Computational Tools for the Untargeted Assignment of FT-MS Metabolomics Datasets

Metabolomics is the study of metabolomes, the sets of metabolites observed in living systems. Metabolism interconverts these metabolites to provide the molecules and energy necessary for life processes. Many disease processes, including cancer, have a significant metabolic component that manifests as differences in what metabolites are present and in what quantities they are produced and utilized. Thus, using metabolomics, differences between metabolomes in disease and non-disease states can be detected and these differences improve our understanding of disease processes at the molecular level. Despite the potential benefits of metabolomics, the comprehensive investigation of metabolomes remains difficult. A popular analytical technique for metabolomics is mass spectrometry. Advances in Fourier transform mass spectrometry (FT-MS) instrumentation have yielded simultaneous improvements in mass resolution, mass accuracy, and detection sensitivity. In the metabolomics field, these advantages permit more complicated, but more informative experimental designs such as the use of multiple isotope-labeled precursors in stable isotope-resolved metabolomics (SIRM) experiments. However, despite these potential applications, several outstanding problems hamper the use of FT-MS for metabolomics studies. First, artifacts and data quality problems in FT-MS spectra can confound downstream data analyses, confuse machine learning models, and complicate the robust detection and assignment of metabolite features. Second, the assignment of observed spectral features to metabolites remains difficult. Existing targeted approaches for assignment often employ databases of known metabolites; however, metabolite databases are incomplete, thus limiting or biasing assignment results. Additionally, FT-MS provides limited structural information for observed metabolites, which complicates the determination of metabolite class (e.g. lipid, sugar, etc. ) for observed metabolite spectral features, a necessary step for many metabolomics experiments. To address these problems, a set of tools were developed. The first tool identifies artifacts with high peak density observed in many FT-MS spectra and removes them safely. Using this tool, two previously unreported types of high peak density artifact were identified in FT-MS spectra: fuzzy sites and partial ringing. Fuzzy sites were particularly problematic as they confused and reduced the accuracy of machine learning models trained on datasets containing these artifacts. Second, a tool called SMIRFE was developed to assign isotope-resolved molecular formulas to observed spectral features in an untargeted manner without a database of expected metabolites. This new untargeted method was validated on a gold-standard dataset containing both unlabeled and 15N-labeled compounds and was able to identify 18 of 18 expected spectral features. Third, a collection of machine learning models was constructed to predict if a molecular formula corresponds to one or more lipid categories. These models accurately predict the correct one of eight lipid categories on our training dataset of known lipid and non-lipid molecular formulas with precisions and accuracies over 90% for most categories. These models were used to predict lipid categories for untargeted SMIRFE-derived assignments in a non-small cell lung cancer dataset. Subsequent differential abundance analysis revealed a sub-population of non-small cell lung cancer samples with a significantly increased abundance in sterol lipids. This finding implies a possible therapeutic role of statins in the treatment and/or prevention of non-small cell lung cancer. Collectively these tools represent a pipeline for FT-MS metabolomics datasets that is compatible with isotope labeling experiments. With these tools, more robust and untargeted metabolic analyses of disease will be possible

The isolation, characterisation and chemotaxonomic significance of secondary metabolites from selected South African Laurencia spp. Rhodophyta

