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Polyketide synthases (PKSs) are large megaenzymes that occur in bacteria, fungi, and plants and produce polyketides, a class of secondary metabolites. Many polyketide natural products exhibit high biological activities e.g. as antibiotics or anti-fungal compounds. The modular architecture of assembly line PKSs makes them exciting targets for engineering approaches via the exchange of whole modules or single domains. Although many engineering attempts have been pursued over the last three decades, the resulting chimeric PKSs often exhibit decreased turnover rates or diminished product yields.
In this thesis, new approaches to engineer chimeric PKSs were explored, each targeting a different aspect of the chimeric system: First the relative contribution of protein-protein and protein-substrate recognition on the turnover of chimeric PKS was assessed, revealing the importance of protein-protein interactions between the acyl carrier protein (ACP) and the ketosynthase (KS) domain in the chain translocation step. Directed evolution experiments followed to optimize the protein-protein interaction across a chimeric interface. Additionally, different junction sites for the generation of chimeric PKSs were compared, showing the ability for recombination without interfering with the chain translocation reaction, and highlighting the use of SYNZIP domains to bridge PKS modules. To optimize chimeric PKSs even further, multipoint mutagenesis of KS domains was established, with positive effects on the activity of chimeric systems.
To support engineering attempts, several structure elucidation techniques were combined with in silico modeling to characterize the architecture of a PKS module and the domain-domain interactions within it. Preliminary results show a strong conformational flexibility of the PKS module and the great potential of these techniques to define the multitude of transient interactions in PKS modules.
Transport mechanism of a multidrug resistance protein investigated by pulsed EPR spectroscopy
(2019)
In human several diseases result from malfunctions of ATP-binding cassette (ABC) systems, which form one of the largest transport system superfamily. Many ABC exporters contain asymmetric nucleotide-binding sites (NBSs) and some of them are inhibited by the transported substrate.1 For the active transport of diverse chemically substrates across biological membranes, ABC transport complexes use the energy of ATP binding and subsequent hydrolysis. In this thesis, the heterodimeric ABC exporter TmrAB2,3 from Thermus thermophilus, a functional homolog of the human antigen translocation complex TAP, was investigated by using pulsed electron-electron double resonance (PELDOR/DEER) spectroscopy. In the presence of ATP, TmrAB exists in an equilibrium between inward- and outward-facing conformations. This equilibrium can be modulated by changing the ATP concentration, showing asymmetric behaviour in the open-to-close equilibrium between the consensus and the degenerate NBSs. At the degenerate NBS the closed conformation is more preferred and closure of one of the NBSs is sufficient to open the periplasmic gate at the transmembrane domain (TMD).3 By determining the temperature dependence of this conformational equilibrium, the thermodynamics of the energy coupling during ATP-induced conformational changes in TmrAB were investigated. The results demonstrate that ATP-binding alone drives the global conformational switching to the outward-facing state and allows the determination of the entropy and enthalpy changes for this step. With this knowledge, the Gibbs free energy of this ATP induced transition was calculated. Furthermore, an excess of substrate, meaning trans-inhibition of the transporter is resulting mechanistically in a reverse transition from the outward-facing state to an occluded conformation predominantly.3 This work unravels the central role of the reversible conformational equilibrium in the function and regulation of an ABC exporter. For the first time it is shown that the conformational thermodynamics of a large membrane protein complex can be investigated. The presented experiments give new possibilities to investigate other related medically important transporters with asymmetric NBSs or other similar protein complexes.
Many processes in living cells involve interaction and cooperation of multiple proteins to fulfill a specific function. To understand biological processes in their full complexity, it is not sufficient to only identify the molecules being involved but also to understand the kinetic aspects of a reaction. Mass spectrometry (MS) is a very powerful tool which allows to precisely identify the molecules of a reaction. Usually this is done with tandem-MS experiments for purpose of de-novo peptide sequencing. However, since this involves protein digestion, a statement of the in-vivo constitution of non-covalently bound protein complexes is not possible. In order to detect an intact protein complex it is necessary to analyze the biological system softly and in a near-native environment with native MS. Native MS allows the non-destructive analysis of these non-covalent protein complexes as well as to detect their components. However, up to now native MS does not offer a possibility to resolve the timing of the constitution of protein complexes on a fast time-scale. Therefore, the progress of reactions on fast time-scales is invisible. However, a method which delivers both types of information - identification of the components of a protein complex, as well as time-resolving their interaction - would be of high interest.
A suitable ionization technique for native MS is laser-induced liquid-bead ion desorption (LILBID). LILBID employs well-defined droplets which are irradiated by IR laser pulses to generate gas phase ions. The not-continuous, repetitive nature of ion generation offers itself to the development of a time-resolved (TR) native MS system which is able to investigate protein complexes on a fast time scale. The LILBID-droplets can serve as reaction vessels if they are levitated in an electrodynamic Paul-trap. This new setup would allow sample manipulation and MS analysis on precise and fast reaction time-scales. The first part of this dissertation presents the construction and characterization of a setup for TR-LILBID-MS.
An example for a complex biological system is the self-assembly of beta-amyloid (Aβ). This small peptide is the major component in plaques related to Alzheimer’s disease. Clinically relevant is especially the 42 amino acid peptide Aβ42 which aggregates from monomers to oligomers through to fibrils. The oligomers are the neurotoxic species in this process and thus of high interest. Nevertheless, standard analytical techniques are unable to detect those oligomers which makes MS an optimal tool to study the oligomerization process of Aβ with the focus on disease relevant oligomers. TR-LILBID-MS allows to follow the oligomerization of Aβ enabling to study molecules which influence this kinetic. Combining MS with ion-mobility spectrometry adds an additional dimension - the collision cross section - to the mass-to-charge ratio obtained from MS. Therewith structural alterations induced by ligands can be correlated to differences in the aggregation kinetic. This allows to draw a picture of the aggregation process of Aβ for the development of disease-relevant small oligomers on a molecular level.
Die in der vorliegenden Arbeit gewonnenen Erkenntnisse zur Reaktivität zweifach reduzierter 9,10-Dihydro-9,10-diboraanthracene [A]2– erweitern das Einsatzspektrum von Hauptgruppenverbindungen im Hinblick auf die Aktivierung kleiner Moleküle. Komplementär zu Übergangsmetallkomplexen und FLPs ermöglichen die Salze M2[A] (M+ = Li+, Na+, K+) die Entwicklung neuartiger Synthesestrategien. Als besondere Herausforderung gilt die Aktivierung des stabilen H2-Moleküls, dessen Bindung die Dianionen [A]2– homolytisch in einer konzertierten Reaktion spalten.
Untersuchungen zur Kinetik der H2-Addition an M2[A] stellten die Abhängigkeit dieses Reaktionsschritts vom borgebundenen Substituenten und vom Kation heraus. Eine geringe sterische Abschirmung der Boratome durch kleine borgebundene Substituenten (C≡CtBu, Me, H) begünstigt die H2-Aufnahme gegenüber großen Substituenten (pTol, Xyl, Et). Die maximale Ausbeute an M2[A-H2] wird für M+ = Li+ erst nach mehreren Tagen bei 100 °C erhalten, während einige Stunden bei nur 50 °C für die quantitative Bildung von K2[A-H2] ausreichen.
