp3 has been the most challenging Nsp to characterize. Encoded within Nsp3 is the papain-like protease domain (PLpro) that cleaves not only the viral polypeptide but also K48-linked polyubiquitin and the ubiquitin-like modifier, ISG15, from host cell proteins. We here compare the interactors of PLpro and Nsp3 and find a largely overlapping interactome. Intriguingly, we find that near full length Nsp3 is a more active protease compared to the minimal catalytic domain of PLpro. Using a MALDI-TOF based assay, we screen 1971 approved clinical compounds and identify five compounds that inhibit PLpro with IC50s in the low micromolar range but showed cross reactivity with other human deubiquitinases and had no significant antiviral activity in cellular SARS-CoV-2 infection assays. We therefore looked for alternative methods to block PLpro activity and engineered competitive nanobodies that bind to PLpro at the substrate binding site with nanomolar affinity thus inhibiting the enzyme. Our work highlights the importance of studying Nsp3 and provides tools and valuable insights to investigate Nsp3 biology during the viral infection cycle.
Riboswitch RNAs regulate gene expression by conformational changes induced by environmental conditions and specific ligand binding. The guanidine-II riboswitch is proposed to bind the small molecule guanidinium and to subsequently form a kissing loop interaction between the P1 and P2 hairpins. While an interaction was shown for isolated hairpins in crystallization and electron paramagnetic resonance experiments, an intrastrand kissing loop formation has not been demonstrated. Here, we report the first evidence of this interaction in cis in a ligand and Mg2+ dependent manner. Using single-molecule FRET spectroscopy and detailed structural information from coarse-grained simulations, we observe and characterize three interconvertible states representing an open and kissing loop conformation as well as a novel Mg2+ dependent state for the guanidine-II riboswitch from E. coli. The results further substantiate the proposed switching mechanism and provide detailed insight into the regulation mechanism for the guanidine-II riboswitch class. Combining single molecule experiments and coarse-grained simulations therefore provides a promising perspective in resolving the conformational changes induced by environmental conditions and to yield molecular insights into RNA regulation.
Maximum likelihood estimates of diffusion coefficients from single-particle tracking experiments
(2021)
Single-molecule localization microscopy allows practitioners to locate and track labeled molecules in biological systems. When extracting diffusion coefficients from the resulting trajectories, it is common practice to perform a linear fit on mean-squared-displacement curves. However, this strategy is suboptimal and prone to errors. Recently, it was shown that the increments between the observed positions provide a good estimate for the diffusion coefficient, and their statistics are well-suited for likelihood-based analysis methods. Here, we revisit the problem of extracting diffusion coefficients from single-particle tracking experiments subject to static noise and dynamic motion blur using the principle of maximum likelihood. Taking advantage of an efficient real-space formulation, we extend the model to mixtures of subpopulations differing in their diffusion coefficients, which we estimate with the help of the expectation–maximization algorithm. This formulation naturally leads to a probabilistic assignment of trajectories to subpopulations. We employ the theory to analyze experimental tracking data that cannot be explained with a single diffusion coefficient. We test how well a dataset conforms to the assumptions of a diffusion model and determine the optimal number of subpopulations with the help of a quality factor of known analytical distribution. To facilitate use by practitioners, we provide a fast open-source implementation of the theory for the efficient analysis of multiple trajectories in arbitrary dimensions simultaneously.
Nitrate is an abundant nutrient and electron acceptor throughout Earth’s biosphere. Virtually all nitrate in nature is produced by the oxidation of nitrite by the nitrite oxidoreductase (NXR) multiprotein complex. NXR is a crucial enzyme in the global biological nitrogen cycle, and is found in nitrite-oxidizing bacteria (including comammox organisms), which generate the bulk of the nitrate in the environment, and in anaerobic ammonium-oxidizing (anammox) bacteria which produce half of the dinitrogen gas in our atmosphere. However, despite its central role in biology and decades of intense study, no structural information on NXR is available. Here, we present a structural and biochemical analysis of the NXR from the anammox bacterium Kuenenia stuttgartiensis, integrating X-ray crystallography, cryo-electron tomography, helical reconstruction cryo-electron microscopy, interaction and reconstitution studies and enzyme kinetics. We find that NXR catalyses both nitrite oxidation and nitrate reduction, and show that in the cell, NXR is arranged in tubules several hundred nanometres long. We reveal the tubule architecture and show that tubule formation is induced by a previously unidentified, haem-containing subunit, NXR-T. The results also reveal unexpected features in the active site of the enzyme, an unusual cofactor coordination in the protein’s electron transport chain, and elucidate the electron transfer pathways within the complex.
Under natural conditions, the visual system often sees a given input repeatedly. This provides an opportunity to optimize processing of the repeated stimuli. Stimulus repetition has been shown to strongly modulate neuronal-gamma band synchronization, yet crucial questions remained open. Here we used magnetoencephalography in 30 human subjects and find that gamma decreases across ~10 repetitions and then increases across further repetitions, revealing plastic changes of the activated neuronal circuits. Crucially, changes induced by one stimulus did not affect responses to other stimuli, demonstrating stimulus specificity. Changes partially persisted when the inducing stimulus was repeated after 25 minutes of intervening stimuli. They were strongest in early visual cortex and increased interareal feedforward influences. Our results suggest that early visual cortex gamma synchronization enables adaptive neuronal processing of recurring stimuli. These and previously reported changes might be due to an interaction of oscillatory dynamics with established synaptic plasticity mechanisms.
Cysteine cross-linking in native membranes establishes the transmembrane architecture of Ire1
(2021)
The ER is a key organelle of membrane biogenesis and crucial for the folding of both membrane and secretory proteins. Sensors of the unfolded protein response (UPR) monitor the unfolded protein load in the ER and convey effector functions for maintaining ER homeostasis. Aberrant compositions of the ER membrane, referred to as lipid bilayer stress, are equally potent activators of the UPR. How the distinct signals from lipid bilayer stress and unfolded proteins are processed by the conserved UPR transducer Ire1 remains unknown. Here, we have generated a functional, cysteine-less variant of Ire1 and performed systematic cysteine cross-linking experiments in native membranes to establish its transmembrane architecture in signaling-active clusters. We show that the transmembrane helices of two neighboring Ire1 molecules adopt an X-shaped configuration independent of the primary cause for ER stress. This suggests that different forms of stress converge in a common, signaling-active transmembrane architecture of Ire1.
The structure and flexibility of RNA depends sensitively on the microenvironment. Using pulsed electron-electron double-resonance (PELDOR)/double electron-electron resonance (DEER) spectroscopy combined with advanced labeling techniques, we show that the structure of double-stranded RNA (dsRNA) changes upon internalization into Xenopus lævis oocytes. Compared to dilute solution, the dsRNA A-helix is more compact in cells. We recapitulate this compaction in a densely crowded protein solution. Atomic-resolution molecular dynamics simulations of dsRNA semi-quantitatively capture the compaction, and identify non-specific electrostatic interactions between proteins and dsRNA as a possible driver of this effect.
