Fakultät für Chemie und Pharmazie - Digitale Hochschulschriften der LMU - Teil 02/06 Archiv

The kinetics of the electrophilic substitutions of indoles and pyrroles have been investigated and new sythetic applications (i.e. electrophilic allylations and benzylations of indoles and regio- and stereoselective ring opening reactions of epoxides with indoles and pyrroles) have been found.

Zusammenfassung: Das Ziel dieser Arbeit war es, die Funktion der hochkonservierten, essentiellen ABC-ATPase Rli1p in Saccharomyces cerevisiae aufzuklären. Es konnte gezeigt werden, dass beide Nukleotid-Bindungsdomänen und eine Scharnierregion, welche die beiden ABC-Domänen miteinander verbindet, wichtig für die Funktion von Rli1p sind. Des Weiteren konnte auch das N-terminale Eisen-Schwefel Cluster Bindemotiv als essentielle Domäne identifiziert werden. Durch Überexpression von inaktiven rli1p-Proteinvarianten wurde ein dominant-negativer Phänotyp erzeugt. Es konnte nachgewiesen werden, dass Rli1p im Zytoplasma lokalisiert ist und mit den Polyribosomen, den 80S Ribosomen und hauptsächlich mit der 40S Untereinheit co-fraktioniert. Des Weiteren konnte gezeigt werden, dass Rli1p mit einem Subkomplex des Translationsinitiationsfaktors eIF3 und ribosomalen Proteinen interagiert. Diese Interaktion ist abhängig von RNA und funktionalen ABC-Domänen, sowie von eIF3j/Hcr1p, einer Untereinheit des Translationsinitiationsfaktors eIF3. Um die zelluläre Funktion von Rli1p näher charakterisieren zu können, wurde versucht eine konditional temperatursensitive Mutante herzustellen und Rli1p durch Repression der Transkription in vivo zu depletieren. Beide Ansätze waren nicht erfolgreich. Aus diesem Grund wurden in vitro Depletionssysteme etabliert. Immundepletion oder Inaktivierung eines mit TEV-Protease spaltbarem Rli1p führte zu einer Reduktion der Translationsaktivität und demonstrierte damit einen direkten Einfluss von Rli1p auf die Translation. Eine vollständige Inhibition der Translation einer Reporter-mRNA konnte durch Zugabe eines a-RLI1-Antikörper erreicht werden, wobei die Translationsextrakte einen Zerfall von den Polyribosomen und der 80S Komplexe in 60S und 40S Untereinheiten aufwiesen. Die Immunoinhibition konnte durch die Zugabe von gereinigtem Rli1p rückgängig gemacht werden. Neben einer biochemischen konnte auch eine genetische Interaktion von RLI1 mit HCR1 nachgewiesen werden. Durch Kombination eines Dhcr1 Knockout mit einem C-terminal markierten Rli1p entstand ein synthetisch-temperatursensitiver Phänotyp. Nähere Untersuchungen dieses Stammes zeigten eine Reduktion von Polyribosomen und einen Defekt bei der 60S Bindung an den Prä-Initiationskomplex, während die Ribosomenbiogenese nicht beeinflusst wurde. Für die zelluläre Funktion von Rli1p konnte ein Modell erarbeitet werden, in dem Rli1p den Translationsinitiationsfaktor eIF3 auf dem 40S Ribosom assembliert oder richtig positioniert. Diese Funktion des Rli1p könnte auch in der Anti-Assoziation von 80S durch eIF1, eIF1A und eIF3 von Bedeutung sein. Dieses Modell soll in Folgeexperimenten geprüft werden und die Bindungsstelle von Rli1p im 43S Prä-Initiationskomplex durch Cryo-EM näher analysiert werden.