Unter den Salzen M2[A] eignet sich Li2[68] mit borgebundenen Me-Substituenten besonders gut für den Einsatz als Hydrierungskatalysator. Mit Li2[68] konnten das Imin Ph(H)C=NtBu, das terminale Alken Ph2C=CH2 und Anthracen erfolgreich im NMR-Maßstab hydriert werden (Katalysatorladung 37 mol%, THF-d8, 1 atm H2-Initialdruck, 100 °C, 16 h). Im Reaktionsautoklaven war für die Hydrierung von Ph(H)C=NtBu eine Verringerung der Katalysatorladung auf 10 mol% Li2[68] möglich (THF, 7 atm H2-Initialdruck, 100 °C, 18 h). Konkurrenzreaktionen begründen Einschränkungen in Bezug auf die Substratpalette, da M2[68] (M+ = Li+, Na+) mit elektronenarmen ungesättigten Verbindungen, die C=C-, C≡C-, C=O- oder C=N-Bindungen enthalten, [4+2]-Cycloadditionsprodukte bilden können. Die Reversibilität dieser Reaktion entscheidet, ob Li2[68] als Katalysator fungiert oder irreversibel in den Strukturen gebunden bleibt.
Vielseitiger sind die H2-Aktivierungsprodukte M2[A-H2] als H–-Donoren geeignet: Na2[68-H2] ersetzt Halogenid- durch H–-Substituenten in Bromethan, sowie in Chlorsilanen und PCl3; CO2 wird in Natriumformiat überführt. Unabhängig von der Anzahl der Chlorliganden werden die Produkte immer vollständig hydriert. Eine erneute Reduktion von 68 kann wieder Na2[68] bereitstellen, das H2 aufnimmt und Na2[68-H2] regeneriert, welches für neue H–-Abgaben zur Verfügung steht. Bei der experimentellen Umsetzung des Kreislaufs ist es wichtig, die beschriebenen Reaktionsschritte nacheinander auszuführen und jeweils nur stöchiometrische Mengen des Elektrophils zuzugeben. Bei Abweichungen vom schrittweisen Syntheseprotokoll finden formale nukleophile Substitutionen mit M2[68] statt und monoanionische Spezies entstehen, z. B. wenn Et3SiBr als Elektrophil anwesend ist.
Gegenüber CO2 zeigt Li2[68] eine hohe Reaktivität, durch die selektiv CO und [CO3]2– gebildet werden. Wie zuvor bei den H–-Transferreaktionen ermöglicht die Reduktion der Neutralverbindung 68 die Regeneration von Li2[68].
Die Dianionen [A]2– stechen unter anderen cyclischen Borverbindungen in niedrigen Oxidationsstufen heraus, da mit [A]2– nicht nur die Aktivierung von H2 oder CO2 gelang, sondern erstmalig über die Einbindung der Additionsprodukte in zum Teil katalytische Folgereaktionen berichtet werden konnte.
Multidomain enzymes, such as fatty acid synthases (FASs) or polyketide synthases (PKSs), play a crucial role in the biosynthesis of important natural products. They have a high significance in the development of new pharmaceuticals and various research approaches focus on the engineering of these proteins. For example, human type I FAS is an interesting therapeutic target. Owing to its importance in lipogenesis, upregulation of human type I FAS expression has been observed in numerous cancers. Type I FAS is also regarded as important target in antiobesity treatment. Both multidomain enzyme classes - FASs and PKSs - show high structural and functional similarities. Particularly animal type I FAS is most relevant as evolutionary precursor of the PKS family. Therefore, the well characterized FASs are suitable model proteins for the poorly characterized PKSs, to gain deeper understanding in these megasynthases.
Furthermore, fatty acids are considered to be strategically important platform chemicals accessible through sustainable microbial approaches. The recently acquired structural information on FASs provides an excellent understanding of the molecular basis of fatty acid synthesis. The specific understanding of chain-length control, the characterization of a multitude of substrate-specific thioesterases, and the emerging tools and means for metabolic engineering have fostered targeted approaches for modulating chain length. There is large interest in short-chain fatty acids, since these compounds are biotechnologically valuable platform chemicals and biofuel precursors, and attempts on the synthesis of short-chain fatty acids have been reported during the last years.
Primary focus of this thesis lies on the animal type I FASs, which exhibit large conformational variety, as seen in electron microscopy and high-speed atomic force microscopy. Conformational dynamics facilitate productive protein-protein interactions between catalytic domains within the enzyme and aid acyl carrier protein (ACP)-mediated substrate shuttling during the catalytic cycle of fatty acid biosynthesis. To gain deeper insight into the fundamental processes of ACP-mediated substrate shuttling and the underlying conformational dynamics, spectroscopic methods like Förster resonance energy transfer and electron paramagnetic resonance spectroscopy shall be employed. These spectroscopic methods demand site-specific labeling of proteins with fluorophore or spin labels, which can be accomplished with the amber codon suppression technology. Through amber codon suppression, a non-canonical amino acid (ncAA) with an orthogonal functional group is incorporated site-specifically into the protein sequence, which can be used in chemoselective reactions for protein labeling.
This thesis is at the forefront of employing the technology of amber codon suppression for addressing complex biological questions on megasynthases. The successful production of ncAA-modified FASs is challenging. With the aim of incorporating ncAAs into the multidomain 540 kDa large murine FAS, we by far exceed boundaries of documented application of amber codon suppression. Most of the proteins that are reported by Liu & Schultz in applications of amber codon suppression are in the range of 30kDa - for example the TE domain of human FAS. In the same review, the largest protein amber codon suppression was applied to is a potassium channel with roughly 80 kDa. Thus, to the best of my knowledge no protein exceeding 100 kDa has been used in amber codon suppression so far.
In this thesis a low-complex, well-plate based reporter assay is presented, based on an ACP-GFP fusion protein for fast and efficient screening of ncAA incorporation. Reliability and applicability of the reporter assay is demonstrated by successful upscaling to larger protein constructs and increased expression scale.
As outlined in this thesis, we have carefully set up methods for the modification of murine FAS and made several achievements:
(i) We have created our own toolbox with a multitude of suppressor plasmids and various orthogonal pairs. pACU and pACE plasmids are compatible for fast exchange of cassettes, and cloning procedures are optimized for modification of synthetases by site-directed mutagenesis. (ii) We have organic synthesis of several ncAAs stably running in the lab and synthesis of other ncAAs can be established when required. Therefore, extensive screening at moderate costs is possible. (iii) We have established a reporter assay for screening our own library of vectors for amber codon suppression and for optimizing incorporation of ncAAs. (iv) We successfully incorporated ncAAs into subconstructs and full-length murine FAS, and collected initial promising results for the application of these proteins in spectroscopic methods. Thus, laying the foundation for future studies to address fundamental questions of the ACP-mediated substrate shuttling and other conformational dynamics of these enzymes.