Funktionelle und strukturelle Charakterisierung von SLC-Transportern in eukaryotischen Systemen
(2018)
Die evolutionäre Voraussetzung für die Entwicklung komplexer, differenzierter Organismen bildet die Separierung der Zelle in Reaktionsräume, die so genannte Kompartimentierung. Das Prinzip der Kompartimentierung ermöglicht zahlreiche lebensnotwendige, biochemische Prozesse, wie die Konservierung von Energie durch Protonengradienten in der Atmungskette oder parallele, gegenläufige Stoffwechselwege. Zelluläre Kompartimente werden häufig durch Biomembranen gebildet, welche aus einer zweilagigen Lipidschicht bestehen. Lipidmoleküle in einer Zelle sind meistens amphipathisch, das bedeutet, sie bestehen aus einer polaren, hydrophilen Kopfgruppe und einem unpolaren, hydrophopen Ende (Abbildung 1). Die Lipidzusammensetzung in einer Biomembran ist sehr divers und unterscheidet sich in verschiedenen Organismen und Organellen. Phosphoglyceride bilden den Hauptbestandteil der Lipidschicht. Phosphoglyceride besteht aus einem Glycerin Rückgrat, welches an dem C1- und C2-Atom mit zwei Fettsäuren verestert und an dem C3-Atom mit einem Phosphorsäurediester verbunden ist. ...
High-resolution cryo-EM structures of respiratory complex I: Mechanism, assembly, and disease
(2019)
Respiratory complex I is a redox-driven proton pump, accounting for a large part of the electrochemical gradient that powers mitochondrial adenosine triphosphate synthesis. Complex I dysfunction is associated with severe human diseases. Assembly of the one-megadalton complex I in the inner mitochondrial membrane requires assembly factors and chaperones. We have determined the structure of complex I from the aerobic yeast Yarrowia lipolytica by electron cryo-microscopy at 3.2-Å resolution. A ubiquinone molecule was identified in the access path to the active site. The electron cryo-microscopy structure indicated an unusual lipid-protein arrangement at the junction of membrane and matrix arms that was confirmed by molecular simulations. The structure of a complex I mutant and an assembly intermediate provide detailed molecular insights into the cause of a hereditary complex I-linked disease and complex I assembly in the inner mitochondrial membrane.
Mechanistic understanding of dynamic membrane proteins such as transporters, receptors, and channels requires accurate depictions of conformational ensembles, and the manner in which they interchange as a function of environmental factors including substrates, lipids, and inhibitors. Spectroscopic techniques such as electron spin resonance (ESR) pulsed electron–electron double resonance (PELDOR), also known as double electron–electron resonance (DEER), provide a complement to atomistic structures obtained from x-ray crystallography or cryo-EM, since spectroscopic data reflect an ensemble and can be measured in more native solvents, unperturbed by a crystal lattice. However, attempts to interpret DEER data are frequently stymied by discrepancies with the structural data, which may arise due to differences in conditions, the dynamics of the protein, or the flexibility of the attached paramagnetic spin labels. Recently, molecular simulation techniques such as EBMetaD have been developed that create a conformational ensemble matching an experimental distance distribution while applying the minimal possible bias. Moreover, it has been proposed that the work required during an EBMetaD simulation to match an experimentally determined distribution could be used as a metric with which to assign conformational states to a given measurement. Here, we demonstrate the application of this concept for a sodium-coupled transport protein, BetP. Because the probe, protein, and lipid bilayer are all represented in atomic detail, the different contributions to the work, such as the extent of protein backbone movements, can be separated. This work therefore illustrates how ranking simulations based on EBMetaD can help to bridge the gap between structural and biophysical data and thereby enhance our understanding of membrane protein conformational mechanisms.
Coagulation factor XIII (FXIII) is a plasma-circulating heterotetrameric pro-transglutaminase complex that is composed of two catalytic FXIII-A and two protective/regulatory FXIII-B subunits. FXIII acts by forming covalent cross-links within a preformed fibrin clots to prevent its premature fibrinolysis. The FXIII-A subunit is known to have pleiotropic roles outside coagulation, but the FXIII-B subunit is a relatively unexplored entity, both structurally as well as functionally. Its discovered roles so far are limited to that of the carrier/regulatory protein of its partner FXIII-A subunit. In the present study, we have explored the co-presence of protein excipients in commercial FXIII plasma concentrate FibrogamminP by combination of protein purification and mass spectrometry-based verification. Complement factor H was one of the co-excipients observed in this analysis. This was followed by performing pull down assays from plasma in order to detect the putative novel interacting partners for the FXIII-B subunit. Complement system proteins, like complement C3 and complement C1q, were amongst the proteins that were pulled down. The only protein that was observed in both experimental set ups was alpha-2-macroglobulin, which might therefore be a putative interacting partner of the FXIII/FXIII-B subunit. Future functional investigations will be needed to understand the physiological significance of this association.
Type IV pili are flexible filaments on the surface of bacteria, consisting of a helical assembly of pilin proteins. They are involved in bacterial motility (twitching), surface adhesion, biofilm formation and DNA uptake (natural transformation). Here, we use cryo-electron microscopy and mass spectrometry to show that the bacterium Thermus thermophilus produces two forms of type IV pilus ("wide" and "narrow"), differing in structure and protein composition. Wide pili are composed of the major pilin PilA4, while narrow pili are composed of a so-far uncharacterized pilin which we name PilA5. Functional experiments indicate that PilA4 is required for natural transformation, while PilA5 is important for twitching motility.
Potassium homeostasis is vital for all organisms, but is challenging in single-celled organisms like bacteria and yeast and immobile organisms like plants that constantly need to adapt to changing external conditions. KUP transporters facilitate potassium uptake by the co-transport of protons. Here, we uncover the molecular basis for transport in this widely distributed family. We identify the potassium importer KimA from Bacillus subtilis as a member of the KUP family, demonstrate that it functions as a K+/H+ symporter and report a 3.7 Å cryo-EM structure of the KimA homodimer in an inward-occluded, trans-inhibited conformation. By introducing point mutations, we identify key residues for potassium and proton binding, which are conserved among other KUP proteins.
The plasma membrane (PM) is composed of a complex lipid mixture that forms heterogeneous membrane environments. Yet, how small-scale lipid organization controls physiological events at the PM remains largely unknown. Here, we show that ORP-related Osh lipid exchange proteins are critical for the synthesis of phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2], a key regulator of dynamic events at the PM. In real-time assays, we find that unsaturated phosphatidylserine (PS) and sterols, both Osh protein ligands, synergistically stimulate phosphatidylinositol 4-phosphate 5-kinase (PIP5K) activity. Biophysical FRET analyses suggest an unconventional co-distribution of unsaturated PS and phosphatidylinositol 4-phosphate (PI4P) species in sterol-containing membrane bilayers. Moreover, using in vivo imaging approaches and molecular dynamics simulations, we show that Osh protein-mediated unsaturated PI4P and PS membrane lipid organization is sensed by the PIP5K specificity loop. Thus, ORP family members create a nanoscale membrane lipid environment that drives PIP5K activity and PI(4,5)P2 synthesis that ultimately controls global PM organization and dynamics.
Hydride transfers play a crucial role in a multitude of biological redox reactions and are mediated by flavin, deazaflavin or nicotinamide adenine dinucleotide cofactors at standard redox potentials ranging from 0 to –340 mV. 2-Naphthoyl-CoA reductase, a key enzyme of oxygen-independent bacterial naphthalene degradation, uses a low-potential one-electron donor for the two-electron dearomatization of its substrate below the redox limit of known biological hydride transfer processes at E°’ = −493 mV. Here we demonstrate by X-ray structural analyses, QM/MM computational studies, and multiple spectroscopy/activity based titrations that highly cooperative electron transfer (n = 3) from a low-potential one-electron (FAD) to a two-electron (FMN) transferring flavin cofactor is the key to overcome the resonance stabilized aromatic system by hydride transfer in a highly hydrophobic pocket. The results evidence how the protein environment inversely functionalizes two flavins to switch from low-potential one-electron to hydride transfer at the thermodynamic limit of flavin redox chemistry.