Bioprospection of marine organisms as a potential source for lead drugs is becoming increasingly popular. The secondary metabolome of these organisms consists of structurally diverse molecules possessing unprecedented carbon skeletons, the biosynthesis of which occurs via complex metabolomic pathways driven by specialist enzymes. This structural novelty is highly influential on the favourable bioactivity these compounds display. A prominent example of such a compound is trabectedin marketed as Yondelis®. Registered for the treatment of soft tissue sarcomas, this marine drug was developed from extracts of the tunicate Ecteinascidia turbinata. South Africa is renowned for possessing a highly diverse marine biota including several endemic species of marine red algae belonging to the Laurencia sensu stricto genus, which falls within the Laurencia complex. Despite having a good reputation for fascinating secondary metabolites, the taxonomy of Laurencia natural products is proving challenging for reasons including the presence of cryptic species, as well as individual species displaying morphological variability. The aim of this study was thus to isolate secondary metabolites from various South African Laurencia spp. and subsequently assess their chemotaxonomic significance by analysis of a parallel plastid rbcL phylogeny study of Laurencia spp. This study reports the first phycochemical investigation into Laurencia natalensis Kylin, Laurencia cf. corymbosa J.Agardh, Laurencia complanata (Suhr) Kützing, Laurencia sodwaniensis Francis, Bolton, Mattio and Anderson submitted, Laurencia multiclavata Francis, Bolton, Mattio and Anderson submitted, and a South African specimen of Laurenciella marilzae Gil-Rodríguez, Sentíes, Díaz-Larrea, Cassano and M.T. Fujii (basionym: Laurencia marilzae) originally described from Spain. Additionally, the chemical profiles of previously explored species Laurencia flexuosa Kützing and Laurencia glomerata Kützing were re-investigated. The organic extracts of the above species afforded 31 compounds belonging to a wide array of structural classes including halo-chamigranes, linear C15 acetogenins, indole alkaloids, cuparanes and cyclic bromo-ethers. A new tri-cyclic keto-cuparane (4.4) was isolated from L.cf. corymbosa alongside the new cuparanes 4.1 and 4.7. Algoane (5.9), a unique marker compound isolated from L. natalensis, was previously only reported from a sea-hare. Such marker compounds which are exclusive to an individual algal species increase the ease of their subsequent identification. The feasibility of chemotaxonomy as an additional tool to classify Laurencia spp. Was established as broad predictions of a specimen’s phylogeny, based on representatives of its secondary metabolome, proved viable. The study specimens were shown to possess similar chemical profiles to their sister species e.g. L. complanata, L. sodwaniensis and L. multiclavata produced similar metabolites to their sister species as inferred by an rbcL phylogeny tree. Finally, a 1H NMR profiling study on the crude organic extracts of various Laurencia spp. generated distinctive, reproducible spectra, exposing the value of NMR spectroscopy as a rudimentary species discernment tool

Development and Application of Chemometric Methods for Modelling Metabolic Spectral Profiles

The interpretation of metabolic information is crucial to understanding the functioning of a biological system. Latent information about the metabolic state of a sample can be acquired using analytical chemistry methods, which generate spectroscopic profiles. Thus, nuclear magnetic resonance spectroscopy and mass spectrometry techniques can be employed to generate vast amounts of highly complex data on the metabolic content of biofluids and tissue, and this thesis discusses ways to process, analyse and interpret these data successfully. The evaluation of J -resolved spectroscopy in magnetic resonance profiling and the statistical techniques required to extract maximum information from the projections of these spectra are studied. In particular, data processing is evaluated, and correlation and regression methods are investigated with respect to enhanced model interpretation and biomarker identification. Additionally, it is shown that non-linearities in metabonomic data can be effectively modelled with kernel-based orthogonal partial least squares, for which an automated optimisation of the kernel parameter with nested cross-validation is implemented. The interpretation of orthogonal variation and predictive ability enabled by this approach are demonstrated in regression and classification models for applications in toxicology and parasitology. Finally, the vast amount of data generated with mass spectrometry imaging is investigated in terms of data processing, and the benefits of applying multivariate techniques to these data are illustrated, especially in terms of interpretation and visualisation using colour-coding of images. The advantages of methods such as principal component analysis, self-organising maps and manifold learning over univariate analysis are highlighted. This body of work therefore demonstrates new means of increasing the amount of biochemical information that can be obtained from a given set of samples in biological applications using spectral profiling. Various analytical and statistical methods are investigated and illustrated with applications drawn from diverse biomedical areas

Encoding, Storing and Searching of Analytical Properties and Assigned Metabolite Structures