Paramyxo- and pneumoviruses include many pathogens with great relevance for human and animal health. To identify common host factors involved in the Paramyxo- and Pneumoviridae life cycle as a basis for new insights in the biology of these viruses and the development of rationally designed therapeutics, genome scale siRNA screens with wild-type measles, mumps, and respiratory syncytial viruses in A549 cells, a human lung adenocarcinoma cell line, were performed. A comparative bioinformatics analysis yielded different members of the coatomer complex I, the translation factors ABCE1 and eIF3A, and several RNA binding proteins as cellular proteins with proviral activity for all three viruses. The strongest common hit, ABCE1, an ATP-binding cassette transporter member, was chosen for further study. We found that ABCE1 supports replication of all three viruses, confirming its importance for both virus families. While viral protein kinetics showed that ABCE1 knockdown resulted in a drastic decrease of MeV protein expression, viral mRNA kinetics are not directly affected by a reduction of ABCE1.
The impact of ABCE1 on viral and global cellular translation was investigated using both 35S metabolic labelling and non radioactive fluorescent protein labelling. ABCE1 knockdown strongly inhibited the production of MeV proteins, while only modestly affecting global cellular protein synthesis and showed that ABCE1 is specifically required for efficient viral, but not general cellular, protein synthesis, indicating that paramyxoand pneumoviral mRNAs may exploit specific translation mechanisms.
In a second approach the efficacy of the small-molecule polymerase inhibitor ERDRP-0519 against MeV was assessed in squirrel monkeys. Animals treated with the drug experienced less severe clinical disease compared to untreated controls, and this effect correlated with the onset of drug treatment.
We observed a reduction of levels of PBMC-associated viremia and virus release in the upper airways, illustrating effective inhibition of virus replication by the drug treatment. ERDRP-0519 drug treatment also alleviated MeV-induced immunosuppression. In addition to providing proof-of-concept for the support of MeV eradication efforts by preventing disease and transmission with a small-molecule polymerase inhibitor, this dissertation provides a novel perspective on cellular proteins that impact the replication of MeV, MuV and HRSV and highlights the role of ABCE1 as host factor that is required for efficient paramyxo- and pneumovirus translation.
ATP-binding cassette (ABC) transporters constitute an omnipresent superfamily of integral membrane proteins, which catalyze the translocation of a multitude of chemically diverse substrates across biological membranes. In humans, ABC transporters typically act as highly promiscuous exporters, responsible for many physiological processes, multi-drug resistance, and severe diseases, such as hypercholesterolemia, lipid trafficking disorders, and immune deficiency. In all ABC transporters, ATP-driven movements within two highly conserved nucleotide-binding domains (NBDs) are coupled to conformational changes of two transmembrane domains (TMDs), which provide a framework for substrate binding and release on the opposite side of the membrane and enable the transporter to cycle between inward-facing and outward-facing orientations. Several structures of ABC transporters determined either by X-ray crystallography or single-particle electron cryo-microscopy (cryo-EM) have been reported, mostly exhibiting a variation of the inward-facing state, which highlights their dynamic behavior. However, for a complete understanding of the conformational dynamics, further structural information on intermediates is needed – especially for heterodimeric ABC transporters, which are predominant in humans and for which only limited structural information is available.
One prime example of such human heterodimeric ABC transport complexes is the transporter associated with antigen processing (TAP). TAP is a key player of the adaptive immune response, because it translocates proteasomal degradation products into the ER lumen for loading of MHC I molecules. Many functional aspects of TAP have been disclosed in recent years. However, structural information is lacking far behind and a major challenge in the field of medical relevant transporters. Recently, the heterodimeric ABC export system TmrAB (Thermus thermophilus multidrug resistance proteins A and B) was identified as an ortholog of TAP, by sharing structural homology with TAP and, intriguingly, being able to restore antigen presentation in human TAP-deficient cells. Thus, TmrAB is a biochemically well-characterized ABC exporter that can be regarded as a functional ortholog of TAP and serves as a model system for (heterodimeric) ABC export systems in general.
Thus, to illuminate the molecular basis of substrate translocation by single-particle cryo-EM, one of the main objectives of this work was the generation of stabilizing chaperones (synthetic antibodies, nanobodies, cyclic peptides) to reduce the conformational heterogeneity of TAP and TmrAB. Selected antibodies were analyzed with respect to stable complex formation, conformational trapping, and the ability to serve as alignment tools for structural studies by single-particle cryo-EM. Both antibody types were shown to form sufficiently stable complexes to serve as a rigid body for EM analyses. However, all selected antibodies bound to the inward-facing state exclusively.
Hence, for EM studies, various ligands were added to elucidate the full spectrum of conformational states during the catalytic cycle. For TAP, first attempts by negative-stain EM revealed a homogenous distribution of particles on the grid. Surprisingly, no transporter-like features were observed although various attempts were applied to increase the overall sample quality.
For TmrAB, in contrast, the complete conformational space in a native-like lipid environment under turnover conditions was mapped. Cryo-EM analysis of TmrAB incubated with ATP-Mg2+ and substrate revealed two distinct inward-facing conformations (IFwide and IFnarrow) as well as two asymmetric conformations with dimerized NBDs, which were markedly different from all previously reported structures. Here, the catalytically active site was slightly wider and contained ADP, while ATP was still bound at the catalytically-inactive site within the NBDs, demonstrating an asymmetric post-hydrolysis state. Intriguingly for the inward-facing conformations, a weak additional density close to residues M139TmrB and W297TmrB was observed in the inward-facing conformation, which displayed a higher degree of cytosolic gate opening (IFwide) indicating the presence of substrate. To verify that this density corresponds to substrate, single alanine mutations of M139TmrB and W297TmrB were introduced, leading to a strong reduction in substrate binding and transport. Since substrate release requires the opening of the extracellular gate, the absence of an outward-facing open conformation indicated that the opening must be highly transient. In order to explore the outward-facing open conformation, a cryo-EM analysis of the catalytically-inactive TmrAE523QB mutant upon incubation with ATP-Mg2+ was performed. Remarkably, within the same dataset, two different outward-facing conformations (occluded and open) were resolved, both in an ATP-bound state, which indicated that binding of ATP is sufficient to drive the large-scale conformational transition from inward-facing to outward-facing open. To explore the effect of nucleotide hydrolysis, TmrAB was trapped by vanadate. Again, two populations were observed, representing the outward-facing open and outward-facing occluded conformation.
Based on several structures of key intermediates, determined under turnover conditions or trapped in the pre-hydrolysis and hydrolysis transition state, for the first time the complete description of the ATP hydrolysis and translocation cycle of a heterodimeric ABC transport complex was elucidated in one single study. By mapping the conformational landscape during active turnover, aided by mutational and chemical modulation of kinetic rates, fundamental and so-far hidden steps of the substrate translocation cycle of asymmetric ABC transporters were resolved and a general template for (heterodimeric) ABC exporter-catalyzed substrate translocation was provided.
