Refine
Year of publication
Document Type
- Doctoral Thesis (24)
Has Fulltext
- yes (24)
Is part of the Bibliography
- no (24)
Keywords
- EPR (2)
- PELDOR (2)
- Aptamere (1)
- Conformational Dynamics (1)
- DEER (1)
- DNS (1)
- Electron Paramagnetic Resonance (1)
- Elektronenspinresonanz (1)
- Elektronenspinresonanzspektroskopie (1)
- Multi-domain proteins (1)
Institute
One of the most important tasks in chemistry and especially in structural biology has always been the elucidation of three-dimensional molecular structures - either of small molecules or large biopolymers. Among the (bio)physical methods to acquire structural data at atomic resolution electron paramagnetic resonance (EPR) spectroscopy is the most valuable technique for obtaining structural information about many different kinds of paramagnetic species. In biological systems, either paramagnetic metal ions/clusters, transient paramagnetic intermediates in electron transfer processes or artificially attached stable spin labels can be found. The usual approach to interpret EPR spectra is to perform simulations based on the so-called spin Hamiltonian (SH). This means that the well-defined numerical parameters (tensors) in the SH representing different types of interaction are obtained by fitting the experimental data. The SH parameters include electronic g-values, hyperfine coupling (HFC) and quadrupole coupling (&C) constants, zero-field splittings and constants to describe exchange and dipolar interactions between electron spin systems. However, since the SH only contains spin degrees of freedom, a direct translation of the SH EPR parameters into structural information is not straightforward. Therefore, methods to predict such SH interaction parameters starting from molecular structures are required. In this thesis it was investigated whether quantum chemical calculations of EPR parameters based on density functional theory (DFT) methods may be employed to overcome these problems thus enabling a correlation of experimental EPR data with molecular structure. It was the central goal of this work to point out the potential of a fruitful interplay between quantum chemistry and experiment and to study how both can benefit from each other. For this purpose DFT methods were applied to a variety of organic radical or transition metal systems to calculate different EPR parameters. Using the 'broken symmetry' formalism it was possible to compute the exchange coupling constant for a nitroxide biradical and furthermore decompose the exchange mechanism in different through-bond and through-space interactions. Spin density distributions, 14N and 1H HFC constants as well as dipole moments and polarizabilities were computed for a number of aromatic nitroxides to examine their properties and select promising candidates which may serve as DNA-intercalating spin labels. Systematic investigations of the influence of hydrogen bond geometry on the 14N QC parameters for imidazole-water and methylimidazole-benzosemiquinone complexes lead to the conclusion that especially the imidazole amino nitrogen &C parameters are very sensitive probes of the bond geometry, in particular of the hydrogen bond length. The results of this study may be applied to biological systems, e.g. to gain structural information about quinone binding sites. Moreover, quantum chemical methods were applied to elucidate the structure of a nitrogen-centered radical intermediate in the inhibition process of ribonucleotide reductase (RNR). It was possible to find a molecular structure in accordance with all experimentally available data, thus revealing the longsought structure of the No radical and providing evidence for the trapping of a 3'-ketonucleotide in the reduction process catalyzed by RNR. To test the capability of modern DFT methods to predict g- and molybdenum HFC tensors for MoV complexes, validation studies were carried out. Comparison of computed EPR parameters of a number of MoV compounds with corresponding experimental values showed that g- and HFC tensors could be predicted in good accuracy, although some systematic errors of the computational methods have to be considered for such heavy 4d1 transition meta1 systems. Furthermore, DFT calculations on a Mn2+ binding site model of the hammerhead ribozyme allowed to conclude that the structure of the binding site as studied by EPR spectroscopy in frozen solution is very likely to be identical to the site found occupied by Mn2+ in crystals. Finally, computational methods were employed to aid in the structural characterization of the Mn2+ binding site in Ras (rat sarcoma protein) by providing accurate starting parameters for spectral simulations and furthermore helping to interpret the experimental data. In conclusion, it was demonstrated in this thesis that the combination of sophisticated experimental and quantum chemical methods represents a powerful approach in the field of EPR spectroscopy and that it may be essential to employ EPR parameter computations to extract the full information content from EPR spectra. Therefore, great potential lies in future applications of DFT methods to the large number of systems where detailed and reliable experimental data is available but where an unequivocal correlation of these data with structural information is still lacking.
