In this work 3 different proteins are subjected to investigations on their structural, dynamic and functional properties by SDSL EPR spectroscopy, combined with in silico structure prediction and modeling: the pore-forming bacterial toxin colicin A in its membrane-bound form and its corresponding immunity protein Cai, and the Na+/proline symporter PutP. Colicin A (ColA) is a plasmid-encoded water-soluble pore-forming toxin produced by certain E. coli strains that kills unprotected cells of related strains by inserting a pore-forming subdomain into the cytoplasmic membrane to form voltage-dependent ion channels. Detailed structural data for the membrane-bound channel, in the closed as well as in the open state, is still missing, thus in the present study, the in vitro investigation by site-directed spin labeling and EPR spectroscopy has been substantially extended. The results indicate that a larger fraction of the protein than previously suggested penetrates into the hydrophobic core of the membrane, and distance measurements by pulse and cw EPR spectroscopy provide evidence that ColA in lipid bilayer membranes forms an oligomeric structure. Pulse EPR distance measurements under in vivo conditions reveal clear indications for an oligomeric ColA structure also in vivo. The results of all EPR measurements were combined to construct a dimer model for the colicin A closed channel state conformation. The immunity protein Cai, an integral inner membrane protein, protects the producing E. coli cell from the cytotoxic activity of its corresponding toxin (colicin A), by preventing channel opening by a yet unknown mechanism. ESR measurements for single spin label probes attached to ColA in the presence and absence of the immunity protein Cai reveal a clear influence on the ColA helices of the pore-forming domain in the presence of Cai as previously postulated. The data suggest that Cai induces a conformational change in/for the voltage sensor helix H6 of ColA, forming a “locked” inactive channel conformation that is not capable of voltage sensing and channel opening. Initial experiments with spin labeled wt-Cai in the presence and absence of unlabeled ColA suggest a more compact structure in the presence of ColA. PutP is an integral membrane protein located in the cytoplasmic membrane of E. coli, being responsible for the coupled transport of Na+ and proline in a 1:1 stoichiometry. It belongs to the family of sodium solute symporters (SSSF). Three dimensional structural data for PutP are at the moment not available, but a homology model has been developed based on the crystal structure of another member of this protein family, the Na+/galactose symporter vSGLT of Vibrio parahaemolyticus. The observed periodicity in spin label mobility and polarity measurements suggest a secondary structure of the extracellular Loop eL4 of PutP of two α-helical segments eL4a and eL4b, and imply the idea of eL4 functioning as an external gate to the SSSF. The ligand-induced changes observed in mobility, polarity and accessibility upon substrate binding support this notion, thus providing further insights into the mechanistic basis of sodium solute symport.

The signal transduction pathway of halophilic archaea remains a fascinating example of adaptation to extreme environments. Despite similarities with bacterial taxis systems, its structural and dynamics patterns during signal relay remain debatable. The currently investigated SRII/HtrII phototaxis system of Natronomonas pharaonis shows remarkable similarities with chemoreceptors in its membrane and HAMP domains functioning design. By combining site-directed spin labeling (SDSL) with electron paramagnetic resonance (EPR) spectroscopy we investigate the kinase control domain (i.e. signaling domain) of NpSRII/HtrII both in terms of dynamic and structural properties. Our data, as provided by continuous wave and pulse (DEER) EPR techniques, builds on current dynamics based signaling models for HAMP domains (such as the “frozen–dynamic” or two-state equilibrium models). We present an expanded mechanism for signal propagation throughout the signaling domain, where salt and temperature variations trigger subtle shifts in dynamics. Extreme dynamics motional ranges (compact or highly-dynamic) associate with a specific flagellar signaling state, here the kinase-off response, where a more moderate dynamics motion (dynamic) associates with the kinase-on response. Structurally, we reference our data on PML and ND reconstituted NpSRII/HtrII to the EcTsr crystal structure and the NpHtrII homology model. We show that, despite a difference in packing, NpHtrII oligomerizes in a similar manner as EcTsr, even in the absence of stabilizing structures such as the CheA/CheW baseplate. The presence of trimers-of-dimers but also dimers-of-dimers in membrane sheet samples exposes the high affinity with which NpHtrII signaling domains interact. We hope our structural and dynamics details will push further not just drug design but also environmental preservation efforts where taxis systems drive colonization and virulence of pathogens in plants, animals and humans alike.