Informationen über Metabolite und andere kleine organische Moleküle sind von entscheidender Bedeutung in vielen verschiedenen Bereichen der Naturwissenschaften. Sie spielen z.B. eine entscheidende Rolle in metabolischen Netzwerken und das Wissen über ihre Eigenschaften, hilft komplexe biologische Prozesse und komplette biologische Systeme zu verstehen. Da in biologischen und chemischen Laboren täglich Daten anfallen, welche diese Moleküle beschreiben, existiert eine umfassende Datengrundlage, die sich kontinuierlich erweitert. Um Wissenschaftlern die Verarbeitung, den Austausch, die Archivierung und die Suche innerhalb dieser Informationen unter Erhaltung der semantischen Zusammenhänge zu ermöglichen, sind komplexe Softwaresysteme und Datenformate nötig. Das Ziel dieses Projektes bestand darin, Anwendungen und Algorithmen zu entwickeln, welche für die effiziente Kodierung, Sammlung, Normalisierung und Analyse molekularer Daten genutzt werden können. Diese sollen Wissenschaftler bei der Strukturaufklärung, der Dereplikation, der Analyse von molekularen Wechselwirkungen und bei der Veröffentlichung des so gewonnenen Wissens unterstützen. Da die direkte Beschreibung der Struktur und der Funktionsweise einer unbekannten Verbindung sehr schwierig und aufwändig ist, wird dies hauptsächlich indirekt, mit Hilfe beschreibender Eigenschaften erreicht. Diese werden dann zur Vorhersage struktureller und funktioneller Charakteristika genutzt. In diesem Zusammenhang wurden Programmmodule entwickelt, welche sowohl die Visualisierung von Struktur- und Spektroskopiedaten, die gegliederte Darstellung und Veränderung von Metadaten und Eigenschaften, als auch den Import und Export von verschiedenen Datenformaten erlauben. Diese wurden durch Methoden erweitert, welche es ermöglichen, die gewonnenen Informationen weitergehend zu analysieren und Struktur- und Spektroskopiedaten einander zuzuweisen. Außerdem wurde ein System zur strukturierten Archivierung und Verwaltung großer Mengen molekularer Daten und spektroskopischer Informationen, unter Beibehaltung der semantischen Zusammenhänge, sowohl im Dateisystem, als auch in Datenbanken, entwickelt. Um die verlustfreie Speicherung zu gewährleisten, wurde ein offenes und standardisiertes Datenformat definiert (CMLSpect). Dieses erweitert das existierende CML (Chemical Markup Language) Vokabular und erlaubt damit die einfache Handhabung von verknüpften Struktur- und Spektroskopiedaten. Die entwickelten Anwendungen wurden in das Bioclipse System für Bio- und Chemoinformatik eingebunden und bieten dem Nutzer damit eine hochqualitative Benutzeroberfläche und dem Entwickler eine leicht zu erweiternde modulare Programmarchitektur

CHARACTERIZATION OF THE STRUCTURE, FUNCTION, AND PROTEIN-PROTEIN INTERACTIONS INVOLVED IN THE ASSEMBLY OF THE TYPE III SECRETION SYSTEM TIP COMPLEX AND THE TRANSLOCON OF SALMONELLA AND SHIGELLA

