NMR-basierte strukturelle und dynamische Untersuchungen an nicht-nativen Varianten des Hühnereiweißlysozyms

Die Erforschung nicht-nativer Zustände von Proteinen hat bisher viele Erkenntnisse zum besseren Verständnis der Proteinfaltung und -fehlfaltung erbracht. Die Kernmagnetische Resonanzspektroskopie (NMR) ist die einzige bekannte Methode, die zur Aufklärung der strukturellen und dynamischen Eigenschaften solcher Zustände in atomarer Auflösung beitragen kann. Entfaltete und nicht-native Zustände von Proteinen müssen als Ensemble von sich schnell ineinander umwandelten Konformeren beschrieben werden, ihre beobachteten Eigenschaften sind somit populationsgewichtete Mittelungen. In der vorliegenden Arbeit wurde das Lysozym aus dem Hühnereiweiß (HEWL) und Mutanten davon unter Bedingungen untersucht, unter denen diese Proteine permanent in nicht-nativen Zuständen vorliegen. Dies wurde in einem Ansatz durch die Reduktion der vier Disulfidbrücken und der anschließenden Methylierung der acht Cysteinreste („HEWL-SMe“) dieses Proteins und in einem anderen Ansatz durch das Ersetzen der Cysteinreste durch Alanine erreicht („all-Ala-HEWL“). Unter solchen Bedingungen wurde in diesem Protein residuale Sekundär- und Tertiärstruktur beobachtet, es liegt also, anders als einige andere entfaltete Proteine, nicht als Zufallsknäuel (random coil) vor. Insbesondere die Existenz von hydrophoben Clustern und Wechselwirkungen zwischen in der Peptidkette weit entfernten Resten ist dabei bemerkenswert und war aus kombinierten Relaxations- und Mutationsexperimenten bekannt. Dabei spielen die Tryptophanreste eine wichtige Rolle. Zu den NMR-spektroskopischen Untersuchungen wurde nicht-natives HEWL in E. coli-Zellen in inclusion bodies isotopenmarkiert exprimiert und daraus aufgereinigt. Nach nahezu vollständiger Zuordnung der Rückgrat-Resonanzen konnte aus den sekundären chemischen Verschiebungen auf Bereiche innerhalb der Sequenz — vor allem rund um die sechs Tryptophanreste — geschlossen werden, die erhöhte alpha-Helix-artige Konformationsanteile enthalten. Um eine genauere Untersuchung der für die residuale Struktur in nicht-nativem Lysozym wichtigen Tryptophanreste mittels NMR zu ermöglichen, wurde eine Kombination aus zwei Pulssequenzen entwickelt, mittels derer sich die 1HNepsilon und 15Nepsilon Resonanzen in der Seitenkette über den 13Cgamma-Kern mit Hilfe der bekannten 1HN und 15NH Rückgratresonanzen zuordnen ließen. Die Zuordnung der Indolseitenkettenresonanzen ermöglichte die Interpretation von zweidimensionalen 15N/1H photo-CIDNP-Experimenten, die über die unterschiedliche Zugänglichkeiten der Tryptophanseitenketten für einen Farbstoff (z.B. FMN) im Lösungsmittel berichteten und somit strukturelle Informationen lieferten. Dabei wird mittels einem in das NMR-Röhrchen eingekoppelten Laserstrahl das FMN angeregt und schließlich über die Bildung eines Radikalpaares die Tryptophanseitenkette polarisiert. Die unterschiedlichen relativen Intensitäten der photo-CIDNP-Peaks deuten auf eine unterschiedliche Zugänglichkeit der Tryptophanseitenketten in nicht-nativem Lysozym hin. Dagegen sind die Zugänglichkeiten dieser Reste in der W62G-Mutante von HEWL fast alle ähnlich. Dies bestätigte die zentrale Bedeutung dieses Restes für die hydrophoben Cluster, die aus vorherigen Relaxationsstudien bekannt war. Aus Diffusionsmessungen ist bekannt, dass die W62G-Mutante über einen größeren hydrodynamischen Radius verfügt als die nicht-nativen Zustände des Wildtyps, das konformationelle Ensemble zeigt also ausgedehntere Strukturen und somit sind auch die Tryptophanseitenketten exponierter. Eine Ausnahme scheint das Tryptophan 111 zu bilden, welches anders als das benachbarte W108 auch in der W62G-Mutante eine geringere Zugänglichkeit aufweist. Erstmals konnten auch zweidimensionale 13C/1H photo-CIDNP-Experimente in Lösung durchgeführt werden. Für all-Ala-HEWL ergab die Bestimmung von heteronuklearen R1 und R2 Relaxationsraten und des heteronuklearen 1H15N NOE einen Nachweis der verminderten Mobilität in den hydrophoben Clustern. Die Durchführung von Relaxationsdispersionsmessungen sowie die Messung von R1r Relaxationsraten erbrachten keinerlei Hinweise zu konformationellen Austausch auf der Mikro- bis Millisekunden-Zeitskala. Im Gegensatz zur W62G-Mutante zeigte eine W108G-Mutante keinen bedeutenden Einfluss auf das Gesamtbild der hydrophoben Cluster, sondern modulierte nur Cluster 5, in dem es sich selbst befindet. Die Messung von dipolaren Restkopplungen (RDCs) in partiell in gestreckten Polyacrylamidgelen ausgerichtetem all-Ala-HEWL zeigte erhöhte positive RDCs in den Bereichen der hydrophoben Cluster, vorallem in den Bereichen um die Tryptophane 28, 62/62 und 108/111, dies deutet auf eine Strukturierung in diesen Bereichen hin. In der W62G-Mutante waren die positiven RDCs hingegen größtenteils nicht zu beobachten.The formation and maintenance of a defined three-dimensional