Rhodopsins are the most universal biological light-energy transducers and abundant phototrophic mechanisms that evolved on Earth and have a remarkable diversity and potential for biotechnological applications. Recently, the first sodium-pumping rhodopsin KR2 from Krokinobacter eikastus was discovered and characterized. However, the existing structures of KR2 are contradictory, and the mechanism of Na+ pumping is not yet understood. Here, we present a structure of the cationic (non H+) light-driven pump at physiological pH in its pentameric form. We also present 13 atomic structures and functional data on the KR2 and its mutants, including potassium pumps, which show that oligomerization of the microbial rhodopsin is obligatory for its biological function. The studies reveal the structure of KR2 at nonphysiological low pH where it acts as a proton pump. The structure provides new insights into the mechanisms of microbial rhodopsins and opens the way to a rational design of novel cation pumps for optogenetics.
Mechanism of the electroneutral sodium/proton antiporter PaNhaP from transition-path shooting
(2019)
Na+/H+ antiporters exchange sodium ions and protons on opposite sides of lipid membranes. The electroneutral Na+/H+ antiporter NhaP from archaea Pyrococcus abyssi (PaNhaP) is a functional homolog of the human Na+/H+ exchanger NHE1, which is an important drug target. Here we resolve the Na+ and H+ transport cycle of PaNhaP by transition-path sampling. The resulting molecular dynamics trajectories of repeated ion transport events proceed without bias force, and overcome the enormous time-scale gap between seconds-scale ion exchange and microseconds simulations. The simulations reveal a hydrophobic gate to the extracellular side that opens and closes in response to the transporter domain motion. Weakening the gate by mutagenesis makes the transporter faster, suggesting that the gate balances competing demands of fidelity and efficiency. Transition-path sampling and a committor-based reaction coordinate optimization identify the essential motions and interactions that realize conformational alternation between the two access states in transporter function.
Electron cryo-microscopy analyzes the structure of proteins and protein complexes in vitrified solution. Proteins tend to adsorb to the air-water interface in unsupported films of aqueous solution, which can result in partial or complete denaturation. We investigated the structure of yeast fatty acid synthase at the air-water interface by electron cryo-tomography and single-particle image processing. Around 90% of complexes adsorbed to the air-water interface are partly denatured. We show that the unfolded regions face the air-water interface. Denaturation by contact with air may happen at any stage of specimen preparation. Denaturation at the air-water interface is completely avoided when the complex is plunge-frozen on a substrate of hydrophilized graphene.
Mitochondrial ATP synthases form dimers, which assemble into long ribbons at the rims of the inner membrane cristae. We reconstituted detergent-purified mitochondrial ATP synthase dimers from the green algae Polytomella sp. and the yeast Yarrowia lipolytica into liposomes and examined them by electron cryotomography. Tomographic volumes revealed that ATP synthase dimers from both species self-assemble into rows and bend the lipid bilayer locally. The dimer rows and the induced degree of membrane curvature closely resemble those in the inner membrane cristae. Monomers of mitochondrial ATP synthase reconstituted into liposomes do not bend membrane visibly and do not form rows. No specific lipids or proteins other than ATP synthase dimers are required for row formation and membrane remodelling. Long rows of ATP synthase dimers are a conserved feature of mitochondrial inner membranes. They are required for cristae formation and a main factor in mitochondrial morphogenesis.
F1Fo‐ATP synthase is one of the best studied macromolecular machines in nature. It can be inhibited by a range of small molecules, which include the polyphenols, resveratrol and piceatannol. Here, we introduce Photoswitchable Inhibitors of ATP Synthase, termed PIAS, which were synthetically derived from these polyphenols. They can be used to reversibly control the enzymatic activity of purified yeast Yarrowia lipolyticaATP synthase by light. Our experiments indicate that the PIAS bind to the same site in the ATP synthase F1 complex as the polyphenols in their trans form, but they do not bind in their cis form. The PIAS could be useful tools for the optical precision control of ATP synthase in a variety of biochemical and biotechnological applications.
Lunapark (Lnp) is a conserved membrane protein that localizes to and stabilizes three-way junctions of the tubular ER network. In higher eukaryotes, phosphorylation of Lnp may contribute to the conversion of the ER from tubules to sheets during mitosis. Here, we report on the reconstitution of purified Lnp with phospholipids. Surprisingly, Lnp induces the formation of stacked membrane discs. Each disc is a bicelle, with Lnp sitting in the bilayer facing both directions. The interaction between bicelles is mediated by the cytosolic domains of Lnp, resulting in a constant distance between the discs. A phosphomimetic Lnp mutant shows reduced bicelle stacking. Based on these results, we propose that Lnp tethers ER membranes in vivo in a cell cycle–dependent manner. Lnp appears to be the first membrane protein that induces the formation of stacked bicelles.
Mitochondrial complex I has a key role in cellular energy metabolism, generating a major portion of the proton motive force that drives aerobic ATP synthesis. The hydrophilic arm of the L-shaped ~1 MDa membrane protein complex transfers electrons from NADH to ubiquinone, providing the energy to drive proton pumping at distant sites in the membrane arm. The critical steps of energy conversion are associated with the redox chemistry of ubiquinone. We report the cryo-EM structure of complete mitochondrial complex I from the aerobic yeast Yarrowia lipolytica both in the deactive form and after capturing the enzyme during steady-state activity. The site of ubiquinone binding observed during turnover supports a two-state stabilization change mechanism for complex I.
The electron transferring flavoprotein/butyryl-CoA dehydrogenase (EtfAB/Bcd) catalyzes the reduction of one crotonyl-CoA and two ferredoxins by two NADH within a flavin-based electron-bifurcating process. Here we report on the X-ray structure of the Clostridium difficile (EtfAB/Bcd)4 complex in the dehydrogenase-conducting D-state, α-FAD (bound to domain II of EtfA) and δ-FAD (bound to Bcd) being 8 Å apart. Superimposing Acidaminococcus fermentans EtfAB onto C. difficile EtfAB/Bcd reveals a rotation of domain II of nearly 80°. Further rotation by 10° brings EtfAB into the bifurcating B-state, α-FAD and β-FAD (bound to EtfB) being 14 Å apart. This dual binding mode of domain II, substantiated by mutational studies, resembles findings in non-bifurcating EtfAB/acyl-CoA dehydrogenase complexes. In our proposed mechanism, NADH reduces β-FAD, which bifurcates. One electron goes to ferredoxin and one to α-FAD, which swings over to reduce δ-FAD to the semiquinone. Repetition affords a second reduced ferredoxin and δ-FADH−, which reduces crotonyl-CoA.
Complex I couples the free energy released from quinone (Q) reduction to pump protons across the biological membrane in the respiratory chains of mitochondria and many bacteria. The Q reduction site is separated by a large distance from the proton-pumping membrane domain. To address the molecular mechanism of this long-range proton-electron coupling, we perform here full atomistic molecular dynamics simulations, free energy calculations, and continuum electrostatics calculations on complex I from Thermus thermophilus. We show that the dynamics of Q is redox-state-dependent, and that quinol, QH2, moves out of its reduction site and into a site in the Q tunnel that is occupied by a Q analog in a crystal structure of Yarrowia lipolytica. We also identify a second Q-binding site near the opening of the Q tunnel in the membrane domain, where the Q headgroup forms strong interactions with a cluster of aromatic and charged residues, while the Q tail resides in the lipid membrane. We estimate the effective diffusion coefficient of Q in the tunnel, and in turn the characteristic time for Q to reach the active site and for QH2 to escape to the membrane. Our simulations show that Q moves along the Q tunnel in a redox-state-dependent manner, with distinct binding sites formed by conserved residue clusters. The motion of Q to these binding sites is proposed to be coupled to the proton-pumping machinery in complex I.