The type III secretion system (T3SS) is a macromolecular structure assembled by many Gram-negative bacteria in order to invade target host cells. A functional T3SS contains a syringe-like structural component known as the needle apparatus, which works in concert with an export apparatus that recognizes the cargo and an ATPase complex that energizes the transport of bacterial effector proteins. Effectors transported directly into the host cell cytoplasm modulate host cellular functions such as cytoskeletal dynamics and cellular signaling in order to enable the pathogens to invade, survive, and multiply within the host environment. Gram-negative bacteria harboring the T3SS include Salmonella, Shigella, enteropathogenic E. coli, Yersinia, Burkholderia, Pseudomonas, as well as Chlamydia. These organisms are responsible for infectious diseases in humans and pose a threat to human health worldwide. Inactivation of the T3SS, by knocking out structural or functional proteins, renders pathogens incapable of causing infection. Salmonella and Shigella are responsible for millions of cases of food-borne diarrhea annually throughout the world. In addition, large-scale food recalls due to frequent outbreaks of food poisoning has a negative impact on the food industry in the United States. No preventive vaccines are available against Salmonella and Shigella. Study of the T3SS thus has a scope for the development of strategies to combat these pathogens. The T3SS among different bacterial species share common features but also show unique structural and functional characteristics. Therefore, the T3SS provides a suitable target for the development of specific anti-infectives. The needle apparatus of the T3SS consists of a base followed by an extracellular needle. The needle is attached to a tip complex and a translocon. The tip complex serves as a platform for the assembly of the translocon that punctures a translocation pore within the host cell membrane. The tip complex is assembled from several copies of a hydrophilic tip protein and the translocon is assembled from two hydrophobic translocon proteins. This dissertation describes structural and functional studies, as well as characterization of the protein-protein interactions that are important in the assembly of the tip complex and the translocon of Salmonella and Shigella. In Salmonella, the tip complex is formed by the tip protein SipD. A translocon made up of the translocon proteins SipB and SipC is attached on the tip complex. A combination of X-ray crystallography, Nuclear magnetic resonance (NMR) spectroscopy, site-directed mutagenesis, as well as functional assays were applied to determine the structure of the Salmonella tip protein SipD and show that the C-terminus is crucial for the function and an antiparallel β-sheet is important for the tertiary structure of SipD. The function of the T3SS can be regulated by small molecules such as bile salts. The T3SS of Salmonella is down regulated by bile salts. The tip protein SipD directly binds to the bile salt deoxycholate using an unknown mechanism. The crystal structures of SipD bound to bile salts reported herein show that the interaction between bile salts and SipD is predominantly hydrophobic. Further, deoxycholate induced the degradation of the Salmonella translocon protein SipB. These observations have led to the hypothesis that deoxycholate might interfere with the interaction between SipD and SipB leading to a down regulation of the T3SS in Salmonella. The structure of the translocon and how it is attached to the tip complex is not clear. Preliminary structural characterization of a folded, hydrophilic domain at the N-terminus of SipB was undertaken to locate two fragments of SipB within residues 82-240 and 82-226, which produced well-dispersed 2D-NMR spectra. Paramagnetic relaxation enhancement (PRE) was employed to define the interaction between SipD and the N-terminal hydrophilic domain of SipB. A region within residues Asp207-Asn283 of SipB bound to a mixed α/β region of SipD. PRE was also used to study how the Shigella tip protein IpaD binds to its needle protein MxiH in order to assemble the tip complex in Shigella. MxiH was shown to bind to the lower portion of a coiled coil region in IpaD. Secretion through the T3SS is enabled by extensive cross-talk within its components. The assembly of the needle apparatus requires polymerization of multiple copies of several different proteins. Regulation of secretion is most probably an outcome of conformational changes relayed in sequence through the needle apparatus. Work described in this dissertation shows that weak protein-protein interactions are a common theme in the assembly of the needle apparatus. Further, T3SS proteins contain discreet functional domains. For example, the coiled coil of the tip protein allows the assembly of the tip complex while the mixed α/β region attaches to the translocon. The structure of theT3SS proteins varies depending upon the role it plays. For example, the extracellular needle is assembled from a small polar protein but the translocon proteins contain both hydrophilic and hydrophobic domains. Nevertheless, further studies are required to fully appreciate certain aspects of the T3SS such as how large proteins are transported through the needle, how the T3SS switches between active and inactive states of secretion, how substrate specificity is controlled, and how effector secretion is energized. Complete understanding of theT3SS requires the determination of high resolution structures of the needle apparatus, direct binding studies analyzing how isolated components of the needle apparatus interact with each other and behave in vitro, computational modeling of larger substructures, and functional assays to test the physiological implications of these in vitro studies

The Use of Spectroscopic Techniques in the Characterization of Mycobacterial Metabolites

The mycobacterial cell wall metabolites have always imposed great challenges to researchers due to their unusual complexity and structural diversity. A lot of research efforts have been directed towards the evaluation of these metabolites and the role they play in the pathogenesis and virulence of different serious human pathogens including Mycobacterium tuberculosis the causative agent of tuberculosis (TB). In the genomic era, it is crucial to develop new methodologies to analyze these components from a global perspective in a comprehensive and well-validated manner. Towards this end, we developed a rapid NMR-based approach to produce metabolic profiles in the form of 2D1H-13C HSQC maps. Due to NMR low sensitivity, we used 13C-isotope enrichment strategy to improve the detection threshold of NMR. We developed and investigated the use of three NMR-based profiles. The first is for the cell wall associated lipids, the second is for covalently bound mycolic acids and the third deals with the complex cell wall polysaccharides. Key structural features and major lipid species were readily identifiable using this technique. This approach can be used for observing changes in the cell wall due to drug treatment, gene mutation and changes in the physiological environment, species characterization and screening for virulence factor expression. It also allows for rapid comparative analysis of several cell wall metabolites within the same sample. We have used this approach to evaluate the structural diversity displayed in the cell wall metabolites among different Mycobacteria from different clades. We have successfully used the same technique to monitor changes in the carbohydrate and lipid pools from Mycobacteria grown under laboratory conditions that simulate latency and resuscitation from latency. Moreover, we were also able to use such technique to screen for virulence factor in severalM. tuberculosis clinical isolates. Although we have successfully developed and used an NMR approach for metabolite analysis, NMR however remains less sensitive than other spectroscopic techniques like mass spectroscopy (MS). The high sensitivity of MS makes it one of the best candidates for metabolomic studies. However in case of cellular lipids, the low polarity and the variable ionization potential deter its routine use for lipid analysis. Certain metabolites that have low cellular abundance and good ionizability are better analyzed through an MS-based approach. Mycolactones of M. ulcerans and other closely related species are good examples for such metabolites and in this work we have demonstrated the utility of MS as a tool for screening for mycolactones from both bacterial culture and tissue samples. Metabolomics is currently a rapidly growing field and many researchers have directed their attention in developing the right research tools to explore it. Spectroscopic methods along with chromatographic methods have been in the front of this vigorous research effort. The work presented here represents an innovative attempt to utilize such techniques to investigate the metabolome of some of the most serious human pathogens and discusses the potential area where the application of these techniques can be the most useful