structure is a prerequisite for most proteins in order to fulfill their function in the native context. However, there are proteins, which are intrinsically unstructured and thus natively unfolded. In addition, the misfolding and aggregation of many proteins can lead to severe diseases. The investigation of non-native states of proteins significantly contributes to the understanding of protein folding and misfolding. Nuclear magnetic resonance (NMR) spectroscopy is the only known technique that can provide information on structure and dynamics of non-native states of proteins at atomic resolution. Unfolded and non-native states of proteins have to be treated as ensembles of rapidly interconverting conformers and their observed properties are ensemble and time averaged. In this thesis, hen egg white lysozyme (HEWL) and mutants thereof have been investigated by NMR spectroscopy. The reduction of its four disulfide bridges and the successive methylation of the cysteine residues renders HEWL permanently non-native (‘HEWL-SMe’). Alternatively, the exchange of the eight cysteines for alanines results in very similar states (‘all-Ala-HEWL’). Under these conditions, HEWL-SMe and all-Ala-HEWL do not resemble random coil conformations, but exhibit residual secondary and tertiary structure. The presence of hydrophobic clusters and long-range interactions around the proteins six tryptophan residues and the modulation of these properties by single-point mutants has been observed. For the NMR spectroscopic investigation, HEWL has been isotopically labelled in E. coli by expression into inclusion bodies. After purification, the 1HN, 15NH, 13Calpha, 13Cbeta, 13C’, 1Halpha and 1Hbeta resonances of HEWL-SMe and all-Ala-HEWL have been assigned almost completely using three-dimensional NMR experiments. The analysis of secondary chemical shifts revealed regions in the proteins sequence — particularly around the six tryptophan residues—with significantly populated alpha-helix like conformations. In order to further elucidate the influence of the tryptophan side chains, a set of two new pulse sequences has been developed that allowed for the successful assignment of the 13Cg, 15Ne and 1HNe resonances in these side chains. This knowledge was eventually exploited in the interpretation of two-dimensional 15N-1H photo-CIDNP spectra, which revealed a differential solvent accessibility of the tryptophan residues in all-Ala-HEWL but not in the single point mutant W62G-all-Ala-HEWL. In addition, heteronuclear R2 relaxation rates have been determined for the indole 15Ne nuclei of all-Ala-HEWL and W62G. While in the wild-type like all-Ala-HEWL, the rates are different among the six tryptophan residues, in W62G they are more uniform. Together with relaxation data from the amide backbone, these results indicate the significant destabilization of the hydrophobic clusters in the absence of W62. In contrast, in the W108G mutant the profile of the R2 relaxation rates was not found to be significantly altered. No evidence was found by R1rho relaxation rates and relaxation dispersion measurements for conformational exchange on slower (micro- to millisecond) timescales. Residual dipolar couplings have been determined for non-native HEWL in order to retrieve structural information of these states. The differences of the W62G and the wild-type like non-native HEWL is also picked up in NH-RDCs of these proteins aligned in polyacrylamide gels. Significant positive RDCs are observed in the regions of the hydrophobic clusters in all-Ala-HEWL, but to a much lesser degree in W62G. So far, all attempts to simulate RDCs from generated non-native ensembles failed even when including long-range contacts or specific phi/psi backbone angle propensities. However, the measured RDCs can be used to cross-validate structural ensembles of non-native HEWL generated by molecular dynamics simulations that are based on restraints from the other experimental data, such as the differential solvent accessibilities from the photo-CIDNP experiments and the data on the hydrophobic clustering gained from the combined mutational and relaxation studies. Finally, non-native HEWL has been investigated for the first time using two-dimensional NMR in organic solvents, which are able to induce secondary structures and ultimately lead to amyloid formation. Under these conditions severe line broadening was observed, which was attributed to exchange between different — mostly a-helical— conformations. In summary, in this thesis methods have been developed, optimized and successfully applied for the structural and dynamical characterization of non-native states of proteins and the effect of single-point mutants on the properties of such ensembles has been investigated. Data has been gained that can considerably contribute to the further elucidation of the nature of non-native states of HEWL by molecular dynamics simulations