Die Elektronen-Spin-Resonanz (ESR) Spektroskopie hat sich in Verbindung mit der Spinsondentechnik (SDSL) bereits seit einigen Jahren zur Struktur- und Dynamikanalyse von Biomakromolekülen etabliert. Diese Dissertation beschäftigt sich mit dem Aufbau eines Hochfeld-/Hochfrequenz-ESR-Spektrometers bei 3,4T/95GHz (W-Band) und drei attraktiven Anwendungen auf spinmarkierte Proteine. Als eine Anwendung wird die Bestimmung von Polaritäten und Protizitäten spinmarkierter Positionen des sensorischen Rhodopsin-Transducer-Komplexes (SRII/HtrII) aus Natronomonas pharaonis mit Hilfe von cw-Messungen bei Temperaturen <190 K präsentiert. Es werden Rückschlüsse auf die Struktur der HAMP-Domäne des Proteinkomplexes durchgeführt. Positionsabhängige Unterschiede der Protizität werden auf verschiedene Anteile mit null bis zwei Wasserstoffbrücken zum Nitroxid zurückgeführt. Als nächste Anwendung wird die Gewinnung von Reorientierungspotentialen der Spinträgerseitenkette mit dem SRLS-Model und in Kombination mit X- und Q-Bandmessungen bei Raumtemperatur demonstriert. Für die spinmarkierte Position einer Konformation des SRII/HTrII ergibt sich ein Potential, dass mit Ergebnissen von Molekulardynamiksimulationen eines helikalen Polypeptids gut übereinstimmt. Ebenfalls durchgeführt wurde die Bestimmung des Potentials an Position 166 der Kanal bildenden Domäne des Colicin A aus E. coli. Schließlich werden im Temperaturbereich von 120 bis 220 K die Einflüsse verschiedener (Isotopen-)Derivate des Spinlabels und der Einfluss der Wasserprotonen auf den Hahn-Echozerfall zur Bestimmung orientierungsabhängiger Librationsamplituden an einer weiteren SRII/HtrII-Probe und der gleichen Colicin A-Probe wie oben vorgestellt. Es werden Folgerungen für die Struktur der Proteine gezogen und, im Fall der Colicin A-Probe, ein Bezug zur Kristallstruktur hergestellt.

Understanding the conformational and dynamic changes of biomacromolecular complexes in different states, such as the membrane protein photoreceptor-transducer complex NpSRII/NpHtrII, is a key step to gaining insight into the functional mechanism of these important classes of protein complexes, since ~30 % of the human proteome are membrane proteins, yet they are largely underrepresented in terms of structural information with <1 % of all structures in the protein data bank. Hence for the development of methods suitable to study the conformation and dynamics of such complexes there is a strong demand and a vast potential field of applications. Here we combined method development at the interface between biomolecular simulations and model-based analysis of EPR- and fluorescence spectroscopic data with application studies using state-of-the-art spectroscopic techniques in conjunction with site-directed spin- or fluorescence labeling. In an initial benchmark study on the rigid globular protein complex Rpo4/7, we compared experimental inter fluorescence label distances or spin label distance distributions to a variety of predicted inter label distances based on molecular dynamics simulations, Monte Carlo sampling and a discrete rotamer library analysis. We found that while for the molecular dynamics simulations with explicit solvent considerable sampling challenges have to be overcome to reproduce the experimentally observed inter label distance distributions, the Monte Carlo sampling performed well when compared to the experimental data and was computationally less demanding. Significantly more efficient and equally accurate for our examples was the so-called rotamer library analysis available for the spin labels since it relies on a pre-calculated set of rotational isomers. In general, predictions for the mean distances were in agreement within the error margins while distribution shapes were more challenging to reproduce. Overall this study shows a positive evaluation for the assessed tools and the developed simulation protocols as well as their potential applications. Using the combination of EPR and fluorescence spectroscopy for distance determination we studied the structural influence of RNA binding on Rpo4/7, and showed that the protein complex stays conformationally rigid and thereby serves as a guiding rail for the nascent RNA chain that leaves the RNA polymerase along the Rpo4/7 RNA binding interface. To enhance the interpretation of experimentally determined changes of conformation and dynamics in protein complexes and to discuss the observed changes in terms of structural information, we built models of the two transcription factors TFE and the Spt4/5 complex, as well as of Argonaute, a 713 amino acid four-domain protein nuclease from Methanocaldococcus jannaschii. These