Regulation of protein turnover allows cells to react to their environment and maintain homeostasis. Proteins can show different turnover rates in different tissue, but little is known about protein turnover in different brain cell types. We used dynamic SILAC to determine half-lives of over 5100 proteins in rat primary hippocampal cultures as well as in neuron-enriched and glia-enriched cultures ranging from <1 to >20 days. In contrast to synaptic proteins, membrane proteins were relatively shorter-lived and mitochondrial proteins were longer-lived compared to the population. Half-lives also correlate with protein functions and the dynamics of the complexes they are incorporated in. Proteins in glia possessed shorter half-lives than the same proteins in neurons. The presence of glia sped up or slowed down the turnover of neuronal proteins. Our results demonstrate that both the cell-type of origin as well as the nature of the extracellular environment have potent influences on protein turnover.
Electron transfer in respiratory chains generates the electrochemical potential that serves as energy source for the cell. Prokaryotes can use a wide range of electron donors and acceptors and may have alternative complexes performing the same catalytic reactions as the mitochondrial complexes. This is the case for the alternative complex III (ACIII), a quinol:cytochrome c/HiPIP oxidoreductase. In order to understand the catalytic mechanism of this respiratory enzyme, we determined the structure of ACIII from Rhodothermus marinus at 3.9 Å resolution by single-particle cryo-electron microscopy. ACIII presents a so-far unique structure, for which we establish the arrangement of the cofactors (four iron–sulfur clusters and six c-type hemes) and propose the location of the quinol-binding site and the presence of two putative proton pathways in the membrane. Altogether, this structure provides insights into a mechanism for energy transduction and introduces ACIII as a redox-driven proton pump.
Visualization of cytosolic ribosomes on the surface of mitochondria by electron cryo‐tomography
(2017)
We employed electron cryo‐tomography to visualize cytosolic ribosomes on the surface of mitochondria. Translation‐arrested ribosomes reveal the clustered organization of the TOM complex, corroborating earlier reports of localized translation. Ribosomes are shown to interact specifically with the TOM complex, and nascent chain binding is crucial for ribosome recruitment and stabilization. Ribosomes are bound to the membrane in discrete clusters, often in the vicinity of the crista junctions. This interaction highlights how protein synthesis may be coupled with transport. Our work provides unique insights into the spatial organization of cytosolic ribosomes on mitochondria.
We used electron cryo-tomography and subtomogram averaging to investigate the structure of complex I and its supramolecular assemblies in the inner mitochondrial membrane of mammals, fungi, and plants. Tomographic volumes containing complex I were averaged at ∼4 nm resolution. Principal component analysis indicated that ∼60% of complex I formed a supercomplex with dimeric complex III, while ∼40% were not associated with other respiratory chain complexes. The mutual arrangement of complex I and III2 was essentially conserved in all supercomplexes investigated. In addition, up to two copies of monomeric complex IV were associated with the complex I1III2 assembly in bovine heart and the yeast Yarrowia lipolytica, but their positions varied. No complex IV was detected in the respiratory supercomplex of the plant Asparagus officinalis. Instead, an ∼4.5-nm globular protein density was observed on the matrix side of the complex I membrane arm, which we assign to γ-carbonic anhydrase. Our results demonstrate that respiratory chain supercomplexes in situ have a conserved core of complex I and III2, but otherwise their stoichiometry and structure varies. The conserved features of supercomplex assemblies indicate an important role in respiratory electron transfer.
Na+/H+ antiporters are located in the cytoplasmic and intracellular membranes and play crucial roles in regulating intracellular pH, Na+, and volume. The NhaA antiporter of Escherichia coli is the best studied member of the Na+/H+ exchanger family and a model system for all related Na+/H+ exchangers, including eukaryotic representatives. Several amino acid residues are important for the transport activity of NhaA, including Lys-300, a residue that has recently been proposed to carry one of the two H+ ions that NhaA exchanges for one Na+ ion during one transport cycle. Here, we sought to characterize the effects of mutating Lys-300 of NhaA to amino acid residues containing side chains of different polarity and length (i.e. Ala, Arg, Cys, His, Glu, and Leu) on transporter stability and function. Salt resistance assays, acridine-orange fluorescence dequenching, solid supported membrane-based electrophysiology, and differential scanning fluorometry were used to characterize Na+ and H+ transport, charge translocation, and thermal stability of the different variants. These studies revealed that NhaA could still perform electrogenic Na+/H+ exchange even in the absence of a protonatable residue at the Lys-300 position. However, all mutants displayed lower thermal stability and reduced ion transport activity compared with the wild-type enzyme, indicating the critical importance of Lys-300 for optimal NhaA structural stability and function. On the basis of these experimental data, we propose a tentative mechanism integrating the functional and structural role of Lys-300.
In dieser Arbeit wurde das Protein OR1 ausführlich charakterisiert und die Grundlage für weitere Studien an diesem Protein gelegt. Die Zielsetzung dieser Arbeit bestand primär in der biophysikalischen Analyse eines eukaryotischen Proteorhodopsins, da bislang keine Daten zu diesen vorlagen. Dieser Ansatz ist vergleichbar mit der Studie am BR ähnlichen Rhodopsin aus dem Pilz Leptosphaeria maculans (Waschuk et al. 2005). Auch wenn man aus den Eigenschaften von OR1 keine Signatur für eukaryotische PRs herausfiltern kann, so weist OR1 eine Reihe von Charakteristika auf, die es wert sind weiterbearbeitet zu werden. Zu den hervorzuhebenden Ergebnissen dieser Arbeit zählen:
(1) OR1 zeigte in der methylotrophen Hefe Pichia pastoris ein hohes Expressionsniveau weit über der gewohnten Ausbeute bei Membranproteinen.
(2) OR1 offenbarte sich als Proteorhodopsin mit BR ähnlichen Eigenschaften wie dem niedrigen pKs-Wert des Protonenakzeptors und damit guten Protonenpumpeigenschaften über einen großen pH-Bereich. Auch bindet OR1 keinen zweiten Chromophor, was die nahen Verwandten GR und XR hingegen tun.
(3) OR1 demonstriert, dass die Konfiguration des komplexen Gegenions von Proteorhodopsinen stark variiert und sich anscheinend flexibel den physiologischen Erfordernissen des jeweiligen Organismus anpasst. In diesem Zusammenhang spielt auch das konservierte Histidin eine Rolle, da es den primären Protonenakzeptor beeinflusst. Bei OR1 stellte sich heraus, dass das Histidin den pKs Wert der D100 Position nicht signifikant beeinflusst.
(4) OR1 wurde mit 13C und 15N Atomen erfolgreich markiert und das entwickelte Protokoll für die Rekonstitution bewährte sich. Die Proteoliposomen des Wildtyps gaben sehr gut aufgelöste Festkörper-NMR Spektren. In Zukunft sind somit ausführliche NMR Studien an OR1 möglich.