One of the central research topics in the field of biophysical chemistry is the structure and function of membrane proteins involved in energy transduction. Both, the aerobic and the anaerobic respiration include electron transfer and proton translocation across the mitochondrial and bacterial membranes. These electron transfer processes lead to changes in oxidation states of cofactors some of which are paramagnetic. Therefore, EPR spectroscopy is the method of choice to obtain electronic and structural information directly related to the function of the respiratory chain proteins. In this work, multifrequency continuous wave (CW) and pulsed EPR spectroscopy has been used to characterize the molybdenum active site of polysulfide reductase (Psr) from the anaerobic bacterium Wolinella succinogenes and the protein-protein complex between cytochrome c oxidase (CcO) and cytochrome c from the aerobic bacterium Paracoccus denitrificans. Molybdenum in Psr-Psr is an enzyme essential for the sulfur respiration of Wolinella succinogenes. Biochemical studies suggested that the active site of this enzyme contains a mononuclear Mo center, which catalyzes the reduction of the substrate polysulfide to sulfide. Until now there is no crystal structure available for Psr. Consequently, current characterizations of this enzyme have to rely on biochemical and spectroscopic investigations. Within the present work, CW and modern pulsed EPR techniques were applied to investigate its catalytically active site. In the first part of this thesis, different redox agents have been used to generate paramagnetic states of Psr. Multifrequency CW-EPR spectroscopy was applied to identify the Mo(V) states. Using simulations of the experimental spectra, three spectroscopically distinct states have been identified based on the Mo hyperfine- and g-tensor values. Comparison of their EPR parameters with those of related enzymes indicated five or six sulfur ligands at the Mo center depending on the state. The state generated by addition of polysulfide is suggested to be the catalytically active form, in which the Mo is coordinated by a sulfur of the polysulfide chain as the sixth ligand. 33S (I = 3/2) labeled polysulfide was prepared to probe the proximity of the polysulfide to the molybdenum center via its hyperfine coupling. 1D-ESEEM and 2D122 HYSCORE spectroscopy was used to detect these hyperfine and quadrupole interactions, which are too small to be observed in conventional CW EPR spectra. To date there has been only one pulsed-EPR study involving a 33S nucleus [Finazzo et.al. 2003]. The reasons are that this nucleus has a high nuclear spin of I = 3/2 and a large nuclear quadrupole moment in addition to the low Larmor frequency. All these make the detection of sulfur and the extraction of structural information demanding. However, analysis of the 2D-data led to a Mo(V) 33S distance in a range of about 2 to 2.5 Å. Mo-S distances found in molybdenum enzymes of the same family are in a range of 1.8 to 2.8 Å suggesting that the 33S is indeed the sixth ligand of the Mo(V) center and demonstrating that polysulfide is the actual substrate for this enzyme. Thus HYSCORE experiments have been proved to be a powerful technique to gain further insight into the active site structures of molybdenum enzymes and the trafficking of substrate atoms during catalysis. Density functional theory (DFT) calculations together with quantitative numerical simulations of the 2D-data will help to obtain more structural details about the molybdenum binding site in Psr. CcO:cytochrome c complex Protein-protein complex formation is an important step in energy conversion biological processes such as respiration and photosynthesis. These protein-protein complexes are involved in long range electron transfer reactions and are known to be of transient nature. Within the bacterial and mitochondrial respiratory electron transport chains such a complex is formed between CcO and cytochrome c. Upon complex formation cytochrome c donates the electrons required for the CcO catalyzed reduction of dioxygen to water. Here, the protein-protein complex formation between CcO and cytochrome c from Paracoccus denitrificans was investigated by pulsed EPR spectroscopy. The idea was to use the relaxation enhancement due to the distance and orientation dependent magnetic dipole-dipole interaction between the paramagnetic centers in the different CcO constructs and cytochromes. Two-pulse electron spin echo experiments were carried out on mixtures of the CuA containing soluble subunit II or the full size CcO with the physiological partner cytochrome c552 or horse heart cytochrome c. Significantly enhanced relaxation of CuA due to specific protein-protein complex formation has been observed in all four cases. In contrast the non-binding cytochrome c1 showed only a very weak relaxation enhancement due to unspecific protein-protein interactions. The echo decays of the slowly relaxing observer spin (CuA of CcO) measured in the absence and presence of the fast relaxing spin (Fe(III) of cytochrome c) permitted the extraction of the pure dipolar relaxation contributions for the different complexes. Measurements at different temperatures proved the dipolar nature of the relaxation enhancement. Furthermore, it was demonstrated experimentally that this approach also works for the full-size CcO, which contains four paramagnetic metal centers, in complex with cytochrome c. Quantitative simulations of the data suggest a broad distribution in distances (2 - 4 nm) and orientations between the CuA and Fe(III) in the complex between CcO and cytochrome c. High-field EPR spectroscopy will be useful to further analyze and prove these complex structures. Within the present work, it has been shown that pulsed relaxation enhancement experiments can be used to investigate the distance and relative orientation between paramagnetic metal centers. Furthermore, it has been demonstrated on a qualitative level, that this method can be used complimentary to other biophysical approaches to study transient electron transfer protein-protein complexes. Finally, within this work it has been proven that this method can be applied also to biological systems where more than two paramagnetic centers are present. This is particularly interesting for supercomplexes between membrane proteins.