Proteostasis stressors that destabilize the cellular proteome, like heat shock, trigger transcription and translational reactions leading to the accumulation of heat shock proteins, also called molecular chaperones. During stress, induction of stress response genes is prioritized so that molecular chaperones and other stress response proteins are synthesized to cope with proteome misfolding and aggregation. In order to promote the selective translation of stress-specific genes, translation of others genes that are nonessential for cell survival has to stop. Nonessential protein-coding mRNAs accumulate in the cytosol with the associated proteins to form granular structures called stress granules (SG). These membrane-less organelles are thought to be involved in cell survival, mRNA stabilization and mRNA triage. They were proposed to form via the liquid-liquid phase separation which can be triggered by the high local concentration of RNA-binding proteins. mRNAs were long thought to simply play a scaffolding role by bringing RNA-binding proteins together and allowing their concentration and local aggregation. Recently, the active role of mRNAs in the SG assembly became apparent, too. For example, the spontaneous assembly of total yeast RNA into granules was observed, and these RNA granules showed a large overlap with SG transcriptome. Furthermore, cytosolic mRNAs can be released from polyribosomes under stress and be exposed to the cytosolic contents as free mRNAs. It has been suggested that this massive increase of free mRNA in the cytosol might overload the capacities of RNA-stabilizing proteins. The remaining free mRNA molecules would then become exposed to misfolded and aggregation-prone proteins and trigger granulation.
We investigated the role of free mRNAs in different stress conditions during the early and chronic phases of stress response and explored their involvement in SGs assembly and amlyoidogenesis. We identified and studied the interactome of a free mRNA probe incubated with heat shocked cell lysate by means of quantitative mass spectrometry. Proteomics analysis allowed us to identify 79 interactors of free mRNA. Among these interactors, we focused on the translation initiation factor eIF2α and on the RNA methyltransferase TRMT6/61A. Both interactions were verified biochemically, which confirmed that the association is enhanced in heat shocked lysate. In vitro reconstitution showed that free mRNA and TRMT6 interact directly. Ex vivo pulldowns revealed that eIF2α and TRMT6/61A interact under stress conditions and that this interaction is RNA-dependent.
TRMT6/61A is a tRNA methytransferase responsible for the methylation of the adenosine 58 at the position 1 producing m1A. However, also mRNAs have been recently found to be methylated by TRMT6/61A. Our bioinformatics analyses revealed that significantly more mRNAs enriched in SG contain the motif for methylation than SG-depleted mRNAs. We hypothesized that m1A methylation of mRNAs could constitute a tag for the mRNAs targeting to SGs. TRMT61A knock-down (KD) cell lines were generated using the CRISPR-Cas9 technique. In TRMT61A KD cells, m1A was significantly reduced on mRNAs, which correlated with an increased sensitivity of the cells to proteostasis stress. KD cells also showed defects in SG assembly. In heat shocked cells, an m1A motif-containing mRNA recovered better after returning to normal temperature than a control mRNA with mutated motif. In addition, we could isolate SGs and analyze their m1A and m6A content by mass spectrometry. While m6A content in SG mRNAs was very similar to cytosolic mRNAs, m1A was almost 8 times enriched in SGs. Thus, we could confirm experimentally the results of the bioinformatics analysis and directly support the hypothesis that m1A is a tag to direct mRNAs for sequestration. Finally, we compared amyloidogenesis in wild-type and TRMT61A KD cell lines. Cells with reduced levels of TRMT61A demonstrated an increased accumulation of transfected Aβ and an impaired aggregate clearance. Various assays led us to conclude that the lack of m1A deposition on mRNAs enhanced RNA co-aggregation with amyloids.
Based on our results, we propose a model explaining the fate of free mRNA during proteostasis stress. Upon polysome disassembly, free mRNA is released and becomes free to interact with other proteins, including the methyltransferase TRMT6/61A. TRMT6/61A methylates the freed mRNAs containing the cognate motif. The m1A tag then targets mRNAs to SGs promoting sequestration. Upon stress release, SGs disassemble, thus releasing rescued mRNAs which could now reenter translation and support cell recovery. On the other hand, non-sequestered mRNAs increasingly co-aggregate with aggregating proteins. Thus, deficiency of the N1-adenine methylation of mRNAs due to the lack of TRMT6/61A increases the amount of unpacked mRNAs. The deposition of m1A on mRNAs could then be a way to protect them during exposure to stress, to limit their co-aggregation with misfolded proteins and to allow a faster recovery upon stress release.
Enolase is a glycolytic enzyme, which catalyzes the inter-conversion of 2-phosphoglycerate to phosphoenolpyruvate. Altered expression of this enzyme is frequently observed in cancer and accounts for the Warburg effect, an adaptive response of tumor cells to hypoxia. In addition to its catalytic function, ENO-1 exhibits other activities, which strongly depend on its cellular and extracellular localization. For example, the association of ENO-1 with mitochondria membrane was found to be important for the stability of the mitochondrial membrane, and ENO-1 sequestration on the cell surface was crucial for plasmin-mediated pericellular proteolysis. The latter activity of ENO-1 enables many pathogens but also immune and cancer cells to invade the tissue, leading further to infection, inflammation or metastasis formation. The ability of ENO-1 to conduct so many diverse processes is reflected by its contribution to a high number of pathologies, including type 2 diabetes, cardiovascular hypertrophy, fungal and bacterial infections, cancer, systemic lupus erythematosus, hepatic fibrosis, Alzheimer’s disease, rheumatoid arthritis, and systemic sclerosis. These unexpected non-catalytic functions of ENO-1 and their contributions to diseases are the subjects of this review.