CryoEM structures of membrane pore and prepore complex reveal cytolytic mechanism of Pneumolysin
(2017)
Many pathogenic bacteria produce pore-forming toxins to attack and kill human cells. We have determined the 4.5 Å structure of the ~2.2 MDa pore complex of pneumolysin, the main virulence factor of Streptococcus pneumoniae, by cryoEM. The pneumolysin pore is a 400 Å ring of 42 membrane-inserted monomers. Domain 3 of the soluble toxin refolds into two ~85 Å β-hairpins that traverse the lipid bilayer and assemble into a 168-strand β-barrel. The pore complex is stabilized by salt bridges between β-hairpins of adjacent subunits and an internal α-barrel. The apolar outer barrel surface with large sidechains is immersed in the lipid bilayer, while the inner barrel surface is highly charged. Comparison of the cryoEM pore complex to the prepore structure obtained by electron cryo-tomography and the x-ray structure of the soluble form reveals the detailed mechanisms by which the toxin monomers insert into the lipid bilayer to perforate the target membrane.
β-barrel proteins mediate nutrient uptake in bacteria and serve vital functions in cell signaling and adhesion. For the 14-strand outer membrane protein G of Escherichia coli, opening and closing is pH-dependent. Different roles of the extracellular loops in this process were proposed, and X-ray and solution NMR studies were divergent. Here, we report the structure of outer membrane protein G investigated in bilayers of E. coli lipid extracts by magic-angle-spinning NMR. In total, 1847 inter-residue 1H–1H and 13C–13C distance restraints, 256 torsion angles, but no hydrogen bond restraints are used to calculate the structure. The length of β-strands is found to vary beyond the membrane boundary, with strands 6–8 being the longest and the extracellular loops 3 and 4 well ordered. The site of barrel closure at strands 1 and 14 is more disordered than most remaining strands, with the flexibility decreasing toward loops 3 and 4. Loop 4 presents a well-defined helix.
Secretins form multimeric channels across the outer membrane of Gram-negative bacteria that mediate the import or export of substrates and/or extrusion of type IV pili. The secretin complex of Thermus thermophilus is an oligomer of the 757-residue PilQ protein, essential for DNA uptake and pilus extrusion. Here, we present the cryo-EM structure of this bifunctional complex at a resolution of ~7 Å using a new reconstruction protocol. Thirteen protomers form a large periplasmic domain of six stacked rings and a secretin domain in the outer membrane. A homology model of the PilQ protein was fitted into the cryo-EM map. A crown-like structure outside the outer membrane capping the secretin was found not to be part of PilQ. Mutations in the secretin domain disrupted the crown and abolished DNA uptake, suggesting a central role of the crown in natural transformation.
Ion channel gating is essential for cellular homeostasis and is tightly controlled. In some eukaryotic and most bacterial ligand-gated K+ channels, RCK domains regulate ion fluxes. Until now, a single regulatory mechanism has been proposed for all RCK-regulated channels, involving signal transduction from the RCK domain to the gating area. Here, we present an inactive ADP-bound structure of KtrAB from Vibrio alginolyticus, determined by cryo-electron microscopy, which, combined with EPR spectroscopy and molecular dynamics simulations, uncovers a novel regulatory mechanism for ligand-induced action at a distance. Exchange of activating ATP to inactivating ADP triggers short helical segments in the K+-translocating KtrB dimer to organize into two long helices that penetrate deeply into the regulatory RCK domains, thus connecting nucleotide-binding sites and ion gates. As KtrAB and its homolog TrkAH have been implicated as bacterial pathogenicity factors, the discovery of this functionally relevant inactive conformation may advance structure-guided drug development.
Methanogenic archaea share one ion gradient forming reaction in their energy metabolism catalyzed by the membrane-spanning multisubunit complex N5-methyl-tetrahydromethanopterin: coenzyme M methyltransferase (MtrABCDEFGH or simply Mtr). In this reaction the methyl group transfer from methyl-tetrahydromethanopterin to coenzyme M mediated by cobalamin is coupled with the vectorial translocation of Na+ across the cytoplasmic membrane. No detailed structural and mechanistic data are reported about this process. In the present work we describe a procedure to provide a highly pure and homogenous Mtr complex on the basis of a selective removal of the only soluble subunit MtrH with the membrane perturbing agent dimethyl maleic anhydride and a subsequent two-step chromatographic purification. A molecular mass determination of the Mtr complex by laser induced liquid bead ion desorption mass spectrometry (LILBID-MS) and size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) resulted in a (MtrABCDEFG)3 heterotrimeric complex of ca. 430 kDa with both techniques. Taking into account that the membrane protein complex contains various firmly bound small molecules, predominantly detergent molecules, the stoichiometry of the subunits is most likely 1:1. A schematic model for the subunit arrangement within the MtrABCDEFG protomer was deduced from the mass of Mtr subcomplexes obtained by harsh IR-laser LILBID-MS.
The widespread application of human stem-cell-derived neurons for functional studies is impeded by complicated differentiation protocols, immaturity, and deficient optogene expression as stem cells frequently lose transgene expression over time. Here we report a simple but precise Cre-loxP-based strategy for generating conditional, and thereby stable, optogenetic human stem-cell lines. These cells can be easily and efficiently differentiated into functional neurons, and optogene expression can be triggered by administering Cre protein to the cultures. This conditional expression system may be applied to stem-cell-derived neurons whenever timed transgene expression could help to overcome silencing at the stem-cell level.
The inner boundary and the cristae membrane are connected by pore-like structures termed crista junctions (CJs). The MICOS complex is required for CJ formation and enriched at CJs. Here, we address the roles of the MICOS subunits Mic27 and Mic10. We observe a positive genetic interaction between Mic27 and Mic60 and deletion of Mic27 results in impaired formation of CJs and altered cristae membrane curvature. Mic27 acts in an antagonistic manner to Mic60 as it promotes oligomerization of the F1FO-ATP synthase and partially restores CJ formation in cells lacking Mic60. Mic10 impairs oligomerization of the F1FO-ATP synthase similar to Mic60. Applying complexome profiling, we observed that deletion of Mic27 destabilizes the MICOS complex but does not impair formation of a high molecular weight Mic10 subcomplex. Moreover, this Mic10 subcomplex comigrates with the dimeric F1FO-ATP synthase in a Mic27-independent manner. Further, we observed a chemical crosslink of Mic10 to Mic27 and of Mic10 to the F1FO-ATP synthase subunit e. We corroborate the physical interaction of the MICOS complex and the F1FO-ATP synthase. We propose a model in which part of the F1FO-ATP synthase is linked to the MICOS complex via Mic10 and Mic27 and by that is regulating CJ formation.
Fusion of mitochondrial outer membranes is crucial for proper organelle function and involves large GTPases called mitofusins. The discrete steps that allow mitochondria to attach to one another and merge their outer membranes are unknown. By combining an in vitro mitochondrial fusion assay with electron cryo-tomography (cryo-ET), we visualize the junction between attached mitochondria isolated from Saccharomyces cerevisiae and observe complexes that mediate this attachment. We find that cycles of GTP hydrolysis induce progressive formation of a docking ring structure around extended areas of contact. Further GTP hydrolysis triggers local outer membrane fusion at the periphery of the contact region. These findings unravel key features of mitofusin-dependent fusion of outer membranes and constitute an important advance in our understanding of how mitochondria connect and merge.
Respirasomes are macromolecular assemblies of the respiratory chain complexes I, III and IV in the inner mitochondrial membrane. We determined the structure of supercomplex I1III2IV1 from bovine heart mitochondria by cryo-EM at 9 Å resolution. Most protein-protein contacts between complex I, III and IV in the membrane are mediated by supernumerary subunits. Of the two Rieske iron-sulfur cluster domains in the complex III dimer, one is resolved, indicating that this domain is immobile and unable to transfer electrons. The central position of the active complex III monomer between complex I and IV in the respirasome is optimal for accepting reduced quinone from complex I over a short diffusion distance of 11 nm, and delivering reduced cytochrome c to complex IV. The functional asymmetry of complex III provides strong evidence for directed electron flow from complex I to complex IV through the active complex III monomer in the mammalian supercomplex.