Natural Product from the Deep Sea

After the long history of screening, it is becoming difficult to find novel compounds from microorganisms and plants anywhere in the world. Until now, more than about 30,000 marine natural products have been reported. However, with the development of marine natural products research, the hit rate of new compounds is also decreasing. Scientists are now turning their attention to the deep sea, where a high hit rate of novel compounds is expected. Many small compounds and peptides from microorganisms and sponges are with therapeutic activity are shown in this book. This Special Issue Book, “Natural Products from the Deep Sea”, should be useful for the screening of novel and useful compounds from nature

Dedicated to the 55th Anniversary of G.B. Elyakov Pacific Institute of Bioorganic Chemistry of the Far Eastern Branch of the Russian Academy of Sciences

The G.B. Elyakov Pacific Institute of Bioorganic Chemistry of the Far-Eastern Branch of the Russian Academy of Sciences (PIBOC FEB RAS) was founded in 1964 in Vladivostok in the Far East of Russia. Over many years, we have been carrying out studies on the natural products of both marine and terrestrial origin. In collaboration with many Russian and foreign scientists, we have investigated many hundreds of diverse biomolecules, including steroids and terpenoids, quinoid compounds and alkaloids, polysaccharides and lipids, enzymes and lectins, proteins, and peptides. The Institute has a collection of marine microorganisms (KMM) PIBOC, which includes more than 4000 strains of marine bacteria and more than 1000 strains of marine fungi. The biological activity of natural compounds is also being studied. This book includes the 14 manuscripts which covered almost all aspects of PIBOC research activity in the fields of bioorganic chemistry, biochemistry, organic synthesis of natural compounds, marine microbiology, and genetic engineering, and we hope it will provide interesting new information for scientists working in these fields

Charakterisierung eines Protein:RNA-Komplexes : die Funktion von ribosomal binding factor A

In this thesis the three dimensional solution strucutre of the RbfA protein from Thermotoga maritima was solved using multidimensional heteronuclear NMR spectroscopy. The RbfA protein binds to the helix I region of the 16S rRNA. To gain insights into the binding mode of RbfA to its target, a second RbfA construct from Helicobacter pylori was used. Comparison of the RbfA proteins with the published structure of RbfA from Escherichia coli, led to studies concerning the differences between proteins from thermophile and mesophile systems. In the second part of this thesis the native binding motive of the RbfA protein was identified. The RbfA protein binds to an alternate helix fold within the pre-sequence of the immature 16S rRNA.In der vorliegenden Dissertationsschrift wurde die Raumstruktur eines 120 Aminosäure langen Proteins mittels mehrdimensionaler, heteronuklearer NMR-Spektroskopie bestimmt. Es handelt sich dabei um das Kälteschockprotein ribosomal binding factor A aus Thermotoga maritima. Das RbfA Protein bindet and die Helix I Region der 16S rRNA. Um den Bindungsmodus zwischen RbfA und der 16S rRNA näher zu untersuchen wurde ein zweites Konstrukt von RbfA aus Helicobacter pylori verwendet. Durch den Vergleich mit der schon bekannten Struktur des RbfA Proteins aus Escherichia coli wurden Untersuchungen über die Unterschiede zwischen mesophilen und thermophilen Proteinen durchgeführt. Im zweiten Teil der Arbeit wurde der RNA Ligand für das RbfA Protein identifiziert. Die native Bindungsstelle des RbfA Proteins ist eine alternative Helixfaltung in der Prä-Sequenz der unreifen 16S rRNA