Die Lebensfunktion der Zelle beruht unter anderem auf der Funktion und Wechselwirkung der Nukleinsäuren DNA (2’-Desoxyribonukleinsäure) und RNA (Ribonukleinsäure). Mit Hilfe von PDS (engl. ’pulsed dipolare spectroscopy’)-Techniken, basierend auf der EPR (engl. ’electron paramagnetic resonance’)-Spektroskopie, können Abstände in einem Bereich von 2-10 nm zwischen zwei markierten Positionen einer Nukleinsäure bestimmt werden. Daneben kann mit der Abstandsverteilung auf die Flexibilität des Moleküls geschlossen werden. Durch PDS-Messungen eröffnet sich die Möglichkeit, Bewegungen und Zustandsänderungen zu untersuchen. Die Messungen beruhen auf der dipolaren Kopplung von Radikalen (Spinlabel). Da die gemessenen dipolaren Kopplungen eine anisotrope Wechselwirkung sind, können an starren Systemen neben den Abstandsinformationen auch die Orientierungen der beiden Spinlabel zueinander bestimmt werden. Diese zusätzliche Information ermöglicht es, mittels orientierungsselektiver PDS-Messungen noch genauer die Geometrie und Flexibilität des Systems zu untersuchen. Klassischerweise werden alle Messungen mit der Doppelfrequenztechnik PELDOR (engl. ’pulsed electron-electron double resonance’) durchgeführt. Einzelfrequenzmethoden basieren dagegen auf Breitbandanregung, die mit den technischen Gegebenheiten l nge nicht möglich war. Eine solche Sequenz ist 2D-SIFTER.ImmRahmen dieser Arbeit von PELDOR ausgehende, weiterentwickelte Simulationsprozedur etabliert. Eine große Herausforderung ist die eindeutige Interpretation der sensitiven orientierungsselektiven PELDOR-Messungen. Sie mittels MD (Moleküldynamik)-Simulationen zu beschreiben war bisher nur qualitativ möglich. Allerdings wurden mehrere neue Kraftfelder publiziert. Mit einem quantitativen Vergleich mit orientierungsselektiven PELDOR-Daten kann sichergestellt werden, dass die Flexibilität des Systems durch Kraftfelder richtig beschrieben ist. PELDOR-Zeitspuren, gemessen bei Raumtemperatur und 50 K, unterscheiden sich besonders in ihrer Dämpfung. Der physikalische Unterschied beider Messungen konnte durch MD-Simulationen qualitativ nachvollzogen worden. Eine Schwierigkeit für speziell orientierungsselektive PELDOR-Messungen ist die aufwendige Synthese von mit dem starren Ç-Label markierten Nukleinsäuren. Als Alternative wurde in der Sigurdsson-Gruppe das halbstarre IMU-Label entwickelt. Die Analyse der orientierungsselektiven Daten ergab ein klares Bild der Dynamik dieses Labels. Ein weiterer interessanter Spinlabel ist der `G. Dieser Label ist nicht kovalent gebunden, sondern interkaliert in eine Stelle der Nukleinsäure, in der eine Guanin- Base fehlt. MD-Simulationen im quantitativen Vergleich mit orientierungsselektiven PELDOR-Messungen an verschiedenen Magnetfeldern haben eine hohe Übereinstimmung. Dabei konnte gezeigt werden, dass der Label, interkaliert in eine dsDNA, flippen kann, was zu einer Ausmittelung der Anisotropie führt, allerdings zu keiner Verbreiterung der Abstandsverteilung. Dagegen wird in der dsRNA dieses Flippen um die Einfachbindung sterisch gehindert, so dass neben dem Abstand auch die Orientierung des Labels bestimmt werden kann. Kurze dsRNA-Bausteine tendieren dazu, Oligomere zu bilden, was zu Multispineffekten führte. Zusätzlich beeinflusst diese Aggregation die Dynamik der einzelnen RNAs. Daher musste dieses ’end-to-end’-Stacking verhindert werden. Eine Nukleobasean einem Ende der dsRNA führt zu einer Dimerisierung, während eine Nukleobase an beiden Seiten dieses Stacking vollständig verhindert. Messungen mit unterschiedlichen Salzkonzentrationen konnten zusätzlich zeigen, dass die Interaktion zweier dsRNAs bei höheren Salzkonzentrationen zunimmt.
The three major autoimmune diseases (ADs) of the liver are primary biliary cholangitis (PBC), primary sclerosing cholangitis (PSC), and autoimmune hepatitis (AIH). All of those diseases show an aggressive immune reaction resulting in the destruction of liver tissue and finally to the development of hepatic fibrosis.
PSC is an autoimmune mediated disease of unknown etiology. It is characterized by inflammation of intra- and extrahepatic bile ducts. The progressive destruction of the bile ducts can lead to liver cirrhosis and finally to liver failure. Clinical signs for PSC are increased alkaline phosphatase (AP) and gamma glutamyltransferase (GGT) levels, presence of perinuclear anti-neutrophil cytoplasmic antibodies (pANCA) and bile ducts with characteristic strictures and dilations of the biliary tree as well as onion skin fibrosis surrounding the damaged bile ducts. Currently, there is no established treatment for PSC patients. The administration of ursodeoxycholic acid (UDCA) is being use as a therapy. However, it merely serves a symptomatic treatment to reduce serum AP and GGT as well as the formation of gallstones. In the advanced stage of PSC, liver transplantation is the last therapeutic option. Mdr2-/- mice are an excepted mouse model for human PSC. Such mice show lymphocytes infiltration into the liver, bile duct lesions, as well as the presence of the typical onion skin-like pericholangitis and periductal fibrosis.
AIH is a rare chronic autoimmune disease of the liver that results from the loss of self-tolerance to hepatocytes and leads to destruction of the hepatic parenchyma with the onset of cirrhosis. Clinical signs for AIH are elevated alanine aminotransferase (ALT) and aspartate transaminase (AST) levels, hypergammaglobulinemia and different types of autoantibodies. In addition, interphase hepatitis with lymphocytic and plasmacellular infiltrates in the periportal field are characteristic for AIH. Two different subtypes of AIH exist and depending on their autoantibody profile they can be distinguished into AIH type 1 which is characterized by the presence of anti-nuclear (ANA) and/or anti-smooth muscular (SMA) autoantibodies, and AIH type 2 showing liver/kidney microsomal autoantibodies (LKM-1). LKM-1 recognizes the major autoantigen, the 2D6 isoform of the cytochrome P450 enzyme family (CYP2D6). One mouse model for AIH is the CYP2D6 model in which the injection of Ad-2D6 leads to a breakdown of the immune tolerance by the destruction of hepatocytes.
There are some patients with autoimmune diseases of the liver who have both cholestatic and hepatic liver enzymes and histological features suggestive of two different liver diseases. These patients are diagnosed with an overlap syndrome (OS).
In my thesis I generated an animal model with characteristics of both diseases, which would mimic features of human PSC-AIH OS. Mdr2-/- mice which spontaneously develop PSC were infected with Ad-2D6 to trigger the autoimmune-driven hepatic injury. Pathogenesis of PSC-AIH OS mice was compared to mice with solitary PSC or AIH. Naïve FVB wild type mice have been used as healthy controls. The characterization of the PSC-AIH OS model was done by analyzing serological parameters like ALT, AP, different antibodies like pANCA, LKM-1 like CYP2D6 and total IgG. Additionally, fibrosis and cholangitis were analyzed by immunohistochemistry and Western blotting. Moreover, cellular infiltrations of CD4+ and CD8+ T cells, dendritic cells (DCs), monocytes/macrophages and neutrophils were determined with immunohistochemistry. Finally, the overall immune balance in the liver and the frequency of CYP specific T cells were analyzed via flow cytometry. Our new mouse model indeed represents the characteristics of both PSC and AIH and mimics features of the human PSC-AIH OS. It allows studying the development of a PSC-AIH OS and how the two overlapping diseases are influencing one another. In a second approach I wanted to induce CYP2D6-specific tolerance in AIH mice. Therefore, I tried four different approaches, namely intranasal peptide administration, injection of tolerogenic DCs, antigen-coupled splenocytes, and Ag-coupled nanoparticles (NP) and evaluated their potential to induce CYP2D6 specific Treg with the capacity to prevent AIH in mice. Unfortunately, the intranasal peptide administration and also the injection of tolerogenic DCs did not increase the amount of CYP2D6 specific Treg which would lead to a reduction of the frequency of inflammatory T cells. Surprisingly, the injection of antigen-coupled splenocytes showed the opposite effect characterized by a very strong cytokine secretion in the tolerized mice. The use of NPs led to an increase in CYP2D6 specific Treg as well as in decrease in the frequency of inflammatory T cells and finally has the potential for a therapeutic approach.