Ageing is a progressive decline of intrinsic physiological functions. We examined the impact of ageing on the ultrastructure and function of mitochondria in mouse and fruit flies (Drosophila melanogaster) by electron cryo-tomography and respirometry. We discovered distinct age-related changes in both model organisms. Mitochondrial function and ultrastructure are maintained in mouse heart, whereas subpopulations of mitochondria from mouse liver show age-related changes in membrane morphology. Subpopulations of mitochondria from young and old mouse kidney resemble those described for apoptosis. In aged flies, respiratory activity is compromised and the production of peroxide radicals is increased. In about 50% of mitochondria from old flies, the inner membrane organization breaks down. This establishes a clear link between inner membrane architecture and functional decline. Mitochondria were affected by ageing to very different extents, depending on the organism and possibly on the degree to which tissues within the same organism are protected against mitochondrial damage.
Na+/H+ exchange is essential for survival of all organisms, having a role in the regulation of the intracellular Na+ concentration, pH and cell volume. Furthermore, Na+/H+ exchangers were shown to be involved in the virulence of the bacterium Yersinia pestis, indicating they might be potential targets for novel antibiotic treatments. The model system for Na+/H+ exchangers is the NhaA transporter from Escherichia coli, EcNhaA. Therefore, the general transport mechanism of NhaA exchangers is currently well characterized. However, much less is known about NhaB exchangers, with only a limited number of studies available. The pathogen Klebsiella pneumoniae, which is a major source of nosocomial infection, possesses three electrogenic Na+/H+ exchangers, KpNhaA1, KpNhaA2 and KpNhaB, none of which have been previously investigated. Our aim in this study was to functionally characterize KpNhaB using solid supported membrane-based electrophysiology as the main investigation technique, and thus provide the first electrophysiological investigation of an NhaB Na+/H+ exchanger. We found that NhaB can be described by the same competition-based mechanism that was shown to be valid for electrogenic NhaA and NapA, and for electroneutral NhaP Na+/H+ exchangers. For comparison we also characterized the activity of KpNhaA1 and KpNhaA2 and found that the three exchangers have complementary activity profiles, which is likely a survival advantage for K. pneumoniae when faced with environments of different salinity and pH. This underlines their importance as potential antibiotic drug targets.
Proteins of the secretin family form large macromolecular complexes, which assemble in the outer membrane of Gram-negative bacteria. Secretins are major components of type II and III secretion systems and are linked to extrusion of type IV pili (T4P) and to DNA uptake. By electron cryo-tomography of whole Thermus thermophilus cells, we determined the in situ structure of a T4P molecular machine in the open and the closed state. Comparison reveals a major conformational change whereby the N-terminal domains of the central secretin PilQ shift by ∼30 Å, and two periplasmic gates open to make way for pilus extrusion. Furthermore, we determine the structure of the assembled pilus.
Differentiated neurons can be rapidly acquired, within days, by inducing stem cells to express neurogenic transcription factors. We developed a protocol to maintain long-term cultures of human neurons, called iNGNs, which are obtained by inducing Neurogenin-1 and Neurogenin-2 expression in induced pluripotent stem cells. We followed the functional development of iNGNs over months and they showed many hallmark properties for neuronal maturation, including robust electrical and synaptic activity. Using iNGNs expressing a variant of channelrhodopsin-2, called CatCh, we could control iNGN activity with blue light stimulation. In combination with optogenetic tools, iNGNs offer opportunities for studies that require precise spatial and temporal resolution. iNGNs developed spontaneous network activity, and these networks had excitatory glutamatergic synapses, which we characterized with single-cell synaptic recordings. AMPA glutamatergic receptor activity was especially dominant in postsynaptic recordings, whereas NMDA glutamatergic receptor activity was absent from postsynaptic recordings but present in extrasynaptic recordings. Our results on long-term cultures of iNGNs could help in future studies elucidating mechanisms of human synaptogenesis and neurotransmission, along with the ability to scale-up the size of the cultures.
An optochemokine tandem was developed to control the release of calcium from endosomes into the cytosol by light and to analyze the internalization kinetics of G-protein coupled receptors (GPCRs) by electrophysiology. A previously constructed rhodopsin tandem was re-engineered to combine the light-gated Ca2+-permeable cation channel Channelrhodopsin-2(L132C), CatCh, with the chemokine receptor CXCR4 in a functional tandem protein tCXCR4/CatCh. The GPCR was used as a shuttle protein to displace CatCh from the plasma membrane into intracellular areas. As shown by patch-clamp measurements and confocal laser scanning microscopy, heterologously expressed tCXCR4/CatCh was internalized via the endocytic SDF1/CXCR4 signaling pathway. The kinetics of internalization could be followed electrophysiologically via the amplitude of the CatCh signal. The light-induced release of Ca2+ by tandem endosomes into the cytosol via CatCh was visualized using the Ca2+-sensitive dyes rhod2 and rhod2-AM showing an increase of intracellular Ca2+ in response to light.
Maintenance of mitochondria is achieved by several mechanisms, including the regulation of mitochondrial proteostasis. The matrix protease CLPXP, involved in protein quality control, has been implicated in ageing and disease. However, particularly due to the lack of knowledge of CLPXP's substrate spectrum, only little is known about the pathways and mechanisms controlled by this protease. Here we report the first comprehensive identification of potential mitochondrial CLPXP in vivo interaction partners and substrates using a combination of tandem affinity purification and differential proteomics. This analysis reveals that CLPXP in the fungal ageing model Podospora anserina is mainly associated with metabolic pathways in mitochondria, e.g. components of the pyruvate dehydrogenase complex and the tricarboxylic acid cycle as well as subunits of electron transport chain complex I. These data suggest a possible function of mitochondrial CLPXP in the control and/or maintenance of energy metabolism. Since bioenergetic alterations are a common feature of neurodegenerative diseases, cancer, and ageing, our data comprise an important resource for specific studies addressing the role of CLPXP in these adverse processes.
Cytochrome c oxidases (CcOs), members of the heme-copper containing oxidase (HCO) superfamily, are the terminal enzymes of aerobic respiratory chains. The cbb3-type cytochrome c oxidases (cbb3-CcO) form the C-family and have only the central catalytic subunit in common with the A- and B-family HCOs. In Pseudomonas stutzeri, two cbb3 operons are organized in a tandem repeat. The atomic structure of the first cbb3 isoform (Cbb3-1) was determined at 3.2 Å resolution in 2010 (S. Buschmann, E. Warkentin, H. Xie, J. D. Langer, U. Ermler, and H. Michel, Science 329:327-330, 2010, http://dx.doi.org/10.1126/science.1187303). Unexpectedly, the electron density map of Cbb3-1 revealed the presence of an additional transmembrane helix (TMH) which could not be assigned to any known protein. We now identified this TMH as the previously uncharacterized protein PstZoBell_05036, using a customized matrix-assisted laser desorption ionization (MALDI)-tandem mass spectrometry setup. The amino acid sequence matches the electron density of the unassigned TMH. Consequently, the protein was renamed CcoM. In order to identify the function of this new subunit in the cbb3 complex, we generated and analyzed a CcoM knockout strain. The results of the biochemical and biophysical characterization indicate that CcoM may be involved in CcO complex assembly or stabilization. In addition, we found that CcoM plays a role in anaerobic respiration, as the ΔCcoM strain displayed altered growth rates under anaerobic denitrifying conditions.om Pseudomonas stutzeri, a bacterium closely related to the human pathogen Pseudomonas aeruginosa.