Electrodeposition of Fe70Pd30 nanowires from a complexed ammonium–sulfosalicylic electrolyte with high stability

A highly stable plating bath for the electrodeposition of Fe–Pd nanowires into nanoporous alumina templates has been developed. Complexing of both metal ions and exchanging Fe2+ by Fe3+ avoid chemical reduction of Pd ions and, therefore, undesirable deposition. By using a pulse potential mode and appropriate adjustment of deposition potentials homogeneously filled templates without surface deposits and nanowires close to the desired composition of Fe70Pd30 have been achieved. These alloy nanowires represent a key step towards nanoactuators based on magnetic shape memory alloys. Keywords: Electrodeposition, Nanowires, Fe70Pd30, Ferromagnetic shape memory alloy

Heterologous expression of hen egg white lysozyme and resonance assignment of tryptophan side chains in its non-native states

A new protocol is described for the isotope (15N and 13C,15N) enrichment of hen egg white lysozyme. Hen egg white lysozyme and an all-Ala-mutant of this protein have been expressed in E. coli. They formed inclusion bodies from which mg quantities of the proteins were purified and prepared for NMR spectroscopic investigations. 1H,13C and 15N main chain resonances of disulfide reduced and S-methylated lysozyme were assigned and its residual structure in water pH 2 was characterized by chemical shift perturbation analysis. A new NMR experiment has been developed to assign tryptophan side chain indole resonances by correlation of side chain and backbone NH resonances with the Cgamma resonances of these residues. Assignment of tryptophan side chains enables further residue specific investigations on structural and dynamical properties, which are of significant interest for the understanding of non-natives states of lysozyme stabilized by hydrophobic interactions between clusters of tryptophan residues.</p

Interface-Dominated Topological Transport in Nanograined Bulk Bi2 Te3

3D topological insulators (TI) host surface carriers with extremely high mobility. However, their transport properties are typically dominated by bulk carriers that outnumber the surface carriers by orders of magnitude. A strategy is herein presented to overcome the problem of bulk carrier domination by using 3D TI nanoparticles, which are compacted by hot pressing to macroscopic nanograined bulk samples. Bi2Te3 nanoparticles well known for their excellent thermoelectric and 3D TI properties serve as the model system. As key enabler for this approach, a specific synthesis is applied that creates nanoparticles with a low level of impurities and surface contamination. The compacted nanograined bulk contains a high number of interfaces and grain boundaries. Here it is shown that these samples exhibit metallic-like electrical transport properties and a distinct weak antilocalization. A downward trend in the electrical resistivity at temperatures below 5 K is attributed to an increase in the coherence length by applying the Hikami–Larkin–Nagaoka model. THz time-domain spectroscopy reveals a dominance of the surface transport at low frequencies with a mobility of above 103 cm2 V−1 s−1 even at room temperature. These findings clearly demonstrate that nanograined bulk Bi2Te3 features surface carrier properties that are of importance for technical applications