In summary, the generated PSC-AIH OS model represents many clinical signs which can also be observed in PSC-AIH OS patients. This model can be used to study the etiology of this overlap syndrome and further to test potential therapeutic approaches. The different immune tolerance induction pathways which I tried in the AIH model show that NPs have to potential to induce immune tolerance but this approach has to be refined and the outcome has to be characterized in more detail.
Die CXCR4-CXCL12-Signalachse gilt als eines der bislang am besten studierten Signalsysteme in der Hämatopoese. Allerdings stammt unser Wissen über diesen kritischen Signalweg maßgeblich aus subtraktiven Studien, wie z.B. knock-out Modellen oder pharmakologischer Inaktivierung. Zwar können aus diesen Modellen wichtige Erkenntnisse über die physiologische Rolle dieses Signalwegs abgeleitet werden, aber dennoch bleiben einige Phänomene ungeklärt. So konnte gezeigt werden, dass es sowohl bei CXCR4-Defizienz als auch bei Patienten mit dem WHIM-Syndrom (ausgelöst durch eine überaktive CXCR4-Mutante) zu einer ausgeprägten B-Zellaplasie kommt. Dies scheint intuitiv nicht vereinbar. Daher wurde in der vorliegenden Arbeit ein Modell mit einer überaktiven CXCR4- Mutante (CXCR41013/1013) hinsichtlich der (un)reifen Hämatopoese systematisch untersucht.
Zunächst wurden hämatopoetische Stamm- und Vorläuferzellen (HSPC) hinsichtlich der aberranten CXCR4-Signalweiterleitung ex vivo analysiert. Die CXCR4-Überaktivierung konnte sowohl in frühen Effekten nach Aktivierung des Rezeptors (F-Aktinpolymerisierung, Aktivierung des MAPK- Signalweges), als auch in späten, zellfunktionellen Effekten (Migrationsassay) nachgewiesen werden. Die veränderte CXCR4 Signalintegration hatte auch bereits in der Homöostase organismische Konsequenzen im Mausmodell. So konnte eine massiv vergrößerte HSPC-Population in der Milz von CXCR41013/1013-Tieren detektiert werden, im Sinne einer extramedullären Hämatopoese. Knochenmarks-HSPC aus CXCR41013/1013-Tiere zeigten ein massiv eingeschränktes (serielles) Repopulationspotenzial. Kombiniert mit der oben genannten ausgeprägten extramedullären Hämatopoese in diesen Tiere interpretieren wir diese Beobachtung als starken Hinweis auf eine dysfunktionelle Interaktion der Stammzellen mit der hämatopoetischen Stammzellnische im Knochenmark. In diesem Zusammenhang besonders interessant ist die Tatsache, dass auch ein Kompetitorknochenmark das Überleben einer Sekundärtransplantation nicht sichert. Dabei ist zu diskutieren, ob dieser Effekt durch eine effizientere Besetzung von Stammzellnischen durch CXCR41013/1013-Zellen, eine Akkumulation von CXCL12 in der Knochenmarkflüssigkeit (siehe unten) oder eventuell sogar ein vesikelabhänginger Transport von mutiertem CXCR4 in Kompetitorzellen ausgelöst wird. Ein weiteres Merkmal dieser Dysfunktion könnte ebenfalls die gezeigte Akkumulation von CXCL12 in der Knochenmarkflüssigkeit von CXCR41013/1013-Tiere darstellen. Diese Akkumulation könnte die Suppression co-transplantierter wildtypischer Hämatopoese sowie die verminderte Effizienz der G-CSF-induzierten Stammzellmobilisierung funktionell erklären. Zusätzlich konnte gezeigt werden, dass die Mobilisierung von Stammzellen aus dem Knochenmark durch einen CXCR4- Inhibitor in CXCR41013/1013-Tieren ebenfalls erheblich hinter der in Wildtypmäusen zurückbleibt.
Analog zu Patienten mit WHIM-Syndrom zeichnen sich CXCR41013/1013-Mäuse weiterhin durch eine ausgeprägte Leukopenie, insbesondere durch einen schweren B-Zell-Mangel, aus. Aus diesem Grund wurde die B-Lymphopoese und humorale Immunfunktion genauer analysiert. Eine grundsätzliche humorale Immunkompetenz von CXCR41013/1013-Tieren konnte nachgewiesen werden, jedoch ist die B-Memory-Funktion erheblich eingeschränkt. Durchflusszytometrisch und funktionell konnte eine reduzierte preB/pro-B Population im Knochenmark bei einer gleichzeitig vergrößerten preB/pro-B Population in der Milz (vgl. extramedulläre Hämatopoese) nachgewiesen werden. Ebenfalls konnten wir in dieser Zellpopulation eine stark erhöhte CXCR4-Oberflächenexpression im Vergleich zu wildtypischen Zellen nachweisen. Da diese unreifen B-Zellen keine verstärke Apoptoserate aufweisen, gehen wir derzeit davon aus, dass der Differenzierungsstopp nicht durch selektiven Zelltod, sondern durch aberrante Retention der preB/proB-Zellen in einer primitiven B-Vorläufer- Nische im Knochenmark zustande kommt, beziehungsweise durch eine gestörte Migration in differenzierende Nischen im Knochenmark. Alternativ könnte die Überdosis CXCR4-Signal differenzierenden Signalen entgegenstehen. Beide Hypothesen können das eingangs erwähnte Paradoxon bezüglich einer B-Zellaplasie in CXCR4-defizienten und CXCR4-überaktiven Zellen hinreichend erklären.
As central component of the peptide loading complex, the ABC transporter TAP is a key player in the adaptive immune response. By recognizing and translocating antigenic peptides derived from proteasomal degradation into the ER lumen it connects the processing of harmful intruders and the marking of an infected cell for elimination. This work focused mainly on the interaction between TAP and one of its viral inhibitors. Of the five known TAP inhibitors, ICP47 is the only one that is not anchored in the ER membrane and has a nonomolar affinity to TAP. These properties and its specific architecture make it an interesting protein engineering tool that can be used in a variety of ways to generate functionally arrested TAP complexes. Different lengths of ICP47 were chosen to map the optimal distance between the binding pocket and the N-terminal elbow helix of either TAP1 or TAP2. I demonstrated that the interaction of fused ICP47 with coreTAP inhibits antigen presentation via MHC I. Interestingly, the loss of MHC I surface expression only depended on the presence of the active domain and not on the length of the fused ICP47 fragments. Summarizing it can be said that TAP complexes containing an intact active domain of ICP47 successfully suppressed MHC I surface expression. Considering the MHC I surface expression in the use of free ICP47 fragments it was revealed that the active domain may not be sufficient. All free constructs, except the one that contains exclusively the active domain (1-35), were able to fully arrest peptide translocation, while the fragment 1-35 partially restored MHC I surface expression. This was the first evidence suggesting that more residues might be present in the ICP47 sequence that contribute to the interaction with TAP.