Background: In vertebrates, several anatomical regions located within the nasal cavity mediate olfaction. Among these, the main olfactory epithelium detects most conventional odorants. Olfactory sensory neurons, provided with cilia exposed to the air, detect volatile chemicals via an extremely large family of seven-transmembrane chemoreceptors named odorant receptors. Their genes are expressed in a monogenic and monoallelic fashion: a single allele of a single odorant receptor gene is transcribed in a given mature neuron, through a still uncharacterized molecular mechanism known as odorant receptor gene choice.
Aim: Odorant receptor genes are typically arranged in genomic clusters, but a few are isolated (we call them solitary) from the others within a region broader than 1 Mb upstream and downstream with respect to their transcript's coordinates. The study of clustered genes is problematic, because of redundancy and ambiguities in their regulatory elements: we propose to use the solitary genes as simplified models to understand odorant receptor gene choice.
Procedures: Here we define number and identity of the solitary genes in the mouse genome (C57BL/6J), and assess the conservation of the solitary status in some mammalian orthologs. Furthermore, we locate their putative promoters, predict their homeodomain binding sites (commonly present in the promoters of odorant receptor genes) and compare candidate promoter sequences with those of wild-caught mice. We also provide expression data from histological sections.
Results: In the mouse genome there are eight intact solitary genes: Olfr19 (M12), Olfr49, Olfr266, Olfr267, Olfr370, Olfr371, Olfr466, Olfr1402; five are conserved as solitary in rat. These genes are all expressed in the main olfactory epithelium of three-day-old mice. The C57BL/6J candidate promoter of Olfr370 has considerably varied compared to its wild-type counterpart. Within the putative promoter for Olfr266 a homeodomain binding site is predicted. As a whole, our findings favor Olfr266 as a model gene to investigate odorant receptor gene choice.
ATP synthases are multi-subunit membrane enzymes, which utilize the energy stored in a transmembrane electrochemical ion gradient to produce adenosine-5´-triphosphate (ATP), the universal energy carrier in biological systems. Research on these important enzymes goes back more than 50 years and has produced innumerable studies. The F-type ATP synthase consists of two functionally distinct, but tightly coupled subcomplexes, the water-soluble F1 and the membrane-embedded Fo complex. In its simplest form, F1 consists of five different subunits with a stoichiometry of α 3β3γδε, and harbors three catalytic centers in the α 3β3-headpiece, while Fo consists of three different subunits in a stoichiometry of ab2cn, where n varies between 8 to 15 depending on the species. From a mechanistic standpoint, the complex can also be divided into two different units, namely a stator, α3β3δ-ab2, and a rotor, γε-cn. The enzyme utilizes the energy stored in a transmembrane electrochemical gradient of protons, or in some cases Na+, to drive ATP synthesis. In particular, the downhill translocation of these ions across the Fo complex drives rotation of the γε-cn unit, which is then transduced to the active centers, catalyzing the phosphorylation of adenosine-5`-diphosphate (ADP) with inorganic phosphate (Pi), and the release of ATP....
Structure and regulatory interactions of the cytoplasmic terminal domains of serotonin transporter
(2014)
Uptake of neurotransmitters by sodium-coupled monoamine transporters of the NSS family is required for termination of synaptic transmission. Transport is tightly regulated by protein–protein interactions involving the small cytoplasmic segments at the amino- and carboxy-terminal ends of the transporter. Although structures of homologues provide information about the transmembrane regions of these transporters, the structural arrangement of the terminal domains remains largely unknown. Here, we combined molecular modeling, biochemical, and biophysical approaches in an iterative manner to investigate the structure of the 82-residue N-terminal and 30-residue C-terminal domains of human serotonin transporter (SERT). Several secondary structures were predicted in these domains, and structural models were built using the Rosetta fragment-based methodology. One-dimensional 1H nuclear magnetic resonance and circular dichroism spectroscopy supported the presence of helical elements in the isolated SERT N-terminal domain. Moreover, introducing helix-breaking residues within those elements altered the fluorescence resonance energy transfer signal between terminal cyan fluorescent protein and yellow fluorescent protein tags attached to full-length SERT, consistent with the notion that the fold of the terminal domains is relatively well-defined. Full-length models of SERT that are consistent with these and published experimental data were generated. The resultant models predict confined loci for the terminal domains and predict that they move apart during the transport-related conformational cycle, as predicted by structures of homologues and by the “rocking bundle” hypothesis, which is consistent with spectroscopic measurements. The models also suggest the nature of binding to regulatory interaction partners. This study provides a structural context for functional and regulatory mechanisms involving SERT terminal domains.
Channelrhodopsin-2 (ChR2) is a cation-selective light-gated channel from Chlamydomonas reinhardtii (Nagel G, Szellas T, Huhn W, Kateriya S, Adeishvili N, Berthold P, et al. Channelrhodopsin-2, a directly light-gated cation-selective membrane channel. Proc Natl Acad Sci USA 2003;100:13940-5), which has become a powerful tool in optogenetics. Two-dimensional crystals of the slow photocycling C128T ChR2 mutant were exposed to 473 nm light and rapidly frozen to trap the open state. Projection difference maps at 6Å resolution show the location, extent and direction of light-induced conformational changes in ChR2 during the transition from the closed state to the ion-conducting open state. Difference peaks indicate that transmembrane helices (TMHs) TMH2, TMH6 and TMH7 reorient or rearrange during the photocycle. No major differences were found near TMH3 and TMH4 at the dimer interface. While conformational changes in TMH6 and TMH7 are known from other microbial-type rhodopsins, our results indicate that TMH2 has a key role in light-induced channel opening and closing in ChR2.
Bacteria have adapted their NhaA Na(+)/H(+) exchangers responsible for salt homeostasis to their different habitats. We present an electrophysiological and kinetic analysis of NhaA from Helicobacter pylori and compare it to the previously investigated exchangers from Escherichia coli and Salmonella typhimurium. Properties of all three transporters are described by a simple model using a single binding site for H(+) and Na(+). We show that H.pylori NhaA only has a small acidic shift of its pH-dependent activity profile compared to the other transporters and discuss why a more drastic change in its pH activity profile is not physiologically required.
Na+/H+ antiporters are integral membrane proteins that are present in almost every cell and in every kingdom of life. They are essential for the regulation of intracellular pH-value, Na+-concentration and cell volume. These secondary active transporters exchange sodium ions against protons via an alternating access mechanism, which is not understood in full detail. Na+/H+ antiporters show distinct species-specific transport characteristics and regulatory properties that correlate with respective physiological functions. Here we present the characterization of the Na+/H+ antiporter NhaA from Salmonella enterica serovar Thyphimurium LT2, the causing agent of food-born human gastroenteritis and typhoid like infections. The recombinant antiporter was functional in vivo and in vitro. Expression of its gene complemented the Na+-sensitive phenotype of an E. coli strain that lacks the main Na+/H+ antiporters. Purified to homogeneity, the antiporter was a dimer in solution as accurately determined by size-exclusion chromatography combined with multi-angle laser-light scattering and refractive index monitoring. The purified antiporter was fully capable of electrogenic Na+(Li+)/H+-antiport when reconstituted in proteoliposomes and assayed by solid-supported membrane-based electrophysiological measurements. Transport activity was inhibited by 2-aminoperimidine. The recorded negative currents were in agreement with a 1Na+(Li+)/2H+ stoichiometry. Transport activity was low at pH 7 and up-regulation above this pH value was accompanied by a nearly 10-fold decrease of KmNa (16 mM at pH 8.5) supporting a competitive substrate binding mechanism. K+ does not affect Na+ affinity or transport of substrate cations, indicating that selectivity of the antiport arises from the substrate binding step. In contrast to homologous E. coli NhaA, transport activity remains high at pH values above 8.5. The antiporter from S. Typhimurium is a promising candidate for combined structural and functional studies to contribute to the elucidation of the mechanism of pH-dependent Na+/H+ antiporters and to provide insights in the molecular basis of species-specific growth and survival strategies.