structural models not only allowed a more accurate planning of fluorescence or EPR labeling experiments, but also the models enabled the discussion of the experimental data in structural terms. Based on such an initial structure further computational analysis techniques may be applied to identify putative structural changes or dynamic modes. This was shown for the histidine transporter HisQMP2, where we combined normal mode analysis to model protein flexibility with the rotamer library analysis to screen for possible conformational changes in comparison to experimental inter spin distance data. The most prominent agreement with one mode led to a working hypothesis of a conformational change and provides the basis for validation in future experiments. Due to the inherent synergy effects, we applied a combined experimental and simulation approach for the EPR-based distance determination in the globular DNA-binding protein LexA to probe conformation and dynamics of the N-terminal DNA-binding domains with respect to the C-terminal domains within the LexA homodimer. While the C-terminal dimerization domains exhibit a well-defined conformation that proved to be independent of DNA-binding, large-scale changes in conformation and dynamics were detected for the N-terminal domains. They were only found in a defined conformation when bound to DNA while in its absence a large rotational freedom of the entire N-terminal domains contributed to the conformational ensemble. Combined with a biochemical characterization of the autocatalytic cleavage of LexA, our data explains how LexA induces the SOS response after DNA damage or under latent antibiotic stress. We further studied the membrane photoreceptor-transducer complex NpSRII/NpHtrII that governs the light-dependent swimming behavior in Natronomonas pharaonis by a two-component signaling system. This system comprises extraordinary features of sensitivity, signal amplification, integration and transducer cooperativity, yet the molecular details of these features are poorly understood, as is signal propagation itself. By combining time-resolved cw EPR spectroscopy of NpSRII/NpHtrII variants spin labeled in the HAMP1 domain with time-resolved optical absorbance spectroscopy to report on the receptor signaling state, we found a tight kinetic coupling of receptor and transducer during the relaxation back to the ground state and hence a prolonged activation period, that with ~500 - ~700 ms is sufficiently long to cause phosphorylation bursts of the cognate kinase CheA. This explains signal amplification already on the level of the NpSRII/NpHtrII dimers. We further determined the transient difference spectra from the time-resolved EPR data that show local differences in dynamics and steric restrictions upon light-activation. Comparing these experimentally observed differences to predictions confirms the assumed two-state structural model and shows this transition between the two states for a single HAMP domain in a light-dependent manner. Additionally, our approach integrates a dynamic view into the model, since the two states are shown to exhibit different local dynamics in a fashion described previously as a competing model for signaling by dynamic differences based on biochemical studies. Here we show unification of the two models into one congruent description encompassing a transition between the two previously suggested states by concerted structural and dynamic changes. In an independent analysis using all-atom and coarse grained molecular dynamics of the NpSRII/NpHtrII complex in the minimal unit that can exert kinase control, the trimer of receptor-transducer dimers, we revealed a distinct dynamical pattern encoded in the primary sequence of the coiled-coil heptad-repeats. Upon receptor activation, these segments alter their dynamics in a concerted fashion with regions such as HAMP1 and the adaptation region becoming more compact, while HAMP2 and the tip become more dynamic, leading to dynamic and to limited structural changes at the CheA-kinase binding sites. Together with an extensive validation against experimental data, these findings suggest the altered dynamics as the mechanism for signal propagation along the extended coiled-coil structure of NpHtrII. This working model, that explains the current body of experimental data, allows for further refinement by all-atom molecular dynamics and provides a basis to devise future experiments for validation. The presented studies outline the versatile methodology of combined experimental and simulation approaches to analyze the conformation and dynamics of biomacromolecules including membrane protein complexes.

The photosensitive unit triggering the negative phototaxis in the haloarchaeum Natronomonas pharaonis consists of the receptor sensory rhodopsin II (NpSRII) and its cognate transducer (NpHtrII) in a 2:2 stoichiometry. Upon light excitation, a structural rearrangement in the receptor initiates a displacement/rotation of the transducer helix TM2, which can be considered as starting event for the signal transduction. This signal is further transmitted to the cytoplasmic signaling domain through the signal transduction unit comprising two HAMP domains.Structural information already exists for the transmembrane region of this complex (crystal structure) as well as for the rod shaped cytoplasmic part of NpHtrII due to its high homologies with chemoreceptors. Moreover, the solution NMR structure of the isolated HAMP domain from A. fulgidus recently obtained shows a homodimeric, four-helical, parallel coiled-coil with an unusual interhelical packing, that is thought to propagate a signal by virtue of concerted helix rotations. Here, an electron paramagnetic resonance (EPR) investigation of site-directed spin labeled transducers in the NpSRII/NpHtrII complex has been carried out for structural and functional elucidation of the N. pharaonis HAMP. For this purpose, cw as well as pulse EPR techniques have been used in terms of mobility, accessibility and intra-transducer dimer distance analyses. Conformational changes induced by environmental inputs, namely salt, temperature and pH, give insight into the two-state equilibrium existing between a highly dynamic (dHAMP) and a more compact (cHAMP) conformation of this linker region.

Das haloalkaphile Archaeon Natronomas pharaonis nutzt den Lichtrezeptor, das Sensorische Rhodopsin II (NpSRII), im Komplex mit dem halobakteriellen Transducer (NpHtrII) zur photophoben Antwort auf schädliches grün-blaues Licht und entsprechender Steuerung des Flagellenmotors um optimale Umgebungen zum Überleben aufzusuchen. In einer Membran rekonstituiert bildet der Rezeptor/Transducer Komplex eine 2:2 Stöchiometrie aus, wobei ein Transducer-Dimer von zwei Rezeptor-Molekülen flankiert wird. Durch die Lichtanregung wird ein Photozyklus initiiert, dessen Intermediate sich aufgrund ihrer optischen und/oder strukturellen Eigenschaften unterscheiden. In dieser Studie sind die strukturellen Änderungen des Rezeptors und des Transducers während des Photozyklus mit Hilfe der Elektronenspinresonanz (ESR)-Spektroskopie in Kombination mit der ortsspezifischen Spinmarkierung aufgeklärt worden. Als Methoden wurden dabei die zeitaufgelöste ESR-Spektroskopie und Abstandsmessungen in unterschiedlichen Intermediaten mit Hilfe von cw- und Puls-ESR-Techniken genutzt. Der Signaltransfer nach Initiierung des Photozyklus im Rezeptor, die Weiterleitung des Signals zum Transducer durch die Auswärtsbewegung der Helix F und die damit verbundene Verschiebung des thermodynamischen Gleichgewichts in der HAMP-Domäne des Transducers konnten beobachtet und analysiert werden. Die Methode der ESR-Spektroskopie erweist sich als mächtige biophysikalische Technik, die eine direkte und zeitaufgelöste Analyse von strukturellen Konformationsänderungen in Membranproteinen und die strukturelle Aufklärung unterschiedlicher Intermediate erlaubt.

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Die Elektronen-Spin-Resonanz-Spektroskopie (ESR) in Verbindung mit ortsspezifischer Spinmarkierung stellt eine hervorragende Möglichkeit dar, um die Struktur und Dynamik von Proteinen aufzuklären. In dieser Dissertation wurden mit Hilfe der Hochfeld-ESR-Spektroskopie (W-Band, 95 GHz, T=160 K) für dreizehn spinmarkierte Colicin A Proben die Polarität und die Protizität der Umgebung der Spinlabelbindestelle bestimmt. Wasserzugänglichkeiten und Wasserstoffbrückenbindungen zum Spinlabel wurden mittels Puls-ESR Methoden (3-Puls-D-ESEEM und Hahn-Echozerfall) bestimmt und die Ergebnisse mit den Polaritäts- und Protizitätswerten korreliert. Raumtemperaturspektren dieser Proben im X-Band (9.5 GHz), Q-Band (34 GHz) und W-Band (95 GHz) liefern Informationen über die Spinlabelbewegung. Mit Hilfe von Molekulardynamiksimulationen (MD) der spinmarkierten kanalbildenden Domäne von Colicin A konnten die Konformationen (Rotameranalyse) und die Dynamik der Spinlabelseitenketten in den unterschiedlichen Umgebungen charakterisiert werden. Der Vergleich der experimentellen mit den aus MD-Trajektorien berechneten ESR-Spektren liefert die Beiträge der unterschiedlichen Rotamerübergänge, die für die beobachteten Spektrenformen charakteristisch sind.