Further characterization of the ICP47-coreTAP fusion complexes comprised the determination of their thermostability and melting temperatures. The ICP47-coreTAP fusion complexes revealed a preferred orientation for ICP47. The ICP47(1-65) fragment led to a stable complex only if fused to TAP2, highlighting an interesting asymmetry at the TAP1/TAP2 interface, which suggests a shorter distance of the C-terminus of the stabilizing region to the elbow helix of TAP2 than of TAP1. The shorter fragments 1-35 and 1-50, and the ICP47 linker fragments, which inhibited, but did not trigger any thermostabilizing effects on TAP, revealed a second hint for the presence of other residues important for the ICP47/TAP interaction. To define the thermostability in more detail, the melting temperature of complexes with fused or freely bound ICP47 fragments was determined. Short fused fragments of ICP47 (residues 1-35 or 1-50) did not fully stabilize the TAP complex. Only ICP47 fragments longer than residues 1-50 raised the melting temperature to the full extent and led to a completely stabilized complex, suggesting that the critical melting temperature, which determines whether a complex is fully stabilized or not, is about 44-45°C. By comparing different ICP47 proteins from the herpesviral clade, I further noticed that the 21 residues following the active domain are highly conserved. The residues in this region were exchanged by glycines and alanines to study their impact on the thermostabilization of TAP. I demonstrated that several charged residues, an alanine rich, and a proline rich sequence were mainly responsible for the preservation of high melting temperatures. In summary, these findings reveal a dual inhibition mechanism of ICP47. While the active domain of ICP47 is wedged at the TAP1/2 interface and arrests the complex in an open-inward facing conformation, the highly conserved C-terminal region stabilizes the ICP47/TAP interaction and generates a thermostabilized TAP complex.
The second part of this thesis deals with two alternative expression and stabilization strategies for coreTAP, designed to provide a 1:1 ratio of TAP subunits during protein biosynthesis. Different glycine-serine (GS) linkers and a self cleaving 2A site were im- plemented into the TAP sequence and used for comparison with the classical coreTAP. Despite their functionality in antigen translocation, the utilization of GS linkers proved to be unsuitable due to low expression and scarce purification efficiency caused by the unfeasible orthogonal purification. In contrast, the use of a 2A site allowed orthogonal His10- and SBP-tag purification and yielded comparable amounts to the classical coreTAP. However, the ICP47/coreTAP interaction appeared to be hampered by the modified N-terminus of ICP47, due to the cleavage process.
The third and last part of this work deals with the Thermus thermophilus ABC trans- porter TmrAB, which was identified to be part of the same ABC subfamily as TAP. The structure of TmrAB is similar to that of coreTAP and includes a TMD and an NBD for each subunit. In comparison to TAP, TmrAB has a broader substrate range, but it can transport peptides, which are also transported by TAP. Since the natural substrate, and thus the actual function, of TmrAB has not yet been identified, it is counted among the multidrug resistance ABC transporters, from where it also takes its name. In this work, the question was investigated whether TmrAB can be utilized as a TAP substitute. To compare the function of TmrAB and TAP in a natural cell environment, the N-terminal domains of the TAP subunits called TMD0s were fused to the TmrAB subunits and subsequently expressed as different combinations. I found that especially the hybrid complexes containing a TMD0 of TAP2 were functional in terms of MHC I surface expression. Furthermore, TmrAB with TMD0 co-localized prevalently with the ER marker PDI while complexes without TMD0 did not co-localize. Interestingly, the analysis of the interaction with components of the PLC revealed that interaction with tapasin could only occur when a TMD0 was present. In turn, calreticulin, MHC I, and ERp57 were bound, regardless of the presence of a TMD0. It is remarkable that a bacterial protein, sharing only 27-30% sequence identity with human TAP is able to take over a key function of our adaptive immune system. Yet, TmrAB originates from a hyperthermophilic bacterium and may have assembly and folding difficulties that the human cell seeks to overcome by recruiting chaperones like calreticulin and ERp57. Although further experiments will be necessary to analyze the interaction of TmrAB with the PLC components in more detail, TmrAB appears to be homologous to coreTAP, not only in terms of sequence and structure, but also in terms of function.
Supersaturating formulations are widely used to improve the oral bioavailability of poorly soluble drugs. However, supersaturated solutions are thermodynamically unstable and such formulations often must include a precipitation inhibitor (PI) to sustain the increased concentrations to ensure that sufficient absorption will take place from the gastrointestinal tract. Recent advances in understanding the importance of drug-polymer interaction for successful precipitation inhibition have been encouraging. However, there still exists a gap in how this newfound understanding can be applied to improve the efficiency of PI screening and selection, which is still largely carried out with trial and error-based approaches. The aim of this study was to demonstrate how drug-polymer mixing enthalpy, calculated with the Conductor like Screening Model for Real Solvents (COSMO-RS), can be used as a parameter to select the most efficient precipitation inhibitors, and thus realise the most successful supersaturating formulations. This approach was tested for three different Biopharmaceutical Classification System (BCS) II compounds: dipyridamole, fenofibrate and glibenclamide, formulated with the supersaturating formulation, mesoporous silica. For all three compounds, precipitation was evident in mesoporous silica formulations without a precipitation inhibitor. Of the nine precipitation inhibitors studied, there was a strong positive correlation between the drug-polymer mixing enthalpy and the overall formulation performance, as measured by the area under the concentration-time curve in in vitro dissolution experiments. The data suggest that a rank-order based approach using calculated drug-polymer mixing enthalpy can be reliably used to select precipitation inhibitors for a more focused screening. Such an approach improves efficiency of precipitation inhibitor selection, whilst also improving the likelihood that the most optimal formulation will be realised.
Objectives: The objective of this review is to provide an overview of PK/PD models, focusing on drug-specific PK/PD models and highlighting their value-added in drug development and regulatory decision-making.
Key findings: Many PK/PD models, with varying degrees of complexity and physiological understanding, have been developed to evaluate the safety and efficacy of drug products. In special populations (e.g. pediatrics), in cases where there is genetic polymorphism and in other instances where therapeutic outcomes are not well described solely by PK metrics, the implementation of PK/PD models is crucial to assure the desired clinical outcome. Since dissociation between the pharmacokinetic and pharmacodynamic profiles is often observed, it is proposed that physiologically-based pharmacokinetic (PBPK) and PK/PD models be given more weight by regulatory authorities when assessing the therapeutic equivalence of drug products.
Summary: Modeling and simulation approaches already play an important role in drug development. While slowly moving away from “one-size fits all” PK methodologies to assess therapeutic outcomes, further work is required to increase confidence in PK/PD models in translatability and prediction of various clinical scenarios to encourage more widespread implementation in regulatory decision-making.