The dependence of the Escherichia coli Na+H+ antiporter A (EcNhaA) pH sensor mutant E241C on H+ and Na+ concentrations was tested using a solid supported membrane (SSM) based electrophysiological approach. Proteoliposome preparations with right side out (RSO) oriented carriers were used to investigate the passive downhill uptake mode (physiologically the reverse transport mode) at zero membrane potential. Na+ concentration gradients established with a rapid solution exchange acted as the driving force. When a Na+ concentration gradient was established at symmetrical pH, the transport activity of the E241C EcNhaA variant was similar to that of the wildtype EcNhaA, with no shift of the bell-shaped pH dependence, an increase of the KmNa at acidic pH and a decrease of the KmNa at alkaline pH, supporting the model of a competitive binding of Na+ and H+ to a common binding site.
Poster presentation at 1st International Workshop on Odor Spaces.
Mice are exceptional in their ability to capture their chemical environment, mapping the olfactory world into a basic sensory representation with over one thousand different types of chemical sensors, that is, olfactory sensory neurons (OSNs). OSNs of each type converge in the olfactory bulb onto exclusive distinct physiological areas called glomeruli. The glomeruli constitute the first relay station of olfactory stimulus representation in the mouse brain. Thus, the stimulus induced glomerular input pattern spatially embodies an important part of the sensory representation in the olfactory bulb. Still, topographic organization principles (chemotopy, tunotopy) are under debate. One reason might be that investigation are, due to experimental limitations, only performed on stimuli sets in the size of one hundred odors. But this represents only a tiny snapshot of the vast amount of molecules in the olfactory world and topographic relationships might be disguised in the incomplete representation of molecular receptive ranges (MRR). Therefore we investigated the problem with the MOR18-2 glomerulus as point of reference: First we determined it's MRR. Then, based on a measurement set covering this MRR, we elucidated the topographic embedding. It shows that MOR18-2 is embedded in a hierarchy of patchy tunotopic domains.
pH and Na+ homeostasis in all cells requires Na+/H+ antiporters. The crystal structure, obtained at pH 4, of NhaA, the main antiporter of Escherichia coli, has provided general insights into an antiporter mechanism and its unique pH regulation. Here, we describe a general method to select various NhaA mutants from a library of randomly mutagenized NhaA. The selected mutants, A167P and F267C are described in detail. Both mutants are expressed in Escherichia coli EP432 cells at 70–95% of the wild type but grow on selective medium only at neutral pH, A167P on Li+ (0.1 M) and F267C on Na+ (0.6 M). Surprising for an electrogenic secondary transporter, and opposed to wild type NhaA, the rates of A167P and F267C are almost indifferent to membrane potential. Detailed kinetic analysis reveals that in both mutants the rate limiting step of the cation exchange cycle is changed from an electrogenic to an electroneutral reaction.
We examined substrate-induced conformational changes in MjNhaP1, an archaeal electroneutral Na+/H+-antiporter resembling the human antiporter NHE1, by electron crystallography of 2D crystals in a range of physiological pH and Na+ conditions. In the absence of sodium, changes in pH had no major effect. By contrast, changes in Na+ concentration caused a marked conformational change that was largely pH-independent. Crystallographically determined, apparent dissociation constants indicated ∼10-fold stronger Na+ binding at pH 8 than at pH 4, consistent with substrate competition for a common ion-binding site. Projection difference maps indicated helix movements by about 2 Å in the 6-helix bundle region of MjNhaP1 that is thought to contain the ion translocation site. We propose that these movements convert the antiporter from the proton-bound, outward-open state to the Na+-bound, inward-open state. Oscillation between the two states would result in rapid Na+/H+ antiport.
A new type of Na+-driven ATP synthase membrane rotor with a two-carboxylate ion-coupling motif
(2013)
Abstract: The anaerobic bacterium Fusobacterium nucleatum uses glutamate decarboxylation to generate a transmembrane gradient of Na+. Here, we demonstrate that this ion-motive force is directly coupled to ATP synthesis, via an F1Fo-ATP synthase with a novel Na+ recognition motif, shared by other human pathogens. Molecular modeling and free-energy simulations of the rotary element of the enzyme, the c-ring, indicate Na+ specificity in physiological settings. Consistently, activity measurements showed Na+ stimulation of the enzyme, either membrane-embedded or isolated, and ATP synthesis was sensitive to the Na+ ionophore monensin. Furthermore, Na+ has a protective effect against inhibitors targeting the ion-binding sites, both in the complete ATP synthase and the isolated c-ring. Definitive evidence of Na+ coupling is provided by two identical crystal structures of the c11 ring, solved by X-ray crystallography at 2.2 and 2.6 Å resolution, at pH 5.3 and 8.7, respectively. Na+ ions occupy all binding sites, each coordinated by four amino acids and a water molecule. Intriguingly, two carboxylates instead of one mediate ion binding. Simulations and experiments demonstrate that this motif implies that a proton is concurrently bound to all sites, although Na+ alone drives the rotary mechanism. The structure thus reveals a new mode of ion coupling in ATP synthases and provides a basis for drug-design efforts against this opportunistic pathogen.
Author Summary: Essential cellular processes such as biosynthesis, transport, and motility are sustained by the energy released in the hydrolysis of ATP, the universal energy carrier in living cells. Most ATP in the cell is produced by a membrane-bound enzyme, the ATP synthase, through a rotary mechanism that is coupled to the translocation of ions across the membrane. The majority of ATP synthases are energized by transmembrane electrochemical gradients of protons (proton-motive force), but a number of organisms, including some important human pathogens, use gradients of sodium ions instead (sodium-motive force). The ion specificity of ATP synthases is determined by a membrane-embedded sub-complex, the c-ring, which is the smallest known biological rotor. The functional mechanism of the rotor ring and its variations among different organisms are of wide interest, because of this enzyme's impact on metabolism and disease, and because of its potential for nanotechnology applications. Here, we characterize a previously unrecognized type of Na+-driven ATP synthase from the opportunistic human pathogen Fusobacterium nucleatum, which is implicated in periodontal diseases. We analyzed this ATP synthase and its rotor ring through a multi-disciplinary approach, combining cell-growth and biochemical assays, X-ray crystallography and computer-simulation methods. Two crystal structures of the membrane rotor were solved, at low and high pH, revealing an atypical ion-recognition motif mediated by two carboxylate side-chains. This motif is shared by other human pathogens, such as Mycobacterium tuberculosis or Streptococcus pneumonia, whose ATP synthases are targets of novel antibiotic drugs. The implications of this ion-recognition mode on the mechanism of the ATP synthase and the cellular bioenergetics of F. nucleatum were thus examined. Our results provide the basis for future pharmacological efforts against this important pathogen.