Characterization of proteorhodopsin 2D crystals by electron microscopy and solid state nuclear magnetic resonance

Proteorhodopsin (PR) originally isolated from uncultivated γ-Proteobacterium as a result of biodiversity screens, is highly abundant ocean wide. PR, a Type I retinal binding protein with 26% sequence identity, is a bacterial homologue of Bacteriorhodopsin (BR). The members within this family share about 78% of sequence identity and display a 40 nm difference in the absorption spectra. This property of the PR family members provides an excellent model system for understanding the mechanism of spectral tuning. Functionally PR is a photoactive proton pump and is suggested to exhibit a pH dependent vectorality of proton transfer. This raises questions about its potential role as pH dependent regulator. The abundance of PR in huge numbers within the cell, its widespread distribution ocean wide at different depths hints towards the involvement of PR in utilization of solar energy, energy metabolism and carbon recycling in the Sea. Contrary to BR, which is known to be a natural 2D crystal, no such information is available for PR til date. Neither its functional mechanism nor its 3D structure has been resolved so far. This PhD project is an attempt to gain a deeper insight so as to understand structural and functional characterization of PR. The approach combines the potentials of 2D crystallography, Atomic Force Microscopy and Solid State NMR techniques for characterization of this protein. Wide range of crystalline conditions was obtained as a result of 2D crystallization screens. This hints towards dominant protein protein interactions. Considering the high number of PR molecules reported per cell, it is likely that driven by such interactions, the protein has a native dense packing in the environment. The projection map represented low resolution of these crystals but suggested a donut shape oligomeric arrangement of protein in a hexagonal lattice with unit cell size of 87Å*87Å. Preliminary FTIR measurements indicated that the crystalline environment does not obstruct the photocycle of PR and K as well as M intermediate states could be identified. Single molecule force spectroscopy and atomic force microscopy on these 2D crystals was used to probe further information about the oligomeric state and nature of unfolding. The data revealed that protein predominantly exists as hexamers in crystalline as well as densely reconstituted regions but a small percentage of pentamers is also observed. The unfolding mechanism was similar to the other relatively well-characterized members of rhodopsin family. A good correlation of the atomic force microscopy and the electron microscopy data was achieved. Solid State NMR of the isotopically labeled 2D crystalline preparations using uniformly and selectively labeling schemes, allowed to obtain high quality SSNMR spectra with typical 15N line width in the range of 0.6-1.2 ppm. The measured 15N chemical shift value of the Schiff base in the 2D crystalline form was observed to be similar to the Schiff base chemical shift values for the functionally active reconstituted samples. This provides an indirect evidence for the active functionality of the protein and hence the folding. The first 15N assignment has been achieved for the Tryptophan with the help of Rotational Echo Double Resonance experiments. The 2D Cross Polarization Lee Goldberg measurements reflect the dynamic state of the protein inspite of restricted mobility in the crystalline state. The behavior of lipids as measured by 31P from the lipid head group showed that the lipids are not tightly bound to the protein but behave more like the lipid bilayer. The 13C-13C homonulear correlation experiments with optimized mixing time based on build up curve analysis, suggest that it is possible to observe individual resonances as seen in case of glutamic acid. The signal to noise was good enough to record a decent spectrum in a feasible period. The selective unlabeling is an efficient method for reduction in the spectral overlap. However, more efficient labeling schemes are required for further characterization. The present spectral resolution is