Die Kernspinresonanz(NMR)-Spektroskopie ist ein leistungsstarkes analytisches Werkzeug. Allerdings ist ihre Empfindlichkeit aufgrund geringer Wechselwirkungs-energie zwischen den Kernspins und dem externen Magnetfeld begrenzt. Die dynamische Kernpolarisation (DNP) erhöht DNP die Empfindlichkeit der NMR, indem sie die Polarisation von ungepaarten Elektronenspins auf die benachbarten Kernspins überträgt. In den letzten Jahrzehnten hat die DNP bei hohen Magnetfeldern erneut an Aufmerksamkeit gewonnen, bedingt durch die Verfügbarkeit leistungsstarker Gyrotron-Mikrowellen(mw)-Quellen. Jedoch wurde die Anwendung von DNP für Flüssigkeiten im Vergleich zu Festkörperproben bei niedrigen Temperaturen (≈100 K) weit weniger erforscht. Zwei Gründe können dafür hauptsächlich benennt werden. Bei hohen Magnetfeldern (entsprechend hohen mw-Frequenzen) wird die mw-Strahlung sehr stark von Flüssigkeiten absorbiert, was zu einer starken Erwärmung führt. Darüber hinaus sind die Translations- und Rotationsdynamik der Radikale und Target-Molekülen nicht schnell genug, um Spectraldichten bei den hohen mw-Frequenzen zu erzeugen, die für eine Overhauser-Effekt (OE) DNP Verstärkung benötigt werden. In dieser Arbeit wird gezeigt, Flüssigzustands-DNP bei hohen Magnetfeldern, insbesondere bei 9,4 T, mit hocheffizienten DNP-Probenköpfen möglich ist.
Der von skalaren Hyperfein-Wechselwirkung (hfWW) angetriebene OE ist für Flüssigzustands-DNP-Forschungen von besonderem Interesse, da der von der Theorie vorhergesagte Mechanismus auch bei hohen Magnetfeldern noch effizient ist. In der vorliegenden Arbeit wurde eine Methode zur Vorabprüfung potenzieller DNP-Kandidaten durch Messungen ihrer paramagnetischen NMR-Verschiebungen vorgeschlagen und untersucht. Wir beobachtete signifikante 13C-skalare OE DNP-Verstärkungen bis zu 50 bei den ausgewählten kleinen Biomolekülen, einschließlich Imidazol, Indol, verschiedene Aminosäuren und Kohlenhydraten. Das Lösungssystem wurde auch von organischen Lösungsmitteln auf Wasser erweitert.
Im Kontext von dipolarer OE DNP haben wir den Beitrag der Rotation des Radikals neben der Translationsbewegung zwischen Radikal und Target-Molekül zur OE DNP-Effizienz systematisch untersucht, indem wir verschiedene Nitroxidderivate mit unterschiedlichen Ringgeometrien und Substituenten verwendet haben. Mithilfe eines Models, das eine 'out-sphere' Translationsbewegung und eine 'inner-sphere' Rotationsbewegung des Radikal-Lösungsmittel-Komplexes enthält, konnte unsere Beobachtungen quantitativ simuliert werden. Außerdem wurde ein anderes Model untersucht, das eine Translationsbewegung mit der Rotation von Radikalen, bei denen das ungepaarte Elektron nicht im Zentrum sitzt, kombiniert.
Eine weitere neue Entdeckung in der DNP bei hohen Magnetfeldern waren der beobachtete SE (Solid-Effekt) an Lipidmolekülen mit BDPA-Radikal oberhalb der Lipidphasen-übergangstemperatur. Die neue Anwendung von SE DNP bietet einen alternativen Mechanismus zur OE DNP in Flüssigkeiten bei hohen Magnetfeldern und könnte möglicherweise auf Makromoleküle mit relativ langsamer Rotationsbewegung angewendet werden.
Wir haben zusätzliche Untersuchungen an den Lipiddoppelschichten mit Nitroxid-radikale durchgeführt, basierend auf dem beobachteten 1H DNP-Verstärkungen in einer viskosen Lipidumgebung bei 9,4 T . Durch Messung des Feldprofils wurden DNP-Verstärkungen durch OE und SE in Abhängigkeit ihrer relativen Verschiebungen von der Elektronen-Larmor-Frequenz bestimmt. Die individuelle OE DNP-Effizienzen für Protonen des Wassers, der Lipid-Cholin-Kopfgruppen oder der Lipid-Acylketten wurde bestimmt. Dadurch wird ein quantitativer Vergleich mit MD-Simulationen ermöglicht. Obwohl die von der MD-Simulationen vorhergesagten DNP Kopplungsfaktoren noch deutliche Abweichungen von den experimentellen Beobachtungen aufweisen, wird die schnelle Dynamik nahe der Elektronen-Larmor-Frequenz, die für einen erfolgreichen OE DNP Transfer erforderlich ist, von den MD-Simulationen gut erfasst.