To become biologically active, proteins have to acquire their correct three-dimensional structure by folding, which is frequently followed by assembly into oligomeric complexes. Although all structure relevant information is contained in the amino acid sequence of a polypeptide, numerous proteins require the assistance of molecular chaperones which prevent the aggregation and promote the efficient folding and/or assembly of newly-synthesized proteins. The enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), which catalyzes carbon fixation in the Calvin-Benson-Bassham cycle, requires chaperones in order to acquire its active structure. In plants and cyanobacteria, RuBisCO (type I) is a complex of approximately 550 kDa composed of eight large (RbcL) and eight small (RbcS) subunits. Remarkably, despite the high abundance and importance of this enzyme, the characteristics and requirements for its folding and assembly pathway are only partly understood. It is known that folding of RbcL is accomplished by chaperonin and most likely supported by the Hsp70 system, whereas recent findings indicate the additional need of specific chaperones for assembly. Nevertheless, this knowledge is incomplete, reflected by the fact that in vitro reconstitution of hexadecameric RuBisCO or synthesis of functional plant RuBisCO in E. coli has not been accomplished thus far. In this thesis, attempts to reconstitute type I RuBisCO in vitro did not result in production of active enzyme although a variety of reaction conditions and additives as well as chaperones of different kind, origin and combination were applied. The major obstacle for reconstitution was found to be the incapability to produce RbcL8 cores competent to form RbcL8S8 holoenzyme. It could be shown that the RbcL subunits interact properly with the chaperonin GroEL in terms of binding, encapsulation and cycling. However, they are not released from GroEL in an assembly-competent state, leading to the conclusion that a yet undefined condition or (assembly) factor is required to shift the reaction equilibrium from GroEL-bound RbcL to properly folded and released RbcL assembling to RbcL8 and RbcL8S8, respectively. Cyanobacterial RbcX was found to promote the production of cynanobacterial RbcL8 core complexes downstream of chaperonin-assisted RbcL folding, both in E. coli and in an in vitro translation system. Structural and functional analysis defined RbcX as a homodimeric, arc-shaped complex of approximately 30 kDa, which interacts with RbcL via two distinct but cooperating binding regions. A central hydrophobic groove recognizes and binds a specific motif in the exposed C-terminus of unassembled RbcL, thereby preventing the latter from uncontrolled misassembly and establishing further contacts with the polar peripheral surface of RbcX. These interactions allow optimal positioning and interconnection of the RbcL subunits, resulting in efficient assembly of RbcL8 core complexes. As a result of the highly dynamic RbcL-RbcX interaction, RbcS can displace RbcX from the core-complexes to produce active RbcL8S8 holoenzyme. Species-specific co-evolution of RbcX with RbcL and RbcS accounts for limited interspecies exchangeability of RbcX and for RbcX-supported or -dependent assembly modes, respectively. In summary, this study helped to specify the problem causing prevention of proper in vitro reconstitution of type I RuBisCO. Moreover, the structural and mechanistic properties of RbcX were analyzed, demonstrating its function as specific assembly chaperone for cyanobacterial RuBisCO. Since the latter is very similar to RuBisCO of higher plants, this work may not only augment the general understanding of type I RuBisCO synthesis, but it might also contribute to advancing the engineering of catalytically more efficient crop plant RuBisCO both in heterologous systems and in planta.

Zelladhäsion wird durch definierte Erkennungsepitope der extrazellulären Matrixproteine vermittelt. Diese werden durch Plasmamembranproteine erkannt und gebunden. Unter den Zelladhäsionsrezeptoren dominieren Integrine. Im Mittelpunkt dieser Arbeit stand die Entwicklung eines Modellsystems, das eine detaillierte Untersuchung von Zell-Oberflächen-Interaktionensprozessen ermöglicht.

To become biologically active, most proteins need to fold into precise three dimensional structures. It has been well established that all the folding information is contained within the primary structure of a protein. However, the mechanisms utilized by proteins to avoid sampling the extraordinarily large amount of possible conformations during their folding process are just beginning to be understood. Molecular chaperones assist the folding of newly synthesized and denatured proteins in acquiring their native state in the crowded intracellular environment. As a nascent chain leaves the ribosome, it is captured first by the upstream chaperones and then possibly transferred to the downstream chaperonins. GroEL-GroES, the Hsp-60 of E.coli, is one of the best studied chaperone systems. An appreciable amount of data is available providing information regarding its structure and function. GroEL encapsulates the substrate into the central cavity where folding occurs unimpaired by aggregation and unwanted inter-molecular interactions. Nevertheless, many important aspects of the GroEL mechanism remain to be addressed. Some of the open questions we have addressed in this study include: In what conformation does a substrate protein bind to the apical domains of GroEL; how is it that GroEL is able to accelerate the rate of folding of certain proteins, and how do the conformational properties of the substrate change as it undergoes repeated cycling. By using ensemble FRET and Sp-FRET (Single Pair-Fluorescence Resonance Energy Transfer), we have probed the conformation of the model substrate DM-MBP (Double Mutant Maltose Binding Protein) during different stages of the functional cycle of GroEL. With Sp-FRET coupled to PIE (Pulsed Interleaved Excitation), we have been able to explore the heterogeneity of the GroEL bound substrate protein and observed a bimodal conformational distribution. One of the two populations is as compact as the native state, whereas the other is as extended as the unfolded protein in denaturant. This unfolding is a local phenomena and can also be observed when the substrate is transferred from DnaK/J system (bacterial Hsp70) to GroEL, indicating the possibility of the existence of this conformational heterogeneity in vivo as the protein follows the cellular chaperone pathway. Subsequent to GroEL binding, there is a transient expansion of the protein upon binding of ATP to GroEL, followed by compaction when GroES triggers the encapsulation of the protein inside the chaperonin cage. This transient expansion is however found not to be a necessary event for the rate acceleration of DM-MBP folding, since ADP-AlFx (transition state analogue of ATP hydrolysis) results in a much slower rate of expansion, which does not cause a change in the folding rate. Anisotropy measurements, probing the freedom of motion of different regions of the GroEL bound protein, revealed that there is a segmental release of the substrate protein from the GroEL surface upon binding of ATP and GroES. As a consequence, the hydrophobic collapse of the protein upon encapsulation by GroES follows a step-wise mechanism. In this process, less hydrophobic regions are released upon binding of ATP, prior to more hydrophobic ones which are released only by GroES binding. Thus, the order of Hydrophobic collapse is reversed as compared to spontaneous folding possibly resulting in conformationally different folding intermediates. Evidence that the folding pathway inside the cage differs from that of spontaneous folding was obtained by observing the effect of external perturbations (e.g. mutations in substrate protein and use of different solvent conditions) on the rate of spontaneous and GroEL assisted folding reactions. These two folding reactions respond differently to the introduced perturbations. Kinetic data obtained from ensemble FRET measurements suggest that the conformation of refolding intermediate is altered by the GroEL cavity, which leads to a folding pathway that is different from the spontaneous refolding pathway. In summary, this study revealed significant novel aspects of the GroEL folding mechanism and provided insights into the basis of rate acceleration of the substrate protein by the chaperonin. This work may thus contribute to advance our fundamental knowledge of the chaperonin system and the basic mechanism of protein folding.

Following oxidative stress often two kinds of DNA-lesions can be found: 8-OxodG and the 2,6-Diamino-4-hydroxy-5-formamidopyrimidine (FaPydG). Both derive from the DNA-Nucleobase guanine. The FaPydG-lesion can exist both in the beta-configuration and the alpha configuration. To clarify biological questions concerning the basepairing and the encoding features of both forms it was necessary to synthesise of the alpha-anomer of the carbocyclic FaPydG-DNA-lesion. The synthesis of the beta-cFaPydG-lesion was already finished in 2005 in the Carell group. In a sixteen-step synthesis the alpha-lesion could be prepared in the form of its phosphoramidite component. Oligonucleotides could be prepared on solid support using automated DNA-synthesis. Some of the synthesised DNA-Nucleotides contained the inserted FaPydG-lesion. Others showed an oxidation to alpha-c8-OxodG. This reaction is not known for natural lesion. Just a de-hydration to guanosine under harsh conditions is described. The observations can be ex-plained by formulation of a cyclic intermediate, which is possibly oxidized by the oxidation di-lution during the DNA-synthesis. If the sequence-dependent equilibrium between alpha cFaPydG and alpha c8-Hydro,hydroxydG is at the side of the closed form, an oxidation from this interme-diate directly to 8-OxodG is imaginable. This assumption is proven by the fact that at the same time synthesised and completely identically treated DNA-strands of different sequence didn´t show oxidation products. This could be ascertained by high resolution ESI-FTICR-mesurement. With this analytical method even smallest variations are detectable in a reliable way, whereas the often used MALDI-TOF-mesurements not always show these variations. Thermodynamic studies showed that alpha-cFaPydG doesn´t form a preferred DNA-basepair. All possible basepairs showed a strong destabilisation in comparison to the undamaged, cor-rect basepair. In vitro elongation experiments with S. cerevisiae polymerase Pol eta, G. stearothermophilus DNA-polymerase I and DinB from G. stearothermophilus showed that the alpha-lesion is a definitive block for all mentioned polymerases. Only by applying rigorous re-action conditions an elongation of the primer with BstPol I could be detected. This elongation showed a favored incorporation of dCTP, followed by an incorporation of dATP. In the time to come experiments to avoid the unwanted oxidation of alpha-cFaPydG will be es-sential to promote the cocristallisation of the alpha-cFaPydG-oligomere with the Fpg-protein from Lactococcus lactis and the BstPol I. In the second part of the thesis attempts were taken to prepare tetrafunctional amino acid derivatives, which were planned to be used in a PNA-template directed ligation reaction. Liga-tion takes place between two amino acids, so that for a ligation attempt with this method there is always needed a matching pair of amino acids. This pair requires a connection of the aminoacid to the PNA over the side chain of the amino acid. The N- and C-terminus of the amino acid had to be unprotected for the ultimate ligation reaction. These termini must not react during the solid support PNA-synthesis. After ligation the template should be separated selectively. Thus, the following requirements for the pair of amino acids result: The amino acids have to possess functional side chains, which exist naturally after cleavage of the PNA-template. For the selective cleavage of the template a con-nection orthogonal to acid and base unstable protecting groups is necessary, because these are already needed for the solid support synthesis on the residual three termini of the compound. Imaginable orthogonal cleavable links are allylic compounds, which are cleavable with Pd(0), but also silicon based compounds, which are cleavable with fluoridions, would be appropriate. In the area of the Pd(0)-cleavable link a retrosynthetic cut at this funtionality should be adequate. Here, e.g. a Horner-Wadsworth-Emmons-reaction is imaginable. The aldehydes and phosphorylides needed for this key step could be prepared with good yields, however only one of the two amino acid derivatives was obtained. The synthesis of the other compound didn´t succeed under diverse reaction conditions. Also efforts to prepare analogue Pd(0)-cleavable link molecules through classic Wittig or metathese reactions with Grubbs catalysts of the first and second generation were unsuccessful. The most successful efforts to make a target molecule synthetically accessible for a first liga-tion attempt involved the preparing of a fluorid-cleavable silyl link. Here all requirements stay the same, just instead of allylic compounds silyl ethers are needed. The acid stability of the silyl protecting group depends on the size of the alkyl groups (tert-butyl>isopropyl>ethyl). Firstly, substitution experiments with the compounds Di-tert-butylchlorosilane, Di-tert-butyl-dichlorosilane and Di-tert-butylsilylbis(trifluoromethanesulfonate) were undertaken to estab-lish a connection between PNA and the amino acid through a Di-tert-butyldisilylether. How-ever, the substitutions produced just monosubstituted silanoles, irrespective of the choice of reaction conditions and substitution order. The experiments with the commercially available (3-Cyanopropyl)-diisopropyl-chlorosilane were more successfull. Here the synthesis of one of the target molecules could be achieved.

Übergangsmetall-Komplexe bzw. -Salze von Mangan, Rhodium, Iridium, Palladium und Kupfer wurden mit verschiedenen Aziridinderivaten umgesetzt. Dabei gelang es neutrale und kationische Mono-, Bis-, Tris- und Tetrakis-Aziridin-Komplexe darzustellen. Auch eine übergangsmetallvermittelte Aziridin-Dimerisierung konnte desöfteren beobachtet werden. Dabei entstanden N-(2-Aminoalkyl)aziridin-Komplexe. Durch die gelungene Freisetzung des formal dimerisierten Aziridinliganden, konnte eine übergangsmetallvermittelte Synthese von N-(2-Aminoalkyl)aziridinen beschrieben werden.

Untersuchung öliger Suspensionen als parenterale Depotsysteme für rekombinante Proteine