good for individual amino acid investigation but for uniformly labeled samples, further improvement is required.Proteorhodopsin (PR) wurde ursprünglich aus nicht kultivierten γ-Proteobakterium isoliert und ist in großen Mengen in den Ozeanen enthalten. PR ist wie sein homolog Bakteriorhodopsin (BR) ein TypI Retinal Bindeprotein und die Sequenzen sind zu 26% identisch. Innerhalb der PR Familie haben die Mitglieder eine Sequenzhomologie zu ungefähr 78% und zeigen einen Unterschied von 40 nm im absorptions spektrum. Diese Eigenschaft bietet ein gutes Modelsystem um zu verstehen durch welchen Mechanismus das Absorptionsspektrum moduliert wird. PR ist ein photoaktive Protonenpumpe und es wird angenommen, dass die Richtung des Protonentransfers vom pH-wert abhängt, was auf eine Rolle als ein pH abhängiger Regulator hindeutet. Da PR sowohl in der Zelle in hoher Zahl, als auch in den Ozeanen in unterschiedlichen Tiefen weit verbreitet ist, wird angenommen, dass PR bei der Verwertung von Sonnenlicht, im Energiestoffwechsel und beim Kohlenstoffumsatz beteiligt ist. Im Gegensatz zu BR, welches bekannterweise 2D Kristalle bildet, ist etwas vergleichbares für PR bis heute nicht bekannt. Weder der Mechanismus von PR noch seine 3D Struktur sind bisher gelöst. Die vorliegende Doktorarbeit versucht offene Punkte zum Mechanismus und zur Struktur von PR zu klären. Für die Charakterisierung werden 2D Kristallographie, "Atomic Force Microscopy" und Festkörper NMR verwendet. Für die Bildung von 2D Kristallen konnte eine große Auswahl an Kristallisationbedingungen ermittelt werden, was auf deutliche Protein Protein Wechselwirkungen hindeutet. Zieht man die hohe Zahl an PR Molekülen pro zelle in betracht, ist es wahrscheinlich, dass durch diese Interaktionen auch in der natürlichen Membran eine dichte Packung der Proteine auftritt. Elektronenmikroskopische Aufnahmen mit geringer Auflösung deuten auf eine ringförmige Anordnung der Proteine in einem hexagonalen Gitter mit einer Einheitszelle von 87Å * 87Å. Vorläufige FTIR Messungen deuten darauf hin, dass diese Anordnung den Photozyklus nicht behindert und sowohl K als auch M Zustand konnten identifiziert werden. Um weitere Informationen über den Oligomerisierungszustand der 2D Kristalle zu gewinnen wurden Einzelmolekül - und Rasterkraft Mikroskopie durchgeführt. Hierbei zeigte sich, dass das Protein in kristallinen und dicht rekonstituierten Regionen überwiegend als Hexamer vorliegt. Daneben kann zu einem geringen Anteil auch ein pentamerer Zustand beobachtet werden. Der Mechanismus der Proteinentfaltung war vergleichbar zu anderen, besser untersuchten Mitgliedern der Rhodopsinfamilie. Zwischen den Daten aus der "Atomic Force Microscopy" und der Elektronenmikroskopie zeigt sich eine gute Korrelation. Festkörper NMR an vollständig und selektiv markierten 2D Kristallen ergaben Spektren mit einer typischen 15N Linienbreite von 0,6 bis 1,2 ppm. Die 15N chemische Verschiebung der Schiffschen Base hat im Kristall den gleichen Wert wie funktional aktiv rekonstitutierte Proben, was indirekt die Funktionalität und die korrekte Faltung bestätigt. Die Zuordnung der 15N Signale für Tryptophan wurde durch "Rotational Echo Double Resonance" Experimente vorgenommen. 2D kreuzpolarisation Lee Goldburg Messungen zeigen den dynamischen Zustand des Proteins trotz der eingeschränkten Mobilität im kristallinen Zustand. Das Verhalten der Lipide wurde mit 31P messungen der Lipidkopfgruppe untersucht und zeigt, dass diese nicht fest gebunden sind, sondern sich mehr wie in einer Lipiddoppelschicht verhalten. Für 13C-13C homonukleare korrelations Experimente wurde die Mischzeit durch die Analyse von Aufbaukurven optimiert. Diese Versuche deuten darauf hin, dass es möglich ist einzelne Resonanzen aufzulösen, wie im Fall des Glutamat gezeigt mit einem gutem Signal zu Rauschen Verhältnis. Selektives "unlabeling" ist eine effizente Methode um die Ueberlappung der Signal zu reduzieren. Darüberhinaus sind für eine weitere Chrakterisisierung effizentere Markierungsschemata notwendig. Die bisherige spektrale Auflösung ist gut genug für die Untersuchung einzelner Aminosäuren, für vollständig markierte Proben sind weitere Verbesserungen notwendig