Die Steuerung biochemischer Prozesse oder die Verbesserung von Materialien erfordert zunächst ein tiefgründiges Verständnis über die zugrundeliegenden Systeme. Zur Untersuchung eignet sich Licht als ideales Werkzeug, da hiermit nützliche Informationen über die chemische Struktur, ihre Eigenschaften sowie den zusammenhängenden, schnellen Reaktionsabläufen erhalten werden können. Um die Aufklärung zu erleichtern können kleine, chemische Verbindungen eingeführt werden, welche beispielsweise ein Fluoreszenzmarker, eine photolabile Schutzgruppe oder eine photoschaltbare Verbindung sein können. Von jeweils einem Vertreter dieser Moleküle wurden unterschiedliche Studien durchgeführt, dessen Ergebnisse in dieser Arbeit in insgesamt drei Projekten zusammengefasst werden.
Zunächst wurde die Funktionalität der Helikase RhlB untersucht, die der Familie der DEAD-Box Proteine zugeordnet wird, und RNA-Duplexe in ihre Einzelstränge entwindet. Als RNA-Modellduplex diente JM2h, an dem ein RNA-Einzelstrang fluoreszenzmarkiert war (M2AP6). Die Einführung dieses Markers ermöglichte die Durchführung von statischen Fluoreszenzmessungen sowie von Mischexperimenten, die mit Hilfe der stopped-flow-Technik durchgeführt wurden. In den einleitenden Studien wurde die Helikase weggelassen, wodurch der Fokus auf den Fluoreszenzeigenschaften der RNA gelegt wurde. Die Ergebnisse hierzu zeigten, dass die Fluoreszenzintensität des Einzelstrangs durch Zugabe des komplementären Strangs deutlich abnimmt, wobei das Minimum bei einem äquimolaren Verhältnis erreicht wird. Die dazugehörigen stopped-flow-Messungen zeigten eine Beschleunigung der Hybridisierungsreaktion, wenn höhere Konzentrationen des Gegenstrangs in der Lösung vorhanden waren. Nach anschließender Zugabe der Helikase zur Lösung wurde ein Anstieg der Fluoreszenzintensität erwartet, der vom separierten Einzelstrang M2AP6 herrühren sollte. Dieser Anstieg wurde jedoch erst nach weiterer Zugabe von ATP beobachtet, der auf eine ATP-Abhängigkeit der Entwindungsreaktion von RhlB hindeutet. Diese Abhängigkeit wurde auch bereits für andere Helikasen der DEAD-Box Familie entdeckt. Die korrekte Funktionalität sowie die ATP-Abhängigkeit wurden in stopped-flow-Messungen verfiziert, bei denen der Fluoreszenzanstieg auch zeitaufgelöst betrachtet werden konnte. Für die spektralen Korrekturen der Fluoreszenzspektren wurde ein selbstgeschriebenes MATLAB-Programm namens FluCY verwendet (engl.: Fluorescence Correction & Quantum yield), welches eine schnelle und fehlerfreie Verarbeitung des Datensatzes ermöglichte.
Die zwei im folgenden beschriebenen Projekte handeln von photoaktivierbaren Molekülen. Zum einen photolabile Verbindungen, welche die Funktion z.B. eines Biomoleküls durch eine chemische Modifikation deaktivieren können. Durch eine lichtinduzierte Reaktion kommt es zur Abspaltung der Modifikation und die Funktion ist wiederhergestellt. In dieser Arbeit wurden verschiedene photolabile Schutzgruppen untersucht, die denselben Chromophor BIST (BIsStyryl-Thiophen) tragen. Durch die Einführung dieses Chromophors absorbierten sämtliche untersuchte Verbindungen sehr effizient sichtbares Licht (epsilon(445)=55.700 M^(-1) cm^(-1)), wodurch der photoinduzierte Bindungsbruch mit Wellenlängen durchgeführt werden, die bei einer biologischen Anwendungen keinen Schaden an der Zelle anrichten würden. Hieraufhin wurden in statischen und zeitaufgelösten Absorptionsmessungen Teilschritte der Freisetzungsreaktion untersucht, indem nach Photoanregung die Absorptionsänderungen auf verschiedenen Zeitskalen analysiert wurden. Die ultraschnelle Dynamik im Piko- bis Nanosekundenbereich (10^(-12)-10^(-9) s) wird durch eine spektral breite, positive Absorptionsänderng dominiert. Diese impliziert, dass die Deaktivierung über den Triplettpfad abläuft, der die vergleichsweise niedrigen Freisetzungsausbeuten erklärt (phi(u) < 5). Aufgrund des hohen Extinktionskoeffizienten reichen dennoch bereits niedrige Strahlungsdosen aus, um eine Freisetzung zu initiieren. Der geschwindigkeitsbestimmende Schritt dieser Reaktion ist dem Zerfall des aci-nitro Intermediats zugeordnet. Für ein sekundäres Amin, welches mit BIST geschützt wurde, ist eine Lebensdauer des Intermediats von 71 µs gefunden worden.
In einigen Fällen ist es erwünscht, eine vorliegende Aktivität nicht nur ein-, sondern auch ausschalten zu können, wofür photochrome Verbindungen (oder Photoschalter) verwendet werden. Die in dieser Arbeit untersuchte Verbindung ceCAM ist ein Alken-Photoschalter und vollführt bei Bestrahlung mit Licht eine cis/trans-Isomerisierung. ceCAM ist das Cyanoester-Derivat (ce) von Cumarin-substituierten Allylidenmalonat, von denen beide Konformere sehr effizient sichtbares Licht absorbieren trans: epsilon(489)=50.300 M^(-1) cm^(-1); cis: epsilon(437)=18.600 M^(-1) cm^(-1)). Andere photophysikalische Eigenschaften umfassen u.a. hohe thermische und photochemische Stabilität. Letztere wurde über ein Experiment nachgewiesen, bei dem die lichtinduzierte Isomerisierung alternierend durchgeführt wurde und selbst bei über 250 Zyklen keine signifikate Abnahme der Absorption beobachtet werden konnte. Des Weiteren konnte die Reaktion mit Quantenausbeuten von 39% (trans) und 42% (cis) induziert werden, wobei im photostationären Gleichgewicht auch hohe Isomerenverhältnisse mit bis zu 80% (trans) und 96% (cis) akkumuliert werden konnten. Die Geschwindigkeit der Reaktion wurde mit Hilfe der Ultakurzzeit-Spektroskopie untersucht. Die Dynamik im Zeitbereich von ps-ns zeigte, dass die trans/cis-Isomerisierung unterhalb von 0,5 ns und die umgekehrte Reaktion noch viel schneller (wenige ps) abgeschlossen ist. Durch die Untersuchungen in dieser Arbeit an den BIST-Verbindungen und ceCAM sind viele vorteilhafte, photophysikalische Eigenschaften charakterisiert worden, wodurch sie als verbesserte Alternative zu den bisher bekannten photolabilen Schutzgruppen oder Photoschaltern anzusehen sind.