In der Arbeit wurden auch zwei unterschiedliche Dreifachresonanz-DNP-Experimente durchgeführt. Zum einen wurde 13C OE DNP unter 1H-Entkopplung in wässriger Natriumpyruvatlösung, und zum anderen 13C-NMR von Glycin, verstärkt durch SE DNP an 1H zusammen mit einem 1H-13C INEPT-Polarisationstransfer, im Rahmen dieser Doktorarbeit durchgeführt.
Pulsed dipolar (PD) EPR spectroscopy is an established and reliable tool for the investigation of biomolecules. In terms of long distance and orientation measurements, it is one of the leading methods and further fields of application are constantly being explored. The distances that can be detected with PD EPR also correspond to the range in which almost all important biomolecule interactions occur. In the transition from in vitro spectroscopy to in-cell spectroscopy, the power of PD EPR spectroscopy is particularly evident. It is non-invasive, more sensitive than NMR, and does not exhibit background signals from diamagnetic molecules. In particular, the absence of background signals is of great importance given the high density of molecules within cellular environment. However, like any other spectroscopic method, PD EPR has certain limitations. Owing to the intrinsically fast electron spin echo dephasing at higher temperature, these experiments are commonly carried out in frozen solutions at about 50 K. This temperature is far away from the physiological conditions and the freezing additives used, e.g. glycols, can further influence the structure. To enable measurements with and within living organisms, it is therefore necessary to ascend from the cold depths of the frozen state. At the same time, one has to adapt the spin tags for the desired application. Established nitroxides commonly used for EPR studies are typically susceptible to reduction. Thus, for studies under physiological conditions, e.g. in the cell, one has to fight against the reductive environment in the cell and somehow protect the spin labels. Initial published in-cell experiments within the research group and investigations of homogeneously distributed labeled double-stranded (ds) ‐DNA samples in solid matrices showed promising results and enabled pulsed measurement in the temperature range of 50‐ 295 K. It could also be demonstrated that spherical shielded nitroxides have a significantly longer life span in cellular environments than non-protected ones and first nuclear acids were measured in cell. Based on these results, we have gone further to overcome the standing limitations and developed the use of PD EPR spectroscopy. This work addresses these challenges with the overall goal of advancing the applications of PD EPR spectroscopy for studying biomolecules under physiological conditions.
We have focused on four different approaches. The results of these studies were published in various publications. They are presented and discussed together with further studies and put into the context of research conducted before and after the authors' publications.
In approach 1, we fought against the two main obstacles for using pulsed dipolar spectroscopy at ambient conditions – minimizing phase memory time T2 and averaging of the anisotropic dipolar coupling by rotational diffusion. We focused on an immobilization approach, while using rigid spin labels at same time. Besidesto the distance information, the incorporated rigid spin labels will give additional angular constrains and information about the molecular dynamics.
In approach 2, we focused on the on-site and on-demand formation of nitroxide spin labels using light-sensitive alkyl protection groups. This a very mild and efficient procedure that will hardly interfere with sensitive functional groups present in oligonucleotides or peptides. By establishing this method and using coumarin protecting groups plus two-photon excitation, this property may offer the potential to generate spin labels with very high levels of spatial and temporal resolution.
For approach 3, we used paramagnetic Gd3+ -ions as intrinsically stable labels, which are not reducible within a cellular environment. Easy to mix and bound to encodable lanthanide binding tags within the molecule Interleucin 1β, we were able to measure distances between two tags with PELDOR spectroscopy. We tested the extent to which this system is suitable for in-cell measurements.
Finally, we focus on methods for easier labeling by using non-covalentlabeling techniques. One of these is the novel nitroxide G´ for site-directed spin labeling of nucleic acids, especially for RNA. This spin label is sterically hindered, easy to build and binding occurs in seconds by simply mixing the spin label with the target. For large RNAs, another easy-to-mix and noncovalent spin-labeling strategy will be experimentally accompanied and presented.
The approaches and results described here are intended to demonstrate that the study of the biological functions of biomolecules under physiological conditions by pulsed EPR spectroscopy is feasible and operational. In combination, they will enable the life sciences to make further and faster progress in the search for